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LARVAL FEEDING ECOLOGY OF AN OPHELES QUADRIMACULAIQS
(SAY) AND A_N_. PUNCTIPENNIS (SAY) (DIPTERA: CULICIDAE) IN
SOUTHCENTRAL MICHIGAN PONDS

By

John Robert Wallace

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Entomology

1997

Copyright by
John Robert Wallace
1997

 

LARV
(SAY) l

Fi
feeding (

ABSTRACT

LARVAL FEEDING ECOLOGY OF AN QPHELES QUADRIMAQQQTUS
(SAY) AND AN. BUNCIIIPENNIS (SAY) (DIPTERA: CULICIDAE) IN
SOUTHCENTRAL MICHIGAN PONDS

By

John Robert Wallace

Field and laboratory studies were conducted to investigate the larval
feeding ecology of Anopheles guadrimaculatus (Say) and Anopheles
punctipennis (Say) in southcentral Michigan ponds. Available food resources
for Q. guadrimaculatus found within the surface microlayer of a permanent
pond were very consistent regardless of season or microhabitat. Within the
surface microlayer, bacteria were the most abundant food type, followed by
algae, detritus and invertebrate parts/protozoans (IPP). Larvae consumed
more algae and IPP particles in the algal clump and emergent plant base
microhabitats. A signiﬁcant number of cladocerans were observed in larval
guts, a previously unimportant component anopheline feeding ecology in
nature. Total bacteria numbers were higher in the vernal than the aestival
pond. However, algal, detritus and IPP numbers were in greater abundance in
the surface microlayer of the aestival pond than the vernal pond. Q.
pungtipennis larval diets differed between pond types. Larvae in the vernal
pond consumed more bacteria and algae than larvae in the aestival pond and
more IPP and detritus from the latter.

Diel feeding studies showed that &. guadrimaculatus larvae consumed
more bacteria, algae and detritus at midnight than noon. Microinvertebrate

consumption differed over a diel period. Larvae consumed more cladocerans at

 

midnight 1
vegetated
within the
mites in 0
signiﬁcan

for rotifer

deveIOpn-
in their r
Survivors
microha}

adequate

hiEllest i

 

midnight than noon and more water mites (Elayidae) from open water versus
vegetated habitats. These differences can be explained by increased numbers
within the surface microlayer habitat of cladocerans at midnight and water
mites in open water areas. However, larval consumption of rotifers was
signiﬁcantly greater in open water than vegetated habitats. Habitat analysis
for rotifer abundance indicates no signiﬁcant difference between habitats. A
comparison between guts and habitat dietary components indicated that An.
W larvae appear to preferentially feed on rotifers in open
water habitats.

Q. guadrimaculatus larval habitat signiﬁcantly affected larval growth,
development and survivorship in the ﬁeld. Larval feeding success is reﬂected
in their microhabitats, i.e. larval growth and developmental rates as well as
survivorship and adult size are signiﬁcantly impacted by the larval
microhabitats within a permanent pond. Algal clumps did not provide
adequate refuge from predation by Notgnecta sp., therefore, survivorship was

highest in the non-predator treatments.

This dis
biol<

 

This dissertation is dedicated to the man who encouraged me as a budding
biologist and believed in me as a scientist, Dr. Frederick O. Howard.

 

 

 

 

My
W. Merrit
endeavor.
hold this .
Walker,
their valL
as excelle

Edman f

 

lorgan (I

 

 

ACKNOWLEDGMENTS

My deepest appreciation and gratitude go to my advisor, Dr. Richard
W. Merritt for his guidance and unconditional support throughout this
endeavor. I am extremely thankful for his recognition of the underdog and
hold this quality of his in high regard. My graduate committee, Drs. Ned
Walker, Ed Graﬁus, Mike Kaufman and Don Hall deserve many thanks for
their valuable assistance with all aspects of this research, they have served
as excellent mentors. I thank Drs. Doug Craig, Roger Wotton, and John
Edman for their many helpful suggestions on experimental design and
procedures. Special thanks go to Dr. Heather Proctor of Queen's University
for water mite identiﬁcations and to Ethan Nedeau for the scientiﬁc
illustrations.

I thank the farm manager at Collins Road, Cliff Zehr for his assistance
and access to research property and water management. The tireless efforts
by Dr. George Ayers for his negotiating stamina with Land Management
personnel is greatly appreciated. A sincere acknowledgment to those student
workers who worked in the trenches during this project, especially Tim
Duniec, Lina Lenberg, Jeff Terwin. Lab technicians, 'h‘acy Smith, Bill
Morgan and Terry Davis deserve special mention for their assistance with lab
techniques and computer advice. I thank Jeff White, Audrey Armoudlian,
Mike Alexander, and Dr. Beth Capaldi for their scholarship, friendship and
peer review of all work, (The key to scientiﬁc creativity is through wasabi).
To Drs. Mark Scriber, Jim Miller, Walt Pett, Fred Stehr, Cathy Bristow,
Eileen Van Tassel, in addition to Lissa Leege, Mike Higgins, John Wilterding,

vi

 

Jim Zablc
Mom/Dad
Bishop fa
provoking
the super
Entomolc

I (
NIH Gra:
of Entom
(Inter-mat
support 11

La
Partner 2
Patience,
i“Spirati.

the light

 

 

Jim Zablotny (Skeletor lives), Fred Warner, Bill Sobczak, Bob Steltzer,
Mom/Dad, Shiner/Kelly, J enni/Mark and Allie/Brooke, The Stackpole and
Bishop families, Tod Hull and the Merritt/Walker labs for their thought
provoking discussions and support over the past several years. I also thank
the superb secretarial and adminstrative staff in the Department of
Entomology at MSU.

I obtained funding to conduct this research from a number of sources,
NIH Grant # A121884, Ecology & Evolutionary Biology Program, Department
of Entomology, Dipterology Fund (ESA), Global Young Scholar Grant
(International Study Program). I am extremely grateful for all ﬁnancial
support provided by these entities.

Last, but certainly not least, I wish to thank my wife, Susan. She is my
partner and friend and I could not have completed this program without her
patience, love and understanding during this long process. Much of my
inspiration to ﬁnish came from my smiling, happy son, Harrison. They are

the light that ﬁlls my day.

vii

 

 

LIST OF T.~‘
LIST OF Fl
INIRODU

cnmR
FEE

INT
ME?
REE
DIS
LIT

CHAPTEI
DIE

TABLE OF CONTENTS

Page
LIST OF TABLES ................................................................................. x
LIST OF FIGURES ................................................................................. xiii
INTRODUCTION ................................................................................. 1

CHAPTER 1
FEEDING ECOLOGY OF LARVAL AN OPHELES MOSQUITOES:
A SEASONAL COMPARISON BETWEEN MICROHABITATS
WITHIN PERMANENT AND TEMPORARY PONDS IN

MICHIGAN ................................................................................... 18
ABSTRACT ........................................................................................... 18
INTRODUCTION ................................................................................. 21
METHODS ............................................................................................ 24
RESULTS .............................................................................................. 34
DISCUSSION ........................................................................................ 63
LITERATURE CITED .......................................................................... 70

CHAPTER 2
DIEL PERIODIC FEEDING ACTIVITY OF AN OPHELES
QUADRIMACULATU S (SAY) MOSQUITO LARVAE ............. 78
ABSTRACT ............................................................................................ 78
INTRODUCTION .................................................................................. 80
METHODS ............................................................................................. 81
RESULTS ............................................................................................... 88
DISCUSSION ........................................................................................ 104
LITERATURE CITED .......................................................................... 112

viii

 

CHAPTER
POP

ABS'
INTI
MET
RES‘
DISC
LITE

CONCLUSI

APPENDIC
APPENDIX
APPENDIX

 

 

CHAPTER 3

POPULATION DYNAMICS OF AN OPHELES QUADRIMACULATUS:
THE EFFECTS OF HABITAT, NUTRITION AND PREDATION

ON LARVAL GROWTH AND SURVIVORSHIP ......................... 117
ABSTRACT ............................................................................................. 117
INTRODUCTION ................................................................................... 119
METHODS .............................................................................................. 121
RESULTS ................................................................................................ 126
DISCUSSION .......................................................................................... 145
LITERATURE CITED ............................................................................ 154

CONCLUSIONS AND RECOMMENDATIONS .............................................. 160
APPENDICES

APPENDDI A: Record of Deposition of Voucher Specimens
APPENDDI B: Voucher Specimen Data

 

CHAPTEI
Table 1. M
thre
witl
PLS

Table 2. Cc
(C°)
East

Table 3. A
surfs

pen:

Table 4 Se;

1 ml
1996

Table 5, Se:

 

LIST OF TABLES

CHAPTER 1

Table 1. Mean water chemistry values (Units = ppm, soluble salts = MMHO) for
three microhabitats (open water, algal clump and emergent plant base)
within the permanent pond, East Lansing, MI 1996. AN OVA, Fisher's
PLSD, (SEM) ................................................................................................ 37

Table 2. Comparison between the mean daily maximum/minimum temperatures
(C°) of a temporary vernal and permanent pond, June 14 - August 9, 1996,
East Lansing, MI ........................................................................................ 41

Table 3. An abbreviated list of the algae identifed to generic level from
surface microlayer water samples in open water and vegetated habitats of a
permanent pond (Collins Road pond), East Lansing, Michigan, 1994 ..... 43

Table 4. Seasonal difference between mean number bacteria particles per
1 ml sample of surface microlayer water during June - September, 1995,
1996. Samples were collected from the permanent pond .......................... 45

Table 5. Seasonal difference between mean number algae, IPP and detritus
particles per 1 ml sample of surface microlayer water during June -
September, 1995, 1996. Samples were collected from the permanent pond
....................................................................................................................... 46

Table 6. The mean number (log/ml) of each bacterial food type found within a
vernal and aestival pond, East Lansing, MI 1995 ...................................... 49

Table 7. The mean number (log/ml) of algal, invertebrate parts/protozoans (IPP)
and detritus food types found within a vernal and aestival pond, East
Lansing, MI 1995 ......................................................................................... 50

 

Table 8. C
baci
surf
AN(

vs. c
CHAPTER

Table 1. Me
disse

HOOD

Table 2. Me
samp
micro

Table 3. Me;
insta:
(Third

24 mi(

Table 4 3183

instal-
1995.(

 

 

Table 8. Control experiment testing the mean number of rod, cocci, total
bacteria, algae, invertebrate parts and protozoans observed in the
surface microlayer of inside vs. outside larval microcosms, two factor
AN OVA was used to test microhabitat (N=2), sample location (N=2) (inside
vs. outside), (N=5 samples/location) for 1995 ............................................ 56

CHAPTER 2

Table 1. Mean number of microbes found within a lml sample (N=6 per sample)

dissected from third and fourth instar Q3, We larval guts at
noon and midnight ........................................................................................ 89

Table 2. Mean concentration/ml of microbial particles observed in water
samples collected from the surfacemicrolayer inside vs. outside of the ﬁeld
microcosms, (SEM). N 0 signiﬁcant differences were observed .................. 93

Table 3. Mean number of microinvertebrates dissected from third and fourth
instar An. W3 at noon and midnight, ﬁrst trial, July, 1995.
(Third instars: N =48, 24 noon, 24 midnight; Fourth instars: N=48, 24 noon,
24 midnight ................................................................................................... 97

Table 4. Mean number of microinvertebrates dissected from third and fourth
instar An. mm at noon and midnight, second trial, August,
1995. (Third instars: N=53, 25 noon, 28 midnight; Fourth instars: N=57, 29
noon, 28 midnight ......................................................................................... 99

CHAPTER 3

Table 1. Mean RGR (% mg/mg/day) and dry weight (mg) measurements for An.
mm male and female adults in the ﬁeld growth study ........ 130

Table 2. Mean wing length for female An. W mosquitoes reared in
two habitats (open water and vegetation) and three treatments: algal
clump, algal clump/predator and predator ................................................. 135

 

 

Table 3. 'l
deV
lab

Table 4. L
w

regi
K. k

Table 5. Ar
coefi
food

 

 

Table 3. Temperature, food quality, and quantity effects on An. W
development rate, adult dry weights and female wing length in the
laboratory ..................................................................................................... 141

Table 4. Life table on the effects of temperature, food type and amount on An.

W mortality through immature life stages.Data are
regression coefficients calculated when k-values are regressed on their sum
K. k: mortality for each instar, K = mortality for entire cohort ............... 143

Table 5. Analysis of variance and MAN OVA on individual instar regression

coefﬁcients calculated from key factor analyses to determine temperature,
food type and amount eﬂ‘ects on instar mortality ...................................... 144

xii

LIST OF FIGURES

CHAPTER 1

Figure 1. Historical distribution of Anopheles We (Say) in
Michigan (Modiﬁed from Sabroskey 1946) ................................................ 23

Figure 2. Sketch of ﬂoating microcosm for ﬁeld experiments. a) 4 L plastic
container, b) N ytex 100um mesh bottom and side panels, c) styrofoam
ﬂoats, (1) garden stake for support, e) ﬁsh line to anchor microcosm to
stake, f) water surface, g) cattails, h) substrate ........................................ 31

Figure 3. Daily maximum/minimum surface water temperature (C°) for two
habitats, (open water vs. vegetated areas) within a permanent pond
East Lansing, MI for July and August, 1995 ............................................. 35

Figure 4. Maximum/minimum surface water temperature (C°) for the
permanent pond during June 14 - August 9, 1995, East Lansing,
MI .................................................................................................................. 36

Figure 5. Daily maximum/minimum temperatures (C°) for the temporary
vernal pond, June 14 - August 7, 1996, East Lansing, MI ........................ 39

Figure 6. pH from the temporary vernal pond, May 18 - July 7, 1996, East
Lansing, MI .................................................................................................. 40

Figure 7. Total bacteria, algae, invertebrate parts/protozoans (IPP) and
detritus in surface microlayer water samples on a seasonal basis for
1995 and 1996 in East Lansing, MI. Error bars represent SEM (N=3

samples/month) ............................................................................................ 44
Figure 8. Percentage of microbial particles per milliliter of sample (N=5 per

sample) in three microhabitats: open water, algal clump, and emergent
plant base in a permanent pond A) 1995, B) 1996 ..................................... 48

xiii

 

Fi

Fi,

Fl;

Fi

Fig

Figure 9. Percentage of particles per sample (N=5 per sample) from two
microhabitats (Floating Debris and Tree Base) in a vernal and aestival
pond, East Lansing, MI 1995 ....................................................................... 52

Figure 10. Seasonal differences in percentage of microbial components found
in the surface microlayer of a vernal pond in East Lansing, MI, 1996.
A) ﬂoating debris microhabitat, B) tree base microhabitat (n=5 per
sample) .......................................................................................................... 53

Figure 11. Microbial composition of a ﬂoating debris and tree base micro-
habitat of a vernal pond, East Lansing, MI 1996. Error bars represent
SEM (N=5 samples per microhabitat) ....................................................... 55

Figure 12. Percentage of microbial particles per gut sample from larvae
reared in three microhabitats: open water, algal clump, emergent plant
base in a permanent pond, 1994 (N =5 samples per habitat ...................... 57

Figure 13. Mean number of cladocerans dissected from 4th instar Anophelee
W guts, permanent pond, East Lansing, MI 1994 ............. 59

Figure 14. Percentage of particles found per gut sample (N=12 guts/pond type)
of An. popotipeooie larvae in a vernal and aestival pond, East Lansing,
MI 1995 ......................................................................................................... 60

Figure 15. Percentage of particles found per gut sample of third and fourth
instar An. mm in a vernal pond, East Lansing, MI 1995 N=6
guts per instar .............................................................................................. 61

Figure 16. A comparison of the percentage of microbes found within third and

fourth instar An. pooetjpeooie guts in a vernal pond, East Lansing, MI
A) 1995, B) 1996 (N=6 guts per instar) ..................................................... 62

xiv

 

Fi

Fi'

Fig

CHAPTER 2

Figure 1. Mean number of particles found in An. W larval guts
over a diel (24h) period. Error bars represent SEM. * indicates
signiﬁcant difference, P < 0.05 (N=40 per time period .............................. 90

Figure 2. Percentage of particles/ml of gut content sample from An.

ooedﬁmeomem larvae in four treatments. * indicates signiﬁcant
difference between treatments, P < 0.05 (N =24 per treatment) ................ 91

Figure 3. Photograph of water mite (Eylaidae: Eyleie sp.) with larval head of
fourth instar @1- ooaogimegﬂetpe. Bar indicates 25pm ........................... 94

Figure 4. Preliminary experiment on diel feeding by An. ooodrimoeolﬁos
larvae on microinvertebrates, samples collected at noon and midnight.
Error bars represent SEM. No signiﬁcant differences were observed
(N =12 per time period) ................................................................................. 95

Figure 5. Mean number of microinvertebrates (N=25 guts) dissected from Ag.
Wm larval guts, pre (before starving) and post (after
starving) treatments. Error bars represent SEM. * Signiﬁcantly
different from pre-treatment, P < 0.05 ....................................................... 96

Figure 6. Mean number of microinvertebrates dissected from An.
Wm larval guts at noon(N=24) and midnight (N=24), ﬁrst
and second trials. Error bars represent SEM. * Indicates signiﬁcant
diﬂ'erence, P < 0.05 ...................................................................................... 100

Figure 7. Mean number of microinvertebrates dissected from An.
WM larval guts (N=12 per treatment), comparison
between treatments, ﬁrst and second trials, July and August, 1995.
Error bars represent SEM.* Signiﬁcantly different from vegetated
habitat, P < 0.05 .......................................................................................... 101

 

Figu

Figu

Figui

F igur

Figur

CHAI

Figuh

Flgm'e

Figure 8. Mean number of microinvertebrates found within the surface
microlayer of open water and vegetated habitats at noon (N=10) and
midnight (N=10). Error bars represent SEM. * Signiﬁcantly different
from noon treatment, P < 0.05 ................................................................... 102

Figure 9. Mean number of microhabitats found within the surface micro-
layer over a diel (24h) period from an open water zone (N=48) and a
vegetated area (N=48). Error bars represent SEM. * Signiﬁcantly
different from other treatment, P < 0.05 ................................................... 103

Figure 10. Total number of mites and cladocerans found in 3.
W larval guts from open water (N=3 guts/h) and
vegetated habitats (N=3 guts/h) over a 24 hour period in a permanent
pond, East Lansing, MI 1996 ..................................................................... 105

Figure 11. Total number of rotifers found in An. quadrimaculatus larval guts
from open water (N=3 guts/h) and vegetated habitats (N=3 guts/h)
over a 24 hour period, East Lansing, MI 1996 .......................................... 106

Figure 12. Mean number of mites found in the guts (N=9 guts/treatment) of
dissected 4th instar g1. MW. Error bars represent
SEM ............................................................................................................. 107

CHAPTER 3

Figure 1. Maximum/minimum temperatures (°C) for open water and
vegetated habitats. Error bars represent SEM. * Indicates
signiﬁcant difference from open water habitat ........................................ 127

Figure 2. Treatment effects on relative growth rates for An. W
in ﬁeld growth experiment. Error bars represent SEM. * Indicates
signiﬁcant difference from other treatments. AC = algal clump (N=49),
AC/P = algal clump/predator (N=19), P = predator (N=11) ..................... 128

 

Fi-

Fig

Fig

rig.

Figure 3. Habitat X Treatment effects on relative growth rates (mg/mg/day)
for An._ooe_ddmeeo1etoe reared in three treatments and control.
Habitat key: 0 = open water area; V = vegetated area. Treatment key:
cont. = control; AC = algal clump; AC/P = algal clump/predator; P =
predator. Error bars represent SEM. * Indicates signiﬁcant difference
from control and vegetated habitat ............................................................ 131

Figure 4. Treatment effects on development rate (days) for An.
W. Control (N=49), AC = algal clump (N =49), AC/P =
algal clump/predator (N=19), P = predator (N=11). Error bars are
present but not visible and represent SEM. * Indicates signiﬁcant
difference from control ................................................................................ 132

Figure 5. Treatment effects on adult dry weights for An. quadrimaculatus
in ﬁeld growth experiment. AC = algal clump (N=49),AC/P = algal
clump/predator (N=19), P = predator (N=11), Control = no additions
(N=49). Error bars represent SEM. * Indicates signiﬁcant difference
from control and predator treatment ......................................................... 133

Figure 6. Treatment effects on adult female wing length (mm) for An,
W. Control (N=33), AC = algal clump (N=30), AC/P = algal
clump/predator (N=11),P = predator (N =7 ). Error bars are present but not
visible and represent SEM. * Indicates signiﬁcant diﬂ'erence from
control ........................................................................................................... 134

Figure 7. Treatment effects on larval survivorship for AQW.
Control (N=10), AC = algal clump (N =10), AC/P = algal clump/predator
(N=10), P = predator (N=10). Error bars are present but not visible and
represent SEM. * Indicates signiﬁcant difference from control .............. 136

Figure 8. Relative growth rates for immature stages of An. WEE-1131313
reared in two habitats, open water areas and vegetated zones ................. 138

xvii

Figure 9. Relative growth rates for immature stages of An. Wm.
Error bars are present but not visible and represent SEM ...................... 139

Figure 10. Mean dry weights (mg) for An. W immature life
stages. Error bars are present but not visible and represent SEM. *
Indicates signiﬁcant difference from instar ............................................... 140

Figure 11. Temperature (°C), food type (open water, vegetation) and quantity
(ml) effects on An. Wine larval survivorship (%). Data have
been arcsin transformed from proportions. Error bars represent SEM.

* Indicates signiﬁcant difference from other treatments .......................... 142

Figure 12. Mean dry weights (mg) for male and female adult An.
W from diet experiment with two dietary treatments.
(Males: t-value = 1.899, P = 0.06; Females: t-value = 0.055, P = 0.95).
Error bars represent SEM. N 0 signiﬁcant differences observed .............. 146

Figure 13. Mean development rates (days) for male and female adult An.
W from diet experiment with two dietary treatments.
(Males: t-value = 1.019, P = 0.311; Females: t-value = 0.055, P = 0.812).
Error bars represent SEM. No signiﬁcant differences observed .............. 147

Figure 14. Mean percentage survivorship for ml. mm from diet
experiment with two dietary treatments. (t-value = 1.075, P = 0.31).
Error bars represent SEM. No signiﬁcant differences observed .............. 148

CONCLUSION S/RECOMMENDATIONS

Figure 1. Diagram depicting the time frame of when non-wintering spring
migrants enter a temporary pool and leave before the dry phase for more

permanent habitats (Modiﬁed from Wiggins et a1. 1980) ........................ 163

xviii

 

D110

. Hie

USE

p

INTRODUCTION

Mosquitoes are hosts to a variety of pathogens and parasites including:
viruses, fungi, bacteria, protoctistans and nematodes (Clements 1992). In
number of disease agents they transmit and the magnitude of health problems
these diseases cause to humans and animals worldwide, mosquitoes are
medically the most important group of insects (Service 1989). In fact, in an
attempts to lower the incidence of mosquito-borne diseases, hundreds of
millions of dollars have been spent on mosquito control in global campaigns
against malaria, dengue, yellow fever and other diseases (McClelland 1992).
Mosquito control relies on the knowledge gained through behavioral and
ecological studies of the target species. Until recently however, the need for
vector ecology and behavior studies was unimportant because of the availability
of synthetic insecticides that would kill target species, under a wide range of
conditions (Merritt et a1. 1992).

The development of insecticide resistance has been the primary reason
why vector control has moved from the use of broad spectrum, persistant
chemicals to more speciﬁc control materials, e.g. microbial insecticides,
pathogens and parasites (Brown 1986). Several of the more recent mosquito
biological control agents, such as bacteria lBecilloe thuringieneis var.
imeleneie (Bti) and Beeillue epheerieusl and fungi (e.g. Lagenioium
gigepteom) must be ingested by larvae to be effective (Chapman 1985; de
Barj ac and Sutherland 1990;). Both bacilli species are obligatory stomach
poisons (Aronson et a1. 1980; Davidson & Yousten 1990); zoospores of L,
gigenoeyoo, once inside the larval mouth, generally penetrate the tissues of the
digestive tract (McCray 1985). Therefore, knowledge of the "feeding

1

2
area" or where the larvae forage and optimal particle sizes and food types by
larval mosquitoes in the natural habitats will enhance the success of Bti and
other bacilli as particulate larvicides (Merritt et a1. 1978; Wallace & Merritt
1980; Aly 1983; Dahl 1988). Clearly, research on larval mosquito feeding ecology
and perhaps more speciﬁcally, the nature of their diet has taken on increased
signiﬁcance in recent years.

Several studies emphasize analyses on larval mosquito distributions and
sequential sampling for control decisions (Ikemoto 197 8; Mackey & Hoy 197 8;
Stewart et a1. 1983; Andis & Meek 1984; Service 1985; Sandoski et a1. 1987;
Olds et al. 1989; Orr & Resh 1989; 1992). These studies indicate an increasing
awareness of the importance of quantitative larval ecology to mosquito control.
Moreover, Service (1985) remarks on the ecology of anopheline mosquitoes, to
formulate the best tactic for mosquito control, a more complete understanding
of larval population dynamics is required.

Vector competence (i.e. the internal physiological factors that govern the
infection of human pathogens in a mosquito) (DeFoliart et al. 1987) varies with
the quality of the larval environment. For some mosquitoes, adverse effects on
larval survival, development rates, adult size and adult ﬁtness appear to be
primarily caused by larval stress due to food limitations within habitats (Fish &
Carpenter 1982; Mogi 1984; Reisen et a1. 1984; Hawley 1985; Haramis 1985).
For example, smaller females (which reﬂect reduced larval food availability)
were found to be more competent vectors for arboviruses than larger females
(Grimstad & Haramis 1984; Patrician et a1. 1985; DeFoliart et a1. 1986). An

understanding of the spatial and temporal distribution of the dietary resources

3
available to larval mosquitoes in their natural habitats could clarify the
relationships among food availability and mosquito ﬁtness.

The mosquitoes, AnooheLee omdrimoeoletoe (Say) and A_n. pooeoipeme
are distributed in the eastern half of North America and was the principal
vector of human malaria in this region before eradication of the disease
(Carpenter & LaCasse 1955). They remain important pests, particularly in
more southerly regions of their distributions, e.g. around impoundments in the
Tennessee Valley and in the rice growing areas of Arkansas (Horsfall 1955).

Historically, records of the occurrence of both species show a wide distribution

in Michigan (Sabrosky 1946).

l l e ' EC 10
An. We and g1. poootipenois larvae typically inhabit

permanent, lentic water pools where they feed on microbial particles and
detritus by ﬁlter feeding. Unlike An. pom, Arr. We has
rarely been observed in temporary vernal pools in its temperate distribution.
Anopheles larvae appear to be adapted for feeding at the surface via palmate
hairs on their abdomen which help to maintain position at the surface. Further,
larvae can rotate the head 180° in order to feed on the surface microlayer while
lying horizontally (Christophers & Puri 1929). Why Anopheles; larvae feed at
the surface is poorly known. Merritt et a1. (1992) have noted that larval posture
and movement during feeding modes vary greatly. Speciﬁcally, gophelee
larvae in marsh habitats aggregate at air-water interfaces (surface microlayer)
near plant stems and algal mats (Walker et a1. 1988; Orr & Resh 1989; 1992)
with their bodies ﬂoating parallel to the water surface (Christophers & Puri
1929). Several investigations (Collins et al. 1988; Orr & Resh 1989and 1992)
concluded that this feeding aggregation at the air-water-plant interfaces to be

 

tl
ir

p2

b1
su
fa
ac

ar

\l'
fo

\i

0f

4
as an adaptation to avoid predation, but food resources are probably also more
abundant at these interfaces.

The surface of natural bodies of water is a thin zone (< 1mm) of the water
column, distinct from the epilimnion immediately below it and have named it
the surface microlayer (Norkrans 1980; Hermansson 1990). This air-water
interface is more accurately described as a layer of dissolved substances,
particles and
microorganisms that are brought to the interface by simple diffusion, rising
bubbles (Garrett 1967; Jarvis 1967), convection, upwelling from sediments and
subsurface water, and at the same time, the microlayer serves as a sink for
fallout from the atmosphere (Duce et al. 1976). Thus, it becomes an
accumulation layer, where the concentration of various chemical compounds
and microorganisms (e.g. bacteria and algae) exceeds that of the subsurface
water by orders of magnitude (Parker & Barsom 1970; Hatcher & Parker 1974;
Walker & Merritt 1993). The surface microlayer has been hypothesized to be a
food-rich zone that Anopheles larvae are adapted to exploit via their interfacial
feeding strategy (Merritt et al. 1992; Walker & Merritt 1993).

A qualitative characterization of the microbial components and nutrients
available as food or alimentation for Aoopheks larvae found in this surface
microlayer has been well documented (Coggeshall 1926; Boyd & Foot 1928;
Senor-White 1928; Renn 1941; Odham et al. 1978; Norkrans 1980; Rejmankova
1991; Maki & Hermansson 1994) and conﬁrmed through gut contents analyses
by Coggeshall (1926), Boyd & Foot (1928), Senor-White (1928), Hinman (1930),
Walker et al. (1988), Vasanthi & Hoti (1992) and Walker & Merritt (1993).
These studies have shown that both algae and bacteria are present in the guts

of fourth instars. Although microorganisms are the principal constituent of the

5

larval diet in most species, including Anopheles, little information exists about
the estimated percentage of various microbes in the larval diet. In addition,
little is known about the distribution of Anopheles larvae relative to the
distribution and concentration of bacteria and algae in different microhabitats.

Often the surface microlayer has been characterized as a (1mm - lum)
thick strata consisting of a lipid ﬁlm and polysaccharide-protein layer (Sieburth
et al. 197 6). Golberg and DeMeillon (1948) found that larvae kept in media
lacking protein and amino acids but complete in other respects failed to reach
the second instar. Concomitantly, larvae reared in the absence of lipids did not
develop beyone the third instar (Singh & Brown 1957; Lea & Delong 1958). It is
obvious that protein and lipids are essential nutrients for adequate mosquito
nutrition as observed by the aforementioned studies as well as studies by Dadd
& Kleinjan (1978), Dadd (1980), (1981), and Dadd et al. (1987). Similar to the
microbial component in anopheline larval diets, information regarding nutrient
concentration is lacking. If optimal growth and development are limited by
nutrition, what aspects of the larval mosquito diet are most critical to survival
and in what concentration? Does the type of microhabitat determine microbial
concentrations? Do the microbial concentrations change seasonally?

nv' e t l tor n ulati n D amics

The principal environmental factors that affect rates of mosquito growth
and development are temperature, nutrition, survival and larval density. In
nature, these different factors may interact in a complex way to determine
developmental rates and regulate populations.

Clements (1992) summarized the relationship between temperature and
rate of mosquito growth and development in three statements. First, for any

species, growth and development occur only within a temperature range that is

 

 

deﬁne
Secon

devel
SUpp:
temp
temp
large
h1g3
bald

resi
194-
and
POp

0V9

6
deﬁned by a lower developmental threshold and an upper lethal temperature.
Second, within most of this temperature range the rate of growth and
development is positively correlated with temperature. Huffaker (1944)
supports this second statement with his studies on temperature relations and
Ag. MW development. Finally, growth and developmental
temperature ranges vary with species. Senior-White (1928) noticed that the
temperature in small hoof-mark pools in grass might be 5°C cooler than in a
large pool six inches away. I contend that Senior-White's (1928) hoof-mark pool
in grass and large pool six inches away example is analogous to the discrete
habitats I propose to compare, namely vegetated vs. open water habitats.

The duration of the larval period is an important life-history parameter
because it is a critical component of most life-table and population estimates for
aquatic insects (Sweeney 1984). Life tables allow the ecological role a natural
enemy or other regulatory factor plays in a particular system (i.e. whether it is
a source of regulation contributing to population stability) to be determined
(Bellows et al. 1992). The faster development at high temperatures largely
results from a decrease in the duration of one or several larval stages (Huffaker
1944; Hanec & Brust 1967; Lutz 1974) or from a decrease in both the duration
and number of instars (Ross & Merritt 1978). Suppose that an Anophelee
population containing individuals developing through several stages is sampled
over the developmental period and estimates of the number of individuals in
each stage are available for various points in time. Then, it would be possible to
use the data to estimate stage speciﬁc survival rates and other parameters of
the population (Manly 1974).

Numerous studies have observed high positive correlations between

larval growth rates of natural populations and water temperature (Sweeney

 

 

197.
fact
sen
ten
div
If t
the
eti

se«
qu
th

Di
fo.

7
1978; Mackay 1979; Wallace et al. 1992). Temperature effects on bioenergetic
factors such as growth rates of Aoophelee larvae may be in part due to the
sensitivity of the microorganism community within the surface microlayer to
temperature changes. Dommergues et al. (1978) observed microorganism
diversity within the surface microlayer to be sensitive to temperature changes.
If temperature changes affect microbial abundance and distribution directly,
then An. ooeogimaooleeue growth rates will ﬂuctuate according to the indirect
effect on quantity and quality of food sources in the surface microlayer.

For most anopheline species, the total amount of potential food rarely
seems limiting in natural ecosystems. However, the actual amount of high
quality or preferred food maybe limiting to certain species at speciﬁc times of
the year or in different habitats (or microhabitats) (Sweeney 1984).
Alternatively, food quantity can exert a signiﬁcant effect on mosquito growth.
Available data indicate that larval developmental rates decrease
proportionately with decreased food levels (Wigglesworth 1929; Marcovitch
1960; Moore & Whitacre 1972). Sweeney (1984) suggests that the most
productive experimental approach will be to measure growth or developmental
response to two or more variables simultaneously.

To date, predator-mediated changes in prey behavior that inﬂuence the
fecundity of the prey have yet to be incorporated into population dynamics
theory in any meaningful way (Cappuchino and Price 1995). However, it is clear
from the work in some aquatic systems that predators can act to reduce the
access of their prey to important resources, e.g. habitat or food (Werner et a1
1983; Gilliam and Fraser 1987). For some anopheline mosquito species, the
predator refuge hypothesis has been proposed as the principle regulating factor
for W larval distributions (Orr and Resh 1992). In fact, it is possible

 

that :
anop

hypo

e

aqu
actu

tenr

8

that an alternative hypothesis e.g. enhanced food resources inﬂuence larval
anopheline population dynamics by acting in concert, thus providing a multi-
hypothesis explanation.

This research was designed to investigate the larval feeding ecology of
&, ouedu’mecolatos and punetipennie. Objectives for this research included: 1)
a qualitative and quantitative analysis of the available dietary resources and
actual food items of Ag. ouedg'meeulatue and ponetipenois in permanent and
temporary ponds; 2) an examination of diel feeding activity of third and fourth
instar Q. ouedrimacgatus ; and 3) a ﬁeld and laboratory study of the effects of
temperature, nutrition and predation on An. ooedrimeeoletue growth,

development rates, and survivorship.

 

Aly, (

Andi

Aron

Belle

Boy:

Bro‘

Cap

Car

Cha

Chr

9
LITERATURE CITED

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de

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llarcc

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14
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Va

\Yz

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larva and a possible practical application. Nature, 123: 7.

CHAPTER 1
FEEDING ECOLOGY OF LARVAL W MOSQUITOES:
A SEASONAL COMPARISON BETWEEN MICROHABITATS
WITHIN PERMANENT AND TEMPORARY PONDS IN MICHIGAN

ABSTRACT

A ﬁeld study was conducted to quantify the available food resources
between goggles ouedg’maculatus (Say) and ponctipennie (Say) habitats
and microhabitats. This study examined the food resource distribution
between three microhabitats (open water, algal clumps and emergent plant
bases) within a permanent marsh and two microhabitats (ﬂoating debris and
tree base) in a temporary vernal/aestival pond. A ﬂuorochromatic stain
(DAPI) with DNA binding properties was employed to quantify the available
food resources within the surface microlayer and compare food source types
with gut content analyses.

Available food resources for An. W observed within the
surface microlayer of a permanent pond were very consistent regardless of
season or microhabitat. Within the surface microlayer, bacteria were the
most abundant food type, followed by algae, detritus and invertebrate
parts/protozoans. Seasonal differences across the range of food types were
quite varied.

Contrary to the permanent pond system, surface microlayer analyses

for available food resources in the temporary vernal/aestival pond indicate

18

 

the
ha

805

19
that total bacteria numbers were higher in the vernal pond and that rod
bacteria constituted the primary bacteria type in both pond types. The
aestival pond contained more algal, IPP and detritus particles/ml of surface
microlayer sample than the vernal pond.

Bacteria have been considered to be the most important of the
microorganisms that comprise the food of mosquito larvae and it appears
this may be true for larvae inhabiting open water areas as observed in this
study. Total bacteria abundance was similar for the three microhabitats.
Most interesting was the presence of whole cladoceran (Cladocera) bodies in
larval guts which showed that larvae consume more cladocerans in the open
water habitats than vegetated areas.

Gut content analysis on third and fourth instar &. punctipennis
inhabiting the vernal pond, indicates that larvae consume more bacteria and
algae than larvae from the aestival pond. Larvae appear to consume more
IPP and detritus (close to 25 % more) in the aestival pond. Dietary
differences between Q1. puneﬁipennie instars indicate that larval diet does
not differ quantitatively for bacteria, algae or IPP consumption between
instars. However, fourth instars consumed signiﬁcantly more detritus than
third instars. Larval diets were signiﬁcantly different in the vernal pond
between 1995 and 1996 as algae and detritus consumption differed between
years. Third and fourth instars consumed more algae and less detritus in
1995 than 1996.

In conclusion, I have demonstrated that larval diets differ according
to microhabitat in a permanent pond and most notably that Q.
goodmoagﬂam larvae are able to consume cladocerans, a dietary

component never reported in anopheline guts before. The implications this

uniqi
comp
mosq
ﬁrst '
temp
anOp

IIIOI‘E

20
unique ﬁnding as well as characterization of the bacteria and algal dietary
components may have on biocontrol methods is of potentially great value for
mosquito-borne disease control. Furthermore, I have documented for the
ﬁrst time a quantitative evaluation of anopheline food sources in a
temporary vernal/aestival pond. The exploitation of temporary pools by
anopheline larvae may enhance dispersal and colonization tendencies in

more permanent water habitats.

21
INTRODUCTION

Larval mosquito habitat and diet are fundamental components in the
study of mosquito feeding ecology (Walker et al. 1988a; Laird 1988;
Clements 1992 ). Mosquito larval distribution, growth and feeding success
are largely determined by the distribution and quantity of available food
resources. Moreover, adverse effects on larval survival, development rates,
adult size and ﬁtness appear to be primarily caused by larval stress due to
food limitations within habitats (Fish & Carpenter 1982; Mogi 1984; Reisen
et al. 1984; Hawley 1985; Haramis 1985; Walker et al. 1991).

Many species of Apophelee mosquito larvae inhabit permanent,
lentic water ponds where they aggregate at air-water interfaces (surface
microlayer) near plant stems and algal mats and feed on microbial particles
and detritus using collecting/ﬁltering feeding modes (Merritt et al. 1992).
Some anophelines in North America inhabit smaller, more temporary
aquatic habitats, e.g. An. pom - tree holes, (Darcie & Ward 1981; Laird
1988; Walker et al. 1988b), &. viallseri - temporary vernal pools (Wiggins et
al. 1980). Recently, three anopheline species that typically inhabit
permanent swamps in temperarate regions (Q. ooadrirpaeulotus,
punctipennis and perplexans) have been observed in more ephermeral or
intermittently ﬂooded swamps (Wiggins et al. 1980; Jensen et al. 1994;
1995; Wallace Michigan State University unpublished data). Typically,
within these temporary ponds, Anopheles larvae are found ﬂoating attached
or near ﬂoating debris, e.g. branches, sticks, or leaves, seldom are they found
next to a tree base ( the exception is when the vernal or aestival pond has

been reduced dramatically in size by drought or dessication). In temperate

North America, Wiggins et al. (1980) classiﬁed &. Woe and

22

puneeipeopje as non-wintering spring migrants in temporary pond systems.
To date, virtually nothing is known about the feeding ecology of anophelines
in such seasonally astatic waters. In Michigan and throughout the
temperate ranges of _A_ne ouadrimaculatos and ponctipennis, these species
inhabit permanent marshes or swamps (Figure 1). The air-water interface
of natural bodies of water has been operationally deﬁned in this study as a
thin zone (< 1mm) of the water column, distinct from the epilimnion
immediately below it, and has been classiﬁed as the surface microlayer
(Norkrans 1980; Hermansson 1990, Maki and Hermansson 1994). This air-
water interface is more accurately described as a layer of dissolved
substances, particles and microorganisms that are brought to the interface
by simple diffusion, rising bubbles (Garrett 1967; Jarvis 1967), convection,
upwelling from sediments and subsurface water, and at the same time, the
microlayer serves as a sink for fallout from the atmosphere (Duce et al.
1976). Thus, it becomes an accumulation layer, where the concentration of
various chemical compounds and microorganisms (e.g. bacteria and algae)
exceeds that of the subsurface water by orders of magnitude (Parker &
Barsom 197 0; Hatcher & Parker 1974; Walker & Merritt 1993). Although
several investigations by Collins et al. (1988); Orr & Resh (1989) and (1992)
have shown this feeding aggregation at the air-water-plant interfaces to be
an adaptation to avoid predation, food resources are probably also more
abundant at these interfaces. The reasons that Anopheles larvae feed at the
surface are poorly known and have not been thoroughly investigated.

Qualitative characterizations of the microbial components and
nutrients available as food sources for Anophelee larvae found within the

surface microlayer of larval habitats and in gut contents of larvae have been

 

 

Fig,
Micl

23

#5

 

 

 

 

 

 

 

  

Figure 1. Historical distribution of Anophelee W (Say) in
Michigan (modiﬁed from Sabrosky 1946).

   

._..- 334...... .__._~......r;..u._....._..........1...... .. u _ Z . . . .3 he. tLLr.!.!FH..=Crrr.. . . . . . _ .. .. . ,... . . . . . . _..|I.H..._:h...... 7“ v.5 us£C.Es‘_.h€

24
well documented (See review by Merritt et al. 1992). Studies have shown
that algae and bacteria are present in the surface microlayer as well as in
the guts of fourth instar Anopheles mosquitoes. Although microorganisms
are the principal constituent of the larval diet in most species, including
W, little information exists about the estimated proportion of
various microbes in the larval diet. Walker et al. (1988a) noted that early
studies often conﬂicted as to whether larvae preferred to feed on speciﬁc food
items or sizes, or if larvae fed in a nondescriminatory manner on the
food items available at the time. In addition, little is known about the
distribution of Melee larvae relative to the distribution and
concentration of microorganisms in different microhabitats.

The objectives of this study were to: quantitatively characterize the
availability and variation of food resources between and within &
W and punctipennis larval microhabitats in a permanent
marsh; 2) identify and quantify the food sources consumed by third and
fourth instars of these species and determine if larval diet changes
seasonally or between microhabitats and; 3) characterize the food resources

and larval diet of these species inhabiting a temporary vernal/aestival pond.

MATERIALS AND METHODS

Two sites representing disparate habitats for anopheline mosquito
larvae were chosen for this study based on previous surveys of anopheline
breeding sites. gopheles ouadrimaculeeus (Say) and An. poneeipennie
(Say) mosquito larvae inhabit permanent, standing water/marshes. In 1994,

through a random larval sampling survey, I found a temporary vernal pond

 

 

25
that contained a small population of Anopﬂelee We (Say)
larvae. Thus, in 1995 and 1996, a temporary pond was add to this study to
examine the differences in anopheline diets between a permanent and
temporary habitat.
I. Study Site Deseriptions

The permanent pond is located approximately 8 km south of
Michigan State University at the Collins Road Entomology Field Laboratory
(42°42'00" N Latitude; 84°30'20" W Longitude), Ingham County, Michigan,
U.S.A.. Permanent pond studies were conducted June-September during
1995 and 1996. The permanent pond is approximately 2.25 ha in size. The
pond is bordered with cattails (prloe sp.) and emergent grasses e.g. foxtail
(m sp.).

Vernal/aestival pond studies were conducted April-September 1995
and 1996 in Ingham County, Michigan, U.S.A.. The vernal pond site
(42°45'30" N Latitude; 84°31'15" W Longitude) is ﬁlled with water from snow
melt and spring rains, thus creating a pond that is approximately 3800 m2
in size. The surrounding vegetation at this site include willow (Selig sp.),
cottonwood (Populue sp.) and maple (ﬂ: sp.) tree species along with
several herbaceous plant species, e.g. (Lropetiene sp., ferns and m sp.).In
1995, the vernal pond dried out in early August. After a 7-10 day dry period,
this pond reﬁlled with water from later summer rains to form an aestival
pond. The aestival pond area was approximately 800 m2 in size. The vernal
pond dries out most years, however, in 1996, an unseasonally wet spring

provided enough standing water to prevent drying during this year.

26

11. mm

For the permanent pond, surface water temperatures were measured
daily with a max/min thermometer attached 1 cm deep on the ventral-side of
a styrofoam ﬂoat. Each ﬂoating thermometer was anchored to a stick placed
into the pond. The ﬂoat allowed the thermometer to adjust to ﬂuctuating
water levels. Water chemistry data were analyzed from surface microlayer
samples taken from three distinct microhabitats; open water, algal clump
and emergent plant base areas. Surface microlayer water samples were
collected with a modiﬁed Harvey-Burzell glass plate method (Norkrans
1980), ﬁltered through a 0.22 pm ﬁlter into a steril glass jar and transported
on ice to the Michigan State University Soil Testing lab for analysis. The
following parameters were measured from each sample; calcium,
magnesium, chloride, nitrate, pH, sodium, potassium and soluble salts.

For the vernal pond, surface water temperatures were not collected in
1995 due to equipment failure, however, in 1996, these data were measured
with a max/min thermometer similar to the technique used in the

permanent pond. The pH was measured on a daily basis in 1995 and

biweekly in 1996 with a Hach kit.

1, Hobiteﬁg enalyses: charaeterization of available food resourees for
anophelmelamae.

Selected microhabitats within the two pond types were analyzed to
determine the difference in abundance and concentration of microbial food
resources between microhabitats as well as to examine the change in
microbial numbers through time. Three speciﬁc microhabitats were

compared in this study, an open water zone, algal clump and emergent plant

27
base areas. The open water zone consisted of an area of the pond that was
without ﬂoating or emergent vegetation within 4 m of the sampling point.
Permanent pond habitat.

From J une-September, 1995 and 1996, ﬁve replicate surface
microlayer water samples were collected from three different microhabitats:
open water areas (Open), algal clumps (AC) and the base of emergent plants
(EPB). In this procedure, two sterile microscope slides clamped together
were introduced into the water column of each microhabitat and withdrawn
in a rapid movement (See Walker & Merritt 1993). Water adhering to the
sides of the glass slides was removed and deposited into a sterile, 1 L plastic
container by a rapid shaking motion and neoprene squeegee. With a sterile,
5 ml glass pipet, 2.4 ml of water sample was removed from each plastic
container and preserved in an sterilized 2 dram vial of 4% formalin. Each
vial was stored at 4°C in an ice cooler and then transported to the
laboratory.

Because of the high concentration of bacteria, algae and detritus
found in preliminary studies, 1994 samples were diluted by a factor of 1:5 in
the ﬁeld. Samples collected in 1995 and 1996 were diluted by a factor of 1:3
in the laboratory prior to ﬁltering. In the laboratory, one ml of diluted water
sample was pipeted into ﬁnal sterilized 2 dram vial for staining procedures.
The contents of each vial were stained with 17 drops of 4'6--diamidino--2--
phenylindole (DAPI) (Porter & Feig 1980) of a ﬁnal concentration of 40ug ml-
1 and maintained in the dark for 20 minutes. DAPI is a ﬂuorochromatic
stain with DNA binding properties, In stained samples, bacteria appear

blue, protozoans appear light blue with a distinct nucleus, organic detritus

  

.. . . H . . i . , o a _ v .
.. ....~...:.ou i.e.! .2211 11.5.. . 11...? f. v . . . .. 1.... .. 2.. .113... ... . .: . . . . .. . . I. a. I. E. I.’ mtg.)

28
autoﬂuoresces yellow, chlorophyll in algae autoﬂuoresces dull red and nuclei
are blue (See Kondratieff & Simmons 1985).

Samples were ﬁltered (low vacuum pressure by hand pump) onto
Irgalan black-stained N ucleopore ﬁlters (0.22pm) (Costar, Cambridge MA)
backed by a 0.45pm HA Millipore ﬁlter (Millipore, Bedford, MA) to provide
even distribution of particles. The black ﬁlters were placed onto microscope
slides, smeared with Cargille Type B immersion oil.

Direct counting methods using an epiﬂuorescence microscopy
technique were used to count and identiﬁy bacteria (rod and cocciform),
algae, invertebrate parts/protozoans (IPP) and detritus from each sample.
This technique has been used successfully to identify the microbial
composition within larval mosquito guts and habitats (Walker et al. 1988a,
Walker & Merritt 1993). Algae, invertebrate parts and protozoans were
identiﬁed using keys from (Weber 1971; Prescott 197 8; Thorp & Covich
1991.

Counting was performed on slide preparations with a Leitz Laborlux
11 microscope with epiﬂuorescence ﬁttings, 50-W mercury lamp, UV
excitation ﬁlter (340-389nm), and barrier ﬁlter (430 nm). A minimum of 200
bacteria per morphological group was counted among at least 15 different
ﬁelds on each slide for statistical accuracy as recommended by Kirchman et
al. (1982). Bacteria were counted at a maginiﬁcation of 1000X, whereas,
algae, IPP and detritus were counted at 250X. The number of each
morphological group per ml of original sample was calculated per ﬁeld. A
ﬁeld is equivalent to the number of transects used
to count an item, e.g. a ﬁeld of 10 X 10 was used for bacteria and 5 X 5 for

algae, IPP and detritus.

    

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29
Vernal /Aestival pond.

In 1995, the vernal pond dessicated in early August and then reﬁlled
to form an aestival pond. Therefore, surface microlayer water samples were
collected from two microhabitats; tree base and ﬂoating debris. Rod and
cocciform bacteria, algae, invertebrate parts/protozoans (IPP) and detritus
were quanitiﬁed from these microhabitats.Five replicates were collected on
May 23 for the vernal pond and August 29 for the aestival pond. In 1996,
surface water samples were collected every 2 weeks from April 1 to
September 1 to observe the microbial change over time. During this ﬁeld
season, the vernal pond did not dry out to provide conditions for the
formation of an aestival pond, however, by August 1, it had reduced in size
equivalent to that of the aestival pond in 1995. Since only one sample date
represents each pond type, the 1995 results are a general characterization of
the microbial composition found within these microhabitats. Results for 1996
are ﬁ'om samples collected every two weeks, April 1 - September 1.

Surface microlayer water samples for the two microhabitats were
collected, preserved and diluted with the same technique used with the
permanent pond water samples. Samples were ﬁltered, stained and
enumerated as described above.

a --e° hra t 'z in o_ met anon-get: ' hi 1 l
Anophelesgins.

These experiments were designed to quantify the microbial and
microinvertebrate components found within the food bolus of an An.
goedrimacoletus or W larva. In the permanent pond, the
hypothesis tested was whether food items consumed differed according to

microhabitat, e.g. open water areas, algal clumps and emergent plant type

30
areas. Since virtually nothing is known about larval anopheline diets in
temporary ponds, larval gut contents from the Towar Park vernal pond were
compared to those of the aestival pond (same site) to determine dietary
differences within and between pond types.
Field M icrocosms.

Field microcosms were employed for larval feeding experiments.
Microcosms were constructed of a 4 liter clear, plastic, round container with
dimensions of 20 cm diameter X 25,4 cm length (Figure 2). The bottom and
sides of each container were removed and replaced with a 5 cm X 5 cm piece
of nytex mesh (100nm opening) covering to allow vertical and horizontal
water movement. Each container was ﬁtted with a styrofoam ﬂoat placed
around the top of each container. The ﬂoat allowed each microcosm to adjust
with water level ﬂuctuations and allowed natural light conditions to reach
the interior. Each microcosm was ﬁtted with a screen lid/covering to prohibit
predator and/or competitor recruitment. Microcosms were kept in a speciﬁc
location with a monoﬁlament line tied to a garden stake. Microcosms were
placed randomly approximately 2 m apart in open water areas, algal clumps
and emergent plant type microhabitats in order to simulate areas where
anopheline larvae are present and absent.

Larval gut analyses.

Fourth instar Q1. Woe were collected for gut dissections
and microbial analyses. To ensure that fourth instar guts contained material
from the speciﬁc microhabitats, third instar An. We were
collected ﬁ'om the permanent pond using a 250ml mosquito dipper, placed
inside the microcosms (n =10/microcosm), and allowed to molt before

sampling by removal. Microcosms were checked daily and culled of fourth

 

 

31

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Sketch of ﬂoating microcosm for ﬁeld experiments. a) 4 L plastic
container, b) Nytex 100nm mesh bottom and side panels, c) styrofoam ﬂoats,
d) garden stake for support, e) Fish line to anchor microcosm to stake, f) water
surface, g) cattails, h) substrate.

 

32
instars. Fourth instars were killed in boiling water in the ﬁeld, preserved in
4% formalin at 4°C in order to slow any digestion or other processes that
may have altered the gut contents.

The gut (n = 6 guts per microhabitat) was dissected with minuten
pins and the food bolus removed and separated from the peritrophic
membrane with several washes of distilled water. The contents were
pipeted into a sterilized 2 dram vial. Distilled water was then added to the
vial to bring the total volume to 2.5 ml. Vial contents were placed in a vortex
mixer for 20 seconds to break-up large aggregates of material formed in the
digestive tract, but not long enough to rupture any cells. To identify and
enumerate the microbial components of each larval gut, one milliliter of
sample was removed and stained with DAPI using the same protocol as in
the habitat analyses. Microinvertebrates were counted during dissections
with a binocular dissecting microscope and identiﬁed with several keys, e.g.
Thorp and Covich (1991) or sent to experts for conﬁrmation.

A control study was conducted on surface microlayer water samples
inside versus outside of the microcosms to determine if larval food resources
were affected by the microcosms. Surface water samples from inside (3
replicates) and outside (3 replicates) of the microcosms in open water and
vegetated areas were collected, preserved and analyzed with the same
protocol used in the habitat analyses stated above.

For the vernal and aestival ponds in 1995, third (11 = 7) and fourth
instar (n = 5) Q. puoetipennis were collected for gut content comparisons.
Larvae were collected, preserved, dissected and analyzed as for larvae from
the permanent pond. Although the vernal pond in 1996 never dried up, third
(n = 6) and fourth instar (n = 6) Q. poneje'pennis were collected at

 

 

33
approximately the same time as those larvae were collected in 1995 and
analyzed similarly. Comparisons were conducted within and between years
for the vernal pond study as well as between vernal and aestival ponds in
1995. Larval guts analyzed from mosquito larvae were primarily collected
from a ﬂoating debris type microhabitat.
II tat'stic a l ses

Temperature data was generally displayed as means and SE. except
for the comparison between the vernal and permanent pond
maximum/minimum surface water temperatures. These data were analyzed
with an unpaired t-test with the grouping variable = pond type. A p- level
below 0.05 was considered signiﬁcant for all tests in this study. Hydrogen
ion (pH) data in the permanent pond was analyzed similarly, with the
grouping variable=habitat (open vs. vegetated areas). Water chemistry data
from the permanent pond was analyzed with a one-way AN OVA
(Zar 1984). A Fisher's PLSD specialized t-test was employed with signiﬁcant
F-ratios.

Habitat data from the permanent and vernal/aestival pond
experiments were analyzed with a two-way AN OVA design. Signiﬁcant F-
ratios were further analyzed with Fisher's PLSD specialized t-test of the
means (Super Anova 1989). Larval gut data from the permanent and
vernal/aestival ponds were analyzed with a two-way AN OVA (P < 0.05 is
signiﬁcant). Fisher's PLSD specialized t-test of the means was employed for
signiﬁcant F-ratios. Normality and variance heterogeneity
assumptions were checked and data transformed using Log10 or arcsin

transformations for proportions, when necessary (Zar 1984).

34
RESULTS
The freshwater habitats that I studied were a permanent pond and a
temporary woodland vernal/aestival pond. Both aquatic systems serve as
suitable habitats for anopheline mosquito larvae as well as several other
genera of mosquitoes, e.g.Qdee, (Lula, Mensonie, and Peorophoze.
Anopheline larval density in the vernal pond was very low (approximately

6.1 larvae/m2), however this density was greater than that of Q.

goedziooeeoletoe in the permanent pond at this time.
1, Ehyeieelzghemieal ehegacteg'zation

Permanent pond

In 1995, the surface water temperature for an open water area ranged
from a minimum of 21°C on July 22 to a maximum of 36°C on August 16,
while in the vegetated zone, the surface water temperature ranged from a
minimum of of 20°C on August 1 to a maximum of 36°C on July 16 (Figure
3). Surface water temperatures in 1996 were not compared between open
water and vegetated areas. The surface water temperature in an open water
area ranged from 35°C on three separate dates (June 29,30 and August 7) to
a minimum on July 20 of 13°C (Figure 4). The pH ranged from 6.71 to 7.8
during this ﬁeld season.

To gain a better picture of the chemical composition of the three
microhabitats analyzed for anopheline food resources, a detailed chemical
analysis of the three microhabitats was conducted in 1996. Calcium (ppm),
magnesium (ppm) and soluable salts (MMHO) concentrations differed
signiﬁcantly between microhabitats (AN OVA, Fishers's PLSD p < 0.05) . All
other measurements did not differ between microhabitats (AN OVA, p > 0.05)
(Table 1).

 

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37

Table 1. Mean water chemistry values (Units = ppm, soluble salts = MMHO) for three microhabitats (open water,
algal clump and emergent plant base) within the permanent pond, East Lansing, MI 1996. AN OVA, Fisher's

PLSD, (SEM).

 

 

 

Microhabitat pH (SE) Soluble SaltsKSE) Ca(SE) cuss) Mg(SE) No3(SE) K(SE) Na(SE)
Open 8.6 (0.0) 0.38 (0.0) 20.0 (0.0) 20.3 (1.5) 16.6*(0.7) 2.4 (2.3) 11.6 (4.7) 8.6 (0.7)
Water
Algal 9.4**(O.1) 0.42 (0.0) 20.0 (0.0) 20.3 (1.2) 18.6*(0.7) 0.07(0.1) 5.0 (0.6) 8.0 (0.0)
Clump
Emergent 8.4 (0.0) o.5s*(o.0) 33.3*(4.4) 22.3 (0.3) 22.3*(o.3) 0.07 (0.0) 7.6 (0.7) 8.0 (0.0)

Plant Base

 

1 values in MMHO
* signiﬁcantly different, p < 0.02
** signﬁcantly different p < 0.002

38

Temporary Pond

Surface water temperature data are for the period Anopheles larvae
entered the pond until the time they left. In 1995, surface water
temperatures for the vernal/aestival pond were not collected due to
equipment failure. In 1996, the surface water temperatures ranged from a
minimum of 15°C on July 11 (mean = 18.1°C; S.E.= 0.263) to a maximum of
29°C (mean = 255°C; SE. = 0.355) on July 22 (Figure 5). The pH during the
1995 ﬁeld season ranged from a minimum of 6.65 on May 31 to a maximum
of 8.05 on July 1 (Figure 6).
Permanent/ Temporary pond comparison

The surface water temperature for the vernal pond was measured
from June 14 - August 7, 1996 while in the permanent pond it was measured
from June 14 - August 9, 1996. Mean maximum surface temperature in the
vernal pond (mean = 255°C; SE. = 0.355) was signiﬁcantly lower than the
permanent pond mean daily maximum temperature (mean = 29.4°C; SE. =
0.350). The mean minimum surface water temperature for the vernal pond
(18.1°C; SE. = 0.263) was signiﬁcantly lower than the permanent pond
minimum temperature (mean = 204°C; SE. = 0.335) (Table 2).

nlseszes'tinn nictinfavilbl

resources for anopheline larvae.
_Permanent pond

There were four categories of food resources identiﬁed in open water,
algal clump and emergent plant base microhabitats: bacteria, algae,
invertebrate parts/protozoans (IPP) and detritus. Bacteria were subdivided
into rods, cocci and total bacteria groups. Algae were quantiﬁed as a group

rather than individual species; however, some algae were identiﬁed to the

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41

Table 2. Comparison between the mean daily maximum/minimum temperatures
(°C) of a temporary vernal and permanent pond, June 14 - August 9, 1996, East
Lansing, MI.

 

 

 

Pond Type
Vernal Permanent t-value P-value
N (SE) N (SE)
Maximum 55 25.5 57 29.4 7.905 < 0.0001*
(0.355) (0.35)
Minimum 55 18.1 57 20.4 5.538 < 0.0001*
(0.263) (0.335)

 

* Highly signiﬁcant, p < 0.05 is signiﬁcant

42

generic level from the open water and vegetated zones of the permanent
pond (Table 3). Invertebrate parts and protozoans included an array of
portions or entire organisms ranging from spider exuviae, mosquito larvae,
other diptera parts, copepods, mites, cladocera, and rotifers. Although some
detritus particles were larger than 10 um in size, detritus particles of a
maximal diameter of 10pm were quantiﬁed to facilitate counting procedures.

Seasonal differences in total bacteria, algae, detritus and IPP for 1995
and 1996 in the surface microlayer are presented in Figure 7. Rod and
cocciform bacterial numbers were similar per sample for June and
September but differed signiﬁcantly (P < 0.05) from July and August in
1995 than in June and September (Two-way AN OVA, Fisher's Protected
LSD) (Table 4). In 1996, rod bacterial numbers were lowest during June and
July and highest during August and September. Cocciform bacteria were
signiﬁcantly higher in July and August than in June and September in
1996. Total bacteria numbers were signiﬁcantly highest in the months of
June and September for 1995 and August and September for 1996. In Table
5, algal numbers were signiﬁcantly higher in June and July when compared
to August and September, 1995, whereas in 1996, algal numbers increased
signiﬁcantly on a monthly basis throughout the ﬁeld season. Invertebrate
parts/protozoans (IPP) were signiﬁcantly higher in the beginning of the 1995
ﬁeld season (J une-J uly) compared to August and September but showed the
reverse trend in 1996 (August and September samples contained
signiﬁcantly more IPP). Detritus particles (< 10m in size) were greatest in
abundance during August and September when compared to June and July,

1995. In 1996, detritus abundance remained constant from J une-August,

 

 

43

Table 3. An abbreviated list of the algae identifed to generic level from
surface microlayer water samples in open water and vegetated habitats of a
permanent pond (Collins Road pond), East Lansing, Michigan, 1994.

 

 

Gonium
Scenedesmus
Synedra
Frustulia
Spirogyra
Cystodinium
Actinastrum
Ankistrodesmus
Tabellaria
Achnanthes
Tetrallantos

Chlorosarcinopsis
Gloeothece
Closterium
Cerasterias
Staurastrum
Kirchneriella
Anomoeneis
Scoliopleura
Selenastrum
Enallax
Chrysocapsa ?

Hyalobryon?
Shroederia ?
Hyalotheca ?
Cyanoderma
Gloeocystis
Navicula

 

 

SUI
SUI
.5

1
|
A

F
.r.

..I .41

. . . u. ~. . . . . . ... .. . u.‘ .. .... ...‘..h...~“: ...:
In.» . ... . ELCIE..T:1:13....fﬁi J.......;....E.§:. it... . .. I... E... . .

;. ..a._ *O&._,. VII ..

 

 

 

 

 

 

 

 

 

 

 

A .

.2
a, 7
g 6- 0..... .;:‘:::;.:=.Wﬁf.ﬁ¢
h A-------------A—
cu
in 5 s 0
a 24° ----------
— “‘0.
.2 A 4— ~. LT)
E l ""1
m 31 "0'”
2° 1

2 l I I I

June July August September
Date
B .
2 7
a, D—U/uﬂ
S 6 (a 53:33:176'N‘; ..................... o
(2 ‘3' ......................... 2”“ \‘x T
0) w 0‘ “A
91 a 5 o l
m I-l _l
.2 T
.2 “0
H .. T'.'.' l
:a 4 T ,,,,,,, o
n. c, ....... I
e' i
z 3 I I I I
June July August September

Date

MAL
—c}_ Total Bacteria
........ 0........ Algae
-.....on IPP

IQJLAL
—c}— Total Bacteria
........ 0.....- Algae
...---..o IPP

---¢--- Detritus

Figure 7. Total bacteria, algae, invertebrate parts/protozoans (IPP) and detritus
in surface microlayer water samples on a seasonal basis,(A) 1995; (B) 1996,
East Lansing, MI.Error bars represent S.E.(N = 3 samples per month).

45

Table 4. Seasonal difference between mean log number bacteria particles per
1 ml sample of surface microlayer water during June - September, 1995, 1996.
Samples were collected from the permanent pond a.

 

 

 

Month N Year Rods (SE) Cocci (SE) Total Bacteria (SE)
June 15 1995 6.6 (0.02) 6.1 (0.04) 6.7* (0.02)
15 1996 6.2 (0.08) 5.4 (0.09) 6.3 (0.08)
July 15 1995 6.1 *(0.07) 5.4 *(0.05) 6.2 (0.40)
15 1996 6.2 (0.02) 5.2 *(0.08) 6.3 (0.07)
August 15 1995 6.4 *(0.07) 5.5 *(0.07) 6.4 (0.07)

15 1996 6.6 *(0.01) 5.8 *(0.03) 6.7 (0.01)

 

September 15 1995 6.56 *(0.01) 5.9 (0.03) 6.6 *(0.01)
15 1996 6.7 *(0.01) 5.4 (0.09) 6.7 (0.01)
a Data are Log transformed.

* Signiﬁcantly different p < 0.05

46

Table 5. Seasonal difference between mean log number algae, IPP and detritus

particles per 1 ml sample of surface microlayer water during June - September,

1995, 1996. Samples were collected from the permanent ponda.

 

 

 

 

 

Month N Year Algae (SE) IPP (SE) Detritus (SE)
June 15 1995 6.22 * (0.02) 4.8 *(0.07) 5.8 (0.03)
15 1996 5.5 * (0.06) 3.5 (0.37) 5.9 (0.05)
July 15 1995 6.1 *(0.04) 4.4 *(0.12) 5.8 (0.05)
15 1996 5.9 *(0.13) 3.9 (0.29) 5.9 (0.02)
August 15 1995 6.0 (0.02) 3.2 (0.6) 6.0 (0.05)
15 1996 6.2 *(0.02) 5.1 *(0.08) 6.3 (0.08)
September 15 1995 6.0 (0.02) 3.9 (0.43) 6.1 (0.04)
15 1996 6.0 *(0.01) 4.4 *(0.33) 5.3 *(0.39)
a Data are Log transformed.

* Signiﬁcantly different p < 0.05

47
but was signiﬁcantly lower in September. Between year analyses show that
only cocciform bacteria and algal numbers were signiﬁcantly higher in 1995
than in 1996. All other microbial components did not not differ between
years.

Rod bacteria were signiﬁcantly less abundant in algal clump and
emergent plant base microhabitats than open water zones during 1995 and
1996 (Figure 8). Cocci bacterial numbers did not differ in abundance
between microhabitats in 1995, but were signiﬁcantly greater in the algal
clump and emergent plant base microhabitats than open water zones in
1996. Total bacteria showed a similar trend for 1995 -1996, i.e. algal clump
and emergent plant base microhabitats contained signiﬁcantly more bacteria
than open water zones. Open water zones were signiﬁcantly lower than algal
clump and emergent plant base microhabitats in algal numbers per sample
in 1995 and 1996. Invertebrate parts/protozoans were signiﬁcantly higher in
the emergent plant base and algal clump areas versus the open water zone
in 1995 and 1996. Detritus particle numbers were not signiﬁcantly different
in 1995 but differed signiﬁcantly in all three microhabitats in 1996.

Vernal /Aestival pond

Pond type differences in 1995 for bacteria are represented in Table 6.
Rod bacteria were signiﬁcantly more abundant in the vernal pond. Cocciform
bacteria did not differ between pond types. Total bacteria numbers were
signiﬁcantly higher in the vernal pond. Algal, IPP and detritus abundance in
1995 is displayed in Table 7. Algae numbers were signiﬁcantly greater in the
aestival pond. Invertebrate parts and protozoans were not observed in the

vernal pond samples and were signiﬁcantly greater in the aestival pond. The

A .
El Rods
Emergent Plant Base
Cocci
Algae
Algal Clump I IPP
I Detritus

Open

 

0 25 50 75 100 125
Percentage of particles per sample
B .
:. Rods
Emergent Plant Base Cocci
Algae
Algal Clump IPP

Detritus

Open

 

0 25 50 75 100 125

Percentage of particles per sample

Figure 8. Percentage of microbial particles per millilter of sample (N: 5 per
sample) in three microhabitats: open water; algal clump; and; emergent plant
base in a permanent pond habitat, A) 1995, B) 1996.

49

Table 6. The mean number (log/ml) of each bacterial food type found within a
vernal and aestival pond, East Lansing, MI 1995.8

 

 

 

Pond Type N Bacteria
Rods Cocci Total Bacteria
Vernal 10 6.68* 5.96 6.76*
(0.013) (0.044) (0.015)
Aestival 10 6.61* 5.96 6.70*
(0.009) (0.023) (0.010)

 

a Data are means (SEM)
* Indicates means are signiﬁcantly different, AN OVA, p < 0.05 is signiﬁcant.
Analysis conducted on log transformed data.

50

Table 7. The mean number (log/m1) of algal, invertebrate parts/protozoans

(IPP) and detritus food types found within a vernal and aestival pond, East

Lansing, MI 1995.a

 

 

 

 

Pond Type N Food Type
Algae IPP Detritus
Vernal 10 5.60* 0.00* 5.51*
(0.094) (0.000) (0.098)
Aestival 10 6.00* 2.86* 5.7 7 *
(0.023) (0.629) (0.013)
a Data are means (SEM)

* Indicates means are signiﬁcantly different, AN OVA, p < 0.05 is signiﬁcant.

Analysis conducted on log transformed data.

51
number of detritus particles were signiﬁcantly greater in the aestival pond
than the vernal.

There were two microhabitats within the vernal/aestival ponds that
were analyzed for microbial food resources, ﬂoating debris and tree base
(Figure 9). The percentage of rod bacteria did not differ between
microhabitats. Cocciform bacteria were not signiﬁcantly different between
microhabitats. However, total bacteria numbers were signiﬁcantly higher in
the tree base microhabitat. Algal and IPP numbers did not differ between
microhabitats, however the percentage of detritus particles was signiﬁcantly
higher in the tree base microhabitat. There was a signiﬁcant pond type X
microhabitat interaction for detritus particles, the aestival pond contained
signiﬁcantly more detritus in the ﬂoating debris microhabitat than the
vernal pond.

In 1996, seasonal effects on the microbial composition were
signiﬁcantly different for rod,cocciform, total bacteria, algae, IPP and
detritus food resources (Figure 10). Rod bacteria were signiﬁcantly lower in
number on April 1, July 15 and August 1 and greatest in number on April
15, August 15 and September 1 than any other date. Cocci bacteria followed
a similar trend for these dates, with highest numbers on April 15 and
August 1 respectively. As expected, total bacteria numbers mirrored these
results. Algal numbers per sample tended to increase over time and were
signiﬁcantly higher in the ﬂoating debris microhabitat throughout the
season, especially during the months of July and August. The invertebrate
parts/protozoan numbers were signiﬁcantly lower on April 1 and greatest on
May 31. The IPP food resource availability in this pond increased steadily
from April 1 - May 31 and then began to decline and reach an equilibrium by

 

, _. .... .... .. . ... . . . 3.11“]... 1...! laid-1'
. .. .... ... ....s.. ...:. #1.... ... . m _m........ i. t.~...ov.:;4..:.b _ ...f . . . . .. . a .i‘i
. . .. . . 4 . a .

 

   

 

 

 

 

 

 

   

 

A.
Rods
Tree Base- Cocci
Algae
I IPP
Detritus
Floating Debris 4
I l l l
0 25 50 75 100 125
Percentage of particles per sample
B.
Rods
Tree Base- C0061
Algae
I IPP
Detritus
Floating Debris -
l l l

 

 

 

l
0 25 50 75 100 125

Percentage of particles per sample

Figure 9. Percentage of particles per sample (N = 5 per sample) from
two microhabitats(Floating Debris and Tree Base) in a vernal (A) and
aestival (B) pond,East Lansing, MI 1995.

53

  
 
   
  
  

 

  
   
  
  
   
  
  

A .
Sept. 1 ................................................. Reds
August 15 -I-Z-Z-Z-Z-I-:-:-:-;~;-;.;.-.;.;.;.:.:.:,:,:.:.:_._:.:. .
August 1 ............................................. COCCl
July 15 I-I-Z-Z-Z-:-:-:-;-;-:.;.;.;.;.:. ____ Al
gae
Date July 1 .....................................................
June 15 'i'i'I'Z-1'PI-Zi-Ii-z-ch-z-L;.;.;.;.;.;.;.;. I IPP
May 31 ....................................................... Detritus
May 15 z-:-:-:-:-:-:-;.:.;.;.;.;.;.;.;.;.;.;.;.;,;,;.:,:,:
May 1 ----- ----- : ........................
April 15
April 1 ...............................
0 25 50 75 100 125
% of Food Items/Sample
B .
Sept, 1 ...................................................... ROdS
August 15 -:-:-:-:-:-:-:-:-:.:.;.;.;.;.;.3.;.;.:.:.:,:,:,:_:,.,:_
August 1 .................................... Cocci
July 15 Al ae
Date July 1 ..................................................... g
June 15 '2'2'2'2'1'3'3'3'1'1'1-I-Z-Z':-2-:-:-:-:-:-:-:-:-:.:.:.: I IPP
May 31 ......................................................... Dem-ms
May 15 C-C-Z-Z-Z-:-Z-:-;-:-1.;.j.;.;.:.:.;.:.:_:.:_:_:_:_:_
May 1 .....................................................
April 15
April 1 .._.:.;.: .......................

0 25 50 75 100 125
% of Food Items/Sample

Figure 10. Seasonal differences in percentage of microbial
components found in the surface microlayer of a vernal pond in
1996, East Lansing, MI. A) Floating debris microhabitat, B)
Tree base microhabitat, ( N = 5 samples/habitat/date).

54
August 1. Detritus particles per sample were signiﬁcantly lower on July 15,
August 1 and September 1, essentially the end of the sampling season.
These numbers were relatively constant up to July 15.

Microhabitat differences in the 1996 vernal pond as observed in
Figure 11, indicate that algal numbers were signiﬁcantly higher in the
ﬂoating debris area than the tree base area. All other food resource
categories did not differ in number between microhabitats.

68’ 'e e c ' i _..no Mun iﬁc.tion f91t‘5j On 0'0n ts

AA

fgr angpheline larvae.

Permanent pond

Before larval experiments for microbial food resource analysis were
inititated, the microbial composition of the surface microlayer was tested
between microhabitats and inside vs. outside of the microcosms. As
predicted, no signiﬁcant differences were observed in sample location, i.e.
microbial composition inside the microcosms was no different from outside
(Table 8). Algal, IPP and detritus numbers were signiﬁcantly greater in the
vegetated microhabitat, as expected.

The consumption of rod bacteria did not signiﬁcantly differ between
habitats (Figure 12). Cocciform bacteria consumption was signiﬁcantly less
in the open water zones. Total bacteria numbers were not signiﬁcantly
different between microhabitats. Larvae reared in algal clump and emergent
plant base microhabitats consumed signiﬁcantly more algae than larvae in
open water areas. Furthermore, larvae consumed more invertebrate
parts/protozoans in algal clump and emergent plant base microhabitats than
larvae in open water zones. Finally, detritus particle consumption did not

differ between microhabitats.

 

No. of Particles per sample
Log

    

J

 

 

Floating Debris

 

 

 

Tree Base

Microhabitat

«'7.
mﬁﬁ

Rods

Cocci

Total bacteria
Algae

IPP

Detritus

Figure 11. Microbial composition of a floating debris and tree base
microhabitat in a temporary pond, East Lansing, MI 1996. Error bars
represent SEM, (N = 5 samples per microhabitat).

56

Table 8. Control experiment testing the mean number of rod, cocci, total
bacteria, algae, invertebrate parts and protozoans observed in the surface
microlayer of inside vs. outside larval microcosms, two factor AN OVA was
used to test microhabitat (N = 2), sample location (N = 2, inside vs. outside),
(N = 5 samples/location) for 1995.

 

Factors F-values
Rods Cocci Tot. Bact. Algae IPP Detritus

 

 

df
Microhabitat 1 0.21 6.72* 0.27 3.72 7 .55** 5.60***
Sample Location 1 0.15 0.83 0.38 0.02 0.30 0.01
Interaction 1 0.08 0.88 0.01 0.002 0.02 0.0004
* Signiﬁcant at p < 0.02
** Signiﬁcant at p < 0.02

*** Signiﬁant at p < 0.03

H

-. Rods
Emergent Plant Base

ﬂ

Cocci

Algae
IPP

Algal Clump

 

Detritus

Open

 

0 25 50 75 100 125

Percentage of particles per sample

Figure 12. Percentage of microbial particles per gut sample of
from larvae reared in three microhabitats:open water, algal clump,
emergent plant base in a permanent pond, 1994 (N=5 samples per
habitat).

58

In 1994, preliminary studies revealed an interesting component to the
An. guadg'magulatus fourth instar diet. Larvae reared in open water (N =10)
and vegetated zones (N=8) were dissected and contents examined. A species
of cladoceran (Daphnia sp.; mean size = 20-30 um, N = 10) was identiﬁed
and the consumption of this cladoceran between habitats (open vs. vegetated
areas) was compared with a two-sample ttest (p < 0.05). Larvae reared in
open water microhabitats consumed signiﬁcantly more cladocerans than
larvae reared in vegetated areas (Figure 13).

Vernal lAestival pond

In 1995, third and fourth instar Ag. pungtipennis were collected from
the vernal and aestival ponds on a single date to compare the microbial
composition found within their guts. Rod and cocciform bacteria, total
bacteria, and algae were all signiﬁcantly greater in number in the vernal
pond than the aestival pond (Figure 14). Invertebrate parts/protozoans and
detritus numbers were consumed in signiﬁcantly higher numbers in the
aestival pond than the vernal pond.

The microbial components in gut samples collected from third (N=13)
and fourth (N=11) instar An. punctipennis were compared. The percentage
of particles found per instar gut sample is presented in Figure 15. It appears
that total bacteria, algal and IPP consumption does not signiﬁcantly differ
between fourth instars and third instars. However, fourth instars consumed
signiﬁcantly more detritus than third instars.

To determine if larval diet changed from one year to the next in the
vernal pond, third and fourth instar Q. punctipennis guts were analyzed
and compared in 1995 and 1996 (Figure 16). Between year comparisons

show that larvae consumed signiﬁcantly more rod and cocciform bacteria,

 

 

 

 

 

 

 

 

 

 

 

 

0.8
n=10
E.
u 0.6 -
G
a
g E” 0.4 —
U
4:
5 0.2-
2
0 I
Open Veg
Habitat

Figure 13. Mean number of cladocerans dissected from 4th instar Anopheles
quadrimaculatus guts, permanent pond, East Lansing, MI 1994.

 

 

 

El Rods
Aestival- Cocci
Algae
Pond Type I IPP
Detritus
Vernal-
1 I I

 

 

I
0 25 50 75 100 125

Percentage of particles per gut sample

Figure 14. Percentage of particles found per gut sample (N=12 guts/pond
type) of instar Anpunctipgmis larvae in a vernal and aestival pond, East
Lansing, MI 1995.

 

 

125
Rods
100‘ Cocci
Percentage of m
particles per 75‘ I Algae
gut sample I IPP
50‘ Detritus
25-
0

 

 

 

 

Figure 15. Percentage of particles found per gut sample of third and fourth
instar Q. punctipennis in a vernal and aestival ponds combined, East Lansing,
MI 1995, (N: 6 guts per instar).

A.

 

125
100 -

Percentage of 75-
particles per

 

 

 

 

 

Third

 

Fourth

 

gut sample 50 _
25 -
0
B .
125
100 -

Percentage of
particles per 75-
gut sample

50-

25-

 

 

 

CD

 

Third

 

Elﬁ

Rods
Cocci
Algae
IPP

Detritus

Rods
Cocci
Algae
IPP

Detritus

Figure 16. A comparison of the percentage of microbes found within third
and fourth instar Q. punctipennis in a vernal pond, East Lansing, MI.
A) 1995, B) 1996, (N: 6 guts per instar).

63

total bacteria, and algae in the 1995 vernal pond than the 1996 vernal pond.
The consumption of invertebrate parts/protozoans was greater in the 1996
than in 1995, while detritus consumption did not differ between years. Total
bacteria consumption did not differ between fourth instars and third instars.
However, fourth instars consumed signiﬁcantly more algae than third
instars in 1995. Fourth instars consumed more detritus than third instars
in 1995.

DISCUSSION
Permanent pond

Surface water temperatures were similar for both habitats, with the
exception that minimum temperature was cooler in vegetation areas than in
open water zones. All inorganic ion concentrations were within known
published accounts of tolerable limits for growth and survivorship of &.
W (V rtiska & Pappas 1984; Clements 1992); despite the paucity of
information on chemical habitat data for A_n, Wm, their
widespread distribution suggests they may have broad tolerances for
inorganic ion concentrations. This evidence is supported by the consistent
presence of both species in the permanent pond and
more importantly the temporary vernal/aestival ponds, where inorganic ion
concentrations would be signiﬁcantly different (Iversen 1971).

DAPI has proven to be a reliable staining technique of mosquito food
resources (Walker et al. 1988a; Walker & Merritt 1993 and Hennes & Suttle
1995). Available food resources for Q. quadrimaculatus found within the
surface microlayer of a permanent pond were very consistant regardless of
season or microhabitat. Within the surface microlayer, bacteria were the

most abundant food type, followed by algae, detritus and invertebrate

64
parts/protozoans. Seasonal differences across the range of food types and
microhabitats were quite varied. Other microbial studies have reported a
seasonal component in the abundance of certain bacteria not necessarily
associated with the surface microlayer (Paterek & Paynter 1988), as well as
differing abundances of microbe types within microsites (Hines & Buck
1982; Lopez 1988, Smith et a1 1997). Olds et al. (1989) found cocciform
bacteria to be most numerous compared to other microbers. Rod bacteria
were always the most abundant type of bacteria found within the surface
microlayer although not as numerous as reported by Walker & Merritt
(1993). Greater total bacterial numbers in the algal clump and emergent
plant base microhabitats as compared to the open water microhabitat were
probably due to the aggregation of bacteria around living/dead organic
material.

The diet of fourth instar A_n. W inhabiting a permanent
pond reﬂects the types and abundances found in the microhabitat analyses.
Bacteria have been considered to be the most important of the
microorganisms that comprise the food of mosquito larvae (Christophers
1960; Laird 1956, 1988), and it appears this may be true for larvae
inhabiting open water areas as observed in this study. Cocciform bacteria
concentrations were far less than those found by Walker et al. (1988a) and
Walker & Merritt (1993) for the same species. Indeed, larvae consumed less
cocci bacteria in the open water zones than vegetated areas during this
study. However, Smith et al. (1997) indicate that bacterial diversity differs
between microhabitats with and without vegetation. Microhabitat did not
have an effect on larval consumption of total bacteria, that is, total bacteria

abundance was similar for the three microhabitats.

65

Larvae consumed more algae and IPP particles in the algal clump and
emergent plant base microhabitats than in open water areas. A signiﬁcant
percentage of the larval diet in these microhabitats consists of algae, IPP
and detritus. Larval consumption of detritus did not differ between
microhabitats. These microhabitats may provide not only protection from
predators (Hurlbut 1938; Orr & Resh 1992), but also a signiﬁcant dietary
component for mosquitoes as well as other dipteran larvae. This conclusion
is supported by Campeau et al. (1994), i.e. algal type and abundance may be
important dietary components for chironomid midge production in a
freshwater marsh. .

In the invertebrate parts/protozoan category, most notable was the
presence of cladocerans (Cladocera) in larval guts. Due to the technique
used to dissect larvae and the stage of cladoceran decomposition in larval
guts, no speciﬁc identiﬁcations have been made on this type of cladoceran. It
was suspected that because Angpheles larvae feed within the surface
microlayer, this species of cladoceran may be chphgleberis b11311 , typically
found within this microlayer . However, Thorp & Covich (1991) state that _S.
kingji is the only known distasteful cladoceran and Schwartz et al. (1983)
add that it is not even ingested by HJdra . Recent discussion indicates that
the cladoceran in this study may belong to the genus Daphnia (Snyder
pers.comm.). Early studies on anopheline food types indicated that they
consume an array of microinvertebrates, e.g. rotifers, copepods, ciliates and
even other early instar anopheline larvae (Coggeshall 1925; Boyd & Foot
1926; Senior-White 1927; Hinman 1930), but nowhere in the literature is the

consumption of cladocerans mentioned. This study shows convincingly that

66
larvae consume more cladocerans in the open water habitats than vegetated
areas. The implication of a microinvertebrate food source, e.g. cladocerans
and knowledge of other dietary components may contribute to a better
understanding of future biocontrol methods (Dahl 1988; Thiery et al. 1991;
Porter et a1. 1993).
Vernal /Aestival ponds

To date, the food resources (speciﬁcally bacteria) entrapped in the
surface microlayer of temporary ponds have not been quantiﬁed, but the
algal and protozoan concentrations found in a temporary forest pool have
been discussed by Ameen and Iversen (1978). Surface microlayer analyses
for available food resources in the vernal/aestival pond during the 1995
season indicate that total bacteria numbers were higher in the vernal than
the aestival pond, and that rod bacteria constituted the primary bacteria
type in both pond types. These results are similar to those observed in the
permanent pond. The aestival pond contained more algal, IPP and detritus
particles/m1 of surface microlayer sample than the vernal pond. Minimum
pH (6.65) in the temporary pond may have had an effect on presence of
certain microﬂoral organisms commonly found in anopheline guts e.g.
diatoms (Forged 1964).

In the vernal/aestival ponds, anopheline larvae are usually found
near or next to tree bases and ﬂoating debris. The microhabitat analysis on
food resources within these microhabitats shows that total bacteria numbers
were higher in the tree base microhabitat versus the ﬂoating debris area.
Algae and invertebrate parts/protozoans were found in similar abundance
betwiveen microhabitats. However, the surface microlayer around tree bases

contained more detritus particles than the ﬂoating debris microhabitat.

67

In 1996, the temporary pond remained permanent throughout the
ﬁeld season. While bacteria concentrations ﬂuctuated during the 5 month
sampling period, algal numbers increased over time and were higher in the
ﬂoating debris compared to the tree base microhabitat. Floating debris, such
as branches and sticks provide attachment sites for algae to accumulate.
Aedes gommunis (De Geer) and Q. gantans (Meigen) were observed to
consume a variety of algal types in a Danish temporary pool (Ameen and
Iversen 1978). In my study, invertebrate parts/protozoans increased until
May 31, 1996, afterwhich, numbers began to decline until late summer. This
decline in IPP may be explained by the presence of other culicid ﬁlter
feeders, e.g Qd_e_s canadensis, stimulans, excrucians and m . These
mosquitoes are all present in high densities and most probably consume a
great deal of microorganisms. Detritus particles found within the surface
microlayer declined over time with signiﬁcantly lower numbers at the end of
the 1996 season. Because the vernal pond dried out during the 1995 season,
such a dessication process may have caused the organic detritus to
concentrate proteins and decompose faster (Barlocher et al. 1978). A decline
in detritus abundance during the 1996 season may in part be due to the fast
rate of detritus composition through dessication, thus providing a rich food
source that may have been consumed earlier in the season by the
overwintering spring mosquito recruits such as AW, Mans and
W (Wiggins et al. 1980).

Perhaps due to the ephemeral nature of vernal/aestival ponds or
because Qgpheﬁ larvae are in low densities, there has been no
quantitative description of anopheline diets in such ponds. Gut content

analysis on third and fourth instar Q. pungtipennis inhabiting the vernal

68
pond indicates that larvae consume more bacteria and algae than larvae
from the aestival pond and more IPP and detritus (close to 25 % more) from
the latter. Habitat analyses indicate that more detritus and IPP are
available for larval consumption in the aestival pond, this may explain
greater numbers of these food sources in larval guts. Concommitantly, the
normal ecological succession of algae in these ponds may provide less
palatable types in the aestival pond as observed by Ameen and Iversen
(1978). In addition, IPP diversity may be different in these ponds with more
palatable or available species in the aestival pond.

The dietary components found in Q. pgngtipennis larval guts
indicate that larval diet does not differ quantitatively for bacteria, algae or
IPP consumption between instars. However, fourth instars consumed
signiﬁcantly more detritus than third instars. This difference in detritus
consumption may be a function of larval size, i.e. third instars may not have
the mouth gape large enough to ingest larger detritus particles. Several
authors have demonstrated that mosquito larvae are able to select particles
on the basis of size (Merritt et al. 197 8; Merritt 1987), but only for fairly
broad categories (e.g. less than 2 pm, 2 to 10 pm).

The difference in algal and detritus consumption in the vernal pond
between years may be explained by the date when larvae were sampled, i.e.
late May, 1995 and mid-late June in 1996. Perhaps the dessication of the
aestival pond in 1995 created an abundance of available detritus particles
within the surface microlayer in 1996, hence increased larval consumption of
detritus may be explained. Because of a more enriched food source

(Barlocher et al. 1978), larvae may have selected detritus over algae in 1996.

69
Such dietary selectivity is a novel concept for these as well as most types of
ﬁlter-feeders (Merritt et al. 1992) and warrants further study.

This study has documented for the ﬁrst time a quantitative
evaluation of anopheline food sources in a temporary vernal/aestival pond
and conﬁrmed such ﬁndings with analyses of third and fourth instar guts.
The exploitation of temporary pools by anopheline larvae may enhance
dispersal and colonization tendencies in more permanent water habitats.
Whether this phenomenon is a potential evolutionarily stable strategy or not

requires further study.

70
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CHAPTER 2
DIEL PERIODIC FEEDING ACTIVITY OF AN OPHELES
QUADRIMACULATU S (SAY) MOSQUITO LARVAE

ABSTRACT

Diel feeding activity of third and fourth instar Q. _quadg’maculatus
(Say) was studied in a permanent pond. This ﬁeld study examined the
consumption of microbial and microinvertebrate food resources over a diel (24
h) period within two habitat treatments (open water and vegetated areas-
with/without algal clump additions). A ﬂuorochromatic stain (DAPI) with DNA
binding properties was used to quantify microbial dietary components within
larval guts and habitats.

Within larval guts, microbial analyses show that bacteria were the most
abundant food type, followed by detritus, algae and invertebrate
parts/protozoans (IPP). Diel differences across food types were fairly consistent
across habitats, i.e. more bacteria, algae and detritus were found in larval guts
at midnight than noon. Greater numbers of rod bacteria and IPP were
consumed in vegetated areas than open water zones.

Q. Wars larvae consumed numerous types of
microinvertebrates, e.g. water mites, cladocerans and rotifers over a diel period.
The consumption of water mites is unique and has never been observed before
in nature. Third instars consumed signiﬁcantly less microinvertebrates than
fourth instars. Larval consumption of cladocerans was signiﬁcantly greater at

midnight than noon. Larval gut analyses examined every hour over a 24 h

78

79
period provide signiﬁcant evidence on the approximate time larvae switch
microinvertebrate dietary resources.

Habitat had a signiﬁcant effect on microinvertebrate consumption by Q.
W larvae. Signiﬁcantly more water mites and rotifers were
consumed in the open water areas versus vegetated zones. Surface microlayer
water samples show that larvae do not selectively feed on water mites or
cladocerans, but do feed preferentially on rotifers. Perhaps knowledge of the
"feeding area" or microhabitats, speciﬁcally, where and when larvae optimally
forage as well as particle sizes and food types consumed by larval mosquitoes in
the natural habitats will enhance the success of bacteria and other particulate

larvicides.

80
INTRODUCTION

Mosquitoes are hosts to a variety of pathogens and parasites including:
viruses, fungi, bacteria protoctistans and nematodes (Clements 1992).
Mosquitoes are medically the most important group of insects in the number of
disease agents they transmit and the magnitude of health problems these
diseases cause to humans and animals worldwide (Service 1989). Mosquito
control relies on the knowledge of behavior and ecology of the target species.

Qgpheles ggadljmmlatus (Say) mosquito larvae inhabit permanent,
lentic ponds in eastern North America. Adults were responsible for the
transmission of human malaria prior to disease eradication in this region
(Carpenter and LaCasse 1955). Larval Q. W aggregate at air-
water interfaces (surface microlayer) of plant stems and algal mats (Orr and
Resh 1989; 1992) and feed on food particles from the surface microlayer by
using the collecting-ﬁltering feeding mode (Merritt et al. 1992a). Although
several investigations (Collins et al.1988; Orr and Resh 1989, 1992) have
alluded to this feeding aggregation at the air-water-plant interfaces as a
predator avoidance strategy, food resources may be more abundant at these
interfaces.

An important determinant of mosquito larval distribution and feeding
success is the distribution of larval food resources. Walker and Merritt (1993)
noted that the surface microlayer contains abundant concentrations of non-
aggregated bacteria, plant detritus associated with bacterial aggregates, and
amorphous organic material. A qualitative characterization of the microbial
components and nutrients available as food for Qgphelga larvae found within
the surface microlayer and in gut contents has been well documented (See

review by Merritt et al. 1992a). Recently, Wallace and Merritt (1996) have

81

shown that third and fourth instar Q. W consume an array of
microinvertebrates, e.g., water mites (Eylaidae: Ellaissp), cladocerans, and a
few metazoans e.g. rotifers and copepods, these have been quantiﬁed according
to various microhabitats, e.g. algal mats, emergent plant stems and open water
areas (Wallace and Merritt 1995; Smith et al. 1997).

This study focuses on the diel feeding activity of third and fourth instar
Q. mm in a permanent pond. The objectives were to: 1) identify
and quantify microbial food sources of third and fourth instar Q.
Wins in two habitats (open water areas and vegetated zones) over
a diel period; 2) investigate larval Q. W consumption of
microinvertebrate food items and determine if consumption differs between
habitats; and 3) determine if Q. guadu'magulaﬂrs larvae selectively feed on

such microinvertebrates through ﬁeld habitat assays.

MATERIALS AND METHODS

Experiments were conducted during July - August, 1995 and 1996. Q.
mﬂrjmgﬂatus larvae were used in all feeding experiments during the study
period.
Lﬂudlﬁits

Research was conducted in a permanent pond (approxi. 2.25 ha), located
approximately 8 km south of Michigan State University at the Collins Road
Entomology Field Laboratory, Ingham County, Michigan, USA. Experiments
were performed over three ﬁeld seasons, 1994, 1995 and 1996. The marsh is
bordered with cattails (mug sp.) and emergent grasses e.g. foxtail (Setmja
sp.). Two speciﬁc habitats were compared in this study, an open water zone

and a vegetated area. The open water zone consisted of an area of the pond that

82
was without ﬂoating or emergent vegetation within 4 m of the larval
microcosms. The vegetated area provided ample emergent vegetation, e.g.
grasses (Setaria sp.) and cattails (131133 latifglja) around which microcosms
were placed.
Markham

Q guadg'maculatus larvae were maintained in ﬁeld microcosms for all
diel periodicity (24h) experiments. Microcosms were constructed of a 4 liter
clear, plastic, round container (20 cm diam. X 25.5 cm length). The bottom and
sides of each container were cut out and replaced with a nytex mesh (X 100nm)
covering to allow vertical and horizontal water movement. Each container was
ﬁtted with a styrofoam ﬂoat placed around the top of each container. The ﬂoat
allowed each microcosm to adjust with water level ﬂuctuations and allowed
natural light conditions to reach the interior. Each microcosm was ﬁtted with a
screen lid/covering to prohibit predator and/or competitor recruitment.
Microcosms were kept in a speciﬁc location with a monoﬁliment line tied to a
garden stake. Microcosms were placed randomly approximately 2 m apart in
open water and vegetated areas in order to simulate areas where anopheline
larvae are present and absent (See Figure 2, Chapter 1).

All larvae used in diel experiments were collected in the ﬁeld at noon and
midnight (representing a diel period) with a 250 ml plastic mosquito dipper and
then transported to the laboratory in a plastic collection bucket. In order to
ensure that third and fourth instars had consumed microbial components from
their respective habitat treatments, larvae used in the diel microbial
composition experiment were collected as second instars and placed inside ﬁeld
microcosms (N: 125 larvae/microcosm) prior to the experiment. Second instars

molted into third and later into fourth instars thus providing assurance that all

83
gut contents were voided prior to third and fourth instar collections. Larvae
used in the diel microinvertebrate experiments were collected as early third
instars and transferred to the laboratory for further preparation.

In the laboratory, larvae were placed inside 1 L sterile, plastic containers
with 250 ml of distilled water. The containers were placed inside a Percival®
growth chamber and maintained at a 16:8 (L:D) ratio and constant temperature
of 23 °C. Larvae were maintained inside the growth chambers for 24 h so that
all microinvertebrates larger than 5 um would pass through the digestive
system and not be included with diel microinvertebrate counts from gut
dissections later. The removal of microinvertebrates of 5 pm or larger in size
would ensure that all rotifers, cladocerans and water mites would be expelled
from the larval guts. After a 24 h period, larvae were returned to the ﬁeld and
placed inside the respective microcosm treatments.

There were two habitat treatments for all diel experiments, open water
with/without an algal clump vs. vegetated habitats with/without an algal
clump. The algal clump treatments consisted of a 5 cm X 5 cm piece of ﬂoating
algae cut from a nearby mat, rinsed in distilled water and placed inside each
algal treatment microcosm. A 2 (habitats) X 4 (treatments) factorial design
with 3 replicates was implemented for all diel feeding experiments.

1 1 1° 1 i 1 il' . I

To more fully understand the spatial and temporal contribution of
microbes to larval anopheline diets, a diel feeding experiment was conducted in
July, 1995. Field microcosms were stocked with 125 second instar Q.
qngdn'magulatus. Larvae were maintained inside these microcosms until they
reached the third and fourth instar. Microcosms were checked at noon and

midnight on a daily basis and third and fourth instars were removed. Upon

84
collection, larvae were killed in boiling water in the ﬁeld, preserved in 4%
formalin at 4°C in order to slow any digestion or other processes that may have
altered the gut contents, and transported to the laboratory.

In the laboratory, the food bolus from each gut was dissected with
minuten pins and the gut contents removed and separated from the peritrophic
membrane with several washes of distilled water. The contents were pipeted
into a sterilized 2 dram vial. Distilled water was then added to the vial to bring
the total volume to 2.5 ml. The contents of the vial were mixed with a vortex
mixer for 20 sec to break-up large aggregates of material formed in the
digestive tract, but not long enough to rupture any cells.

To identify and enumerate the microbial components of each larval gut,
one milliliter of sample was removed and stained with DAPI (4'6--diamidino--2-
-phenylindole) (Porter & Feig 1980). For further details on staining and
counting procedures of gut contents, see description in Chapter one. Direct
counting methods using an epiﬂuorescence microscopy technique were used to
enumerate and identiﬁy bacteria (rod and cocciform), algae, invertebrate
parts/protozoans (IPP) and detritus from each sample. This technique has been
used successfully to identify the microbial concentration of 1 ml of sample
within larval mosquito guts and habitats (Walker et al. 1988a, Walker &
Merritt 1993). Algae, invertebrate parts and protozoans were identiﬁed using
keys from (Prescott 1970; Thorp & Covich 1991).

To determine if the ﬁeld microcosms had any effect on the microbial food
resources available to Q. W larvae, a separate ﬁeld assay of the
surface microlayer inside and outside of the microcosms was conducted. Prior to
larval introductions, a surface microlayer water sample was collected,

preserved and analyzed as described above.

85
i ' r t ' e e ' n

These experiments were designed to quantify the microinvertebrate
components found within the food bolus of an Q. W larval guts
and determine if larvae were consuming different microinvertebrates over a 24
h period. To test this objective, I hypothesized that larval consumption of
microinvertebrates would not differ in a diel period between or within
microhabitats. There were two habitats (open water and vegetated areas) and
two microhabitat treatments (control and algal clump additions).

Gut dissections and microinvertebrate analyses were performed on third
and fourth instar Q. W. Therefore, to ensure that fourth instar
guts contained material from the speciﬁc microhabitats, 110 third instar Q.
Wm were collected from the permanent pond using a 250ml
mosquito dipper and transported to the laboratory. Larvae were placed inside 1
L plastic containers ﬁlled with 250 ml of distilled water and set inside climate
controlled growth chambers (16:8, L:D, 23°C) for a period of 24 hours. A 24 hour
period was established as a control in order to clear all microinvertebrates from
the larval guts. A subset of fourth instars (N = 25) were sampled at this time,
killed in boiling water and dissected. Microinvertebrates were identiﬁed and
enumerated at this time using a binocular dissecting microscope.

After 24 h, a second subset of fourth instars (N = 25) was sampled and
dissected for microinvertebrate identiﬁcation and enumeration. The remainder
of the larvae in these containers were transported to the ﬁeld and placed in the
ﬂoating ﬁeld microcosms in open water and vegetated habitats (3
replicates/habitat). Control microcosms in both habitats contained no material
additions, whereas, the microcosms treated with an algal clump contained a 2"

X 2" piece of ﬁlamentous algae (log mean weight = 0.71 mg, SE. = 0.04).

86

Only fourth instars (N: 6/treatment) were collected for gut dissections in
the preliminary experiment of 1994. For the experimental trials in 1995, third
and fourth instars were collected at noon and midnight from the ﬁeld
microcosms over a three day period, killed with boiling water and transported
to the laboratory for dissections. In the ﬁrst trial of 1995, 48 third instars
(12/treatment) and 48 fourth instars (12/treatment) were culled for gut
dissections. During the second trial, 57 third instars (14/treatment) and 53
fourth instars (13/treatment) were examined for microinvertebrate
consumption.

' i n l 'm n s
Habitat analysis

To examine the possibility of larvae actually selecting for speciﬁc
microinvertebrates or randomly browsing for them, I conducted a habitat
survey of the microinvertebrates found within the surface microlayer during a
diel period.
The surface microlayer of the open water and vegetated habitats was sampled
at noon (N =10/habitat) and midnight (N=10/habitat) with a modiﬁed Harvey-
Burzell glass plate technique (Norkrans 1980). Water samples were preserved
in 0.5 liter plastic bags ﬁlled with 70% ethanol and transported to the
laboratory for microinvertebrate identiﬁcation and enumeration. By comparing
the availability of the different microinvertebrates across habitat types over a
diel period, we were able to determine if Q. guadrimagulatus actively select for
speciﬁc dietary components.
Hourly consumption analysis

In 1996, I conducted a ﬁeld experiment to examine the hourly

consumption of microinvertebrates over a 24 h period. Fourth instar Q.

87

W were collected prior to the experiment, starved for 24 h and
then placed into ﬁeld microcosms in open water and vegetated areas. Three
larvae were collected every hour from both habitats during this period, killed in
boiling water, preserved in ethanol and transported to the laboratory for gut
dissections the following day. Microinvertebrates were identiﬁed and
enumerated from each gut sample. The objective for this experiment was to
observe the time frame over a 24 h period in which speciﬁc microinvertebrates
were consumed by fourth instars.
Feeding preference analysis

The observation of microinvertebrate consumption by Qgplrelgs larvae
prompted further investigation. In 1995, I conducted a feeding preference
experiment in the laboratory using fourth instar Q. W and a
semi-terrestrial water mite (Eylaidae: Eylais sp). Fourth instars (N: 18) were
collected in the ﬁeld, transported to the lab and starved for 24 hours (See
protocol described above). After the 24h starvation period, larvae were divided
into two mite treatments, live (N=9, 3/replicate) vs. dead (N=9, 3/replicate).
Water mites were collected in the ﬁeld with plastic pipets, maintained alive in
2 dram vials of pond water and transported to the laboratory. Ten mites per
treatment were placed into each plastic container along with three Q.
W larvae. For the dead mite treatment, mites were killed in
boiling water from a microwave oven and placed into each container. The larvae
were maintained inside the climate controlled growth chambers and allowed to
feed for 24 h. All larvae were killed with boiling water, dissected and examined

for mite consumption.

88
4, Statistical analyses
Differences in microbial and microinvertebrate concentration during the
diel period between habitats were examined by analyses of log10 transformed
data using two-way analysis of variance (2-way AN OVA, P < 0.05 signiﬁcant).
Fisher's PLSD specialized t-test was used for a posteriori multiple comparisons
(Super Anova 1989). A two-sample t-test was used to determine the difference

in live mite vs. dead mite consumption (Zar 1984).

RESULTS
Larval dietary diﬁ'erences on microbes

Four categories of food resources were identiﬁed in third and fourth
instar guts: bacteria (rods and cocciform), algae, invertebrate parts/protozoans
(IPP) and detritus (Table 1). Third and fourth instar diets did not differ
signiﬁcantly for most food items. However, third instars consumed more rod
bacteria and detritus than fourth instars. There was a signiﬁcant Instar X
Time X Treatment interaction in that third instars consumed more detritus at
noon across all treatments than fourth instars . The consumption of cocciform
bacteria, algal and IPP did not differ signiﬁcantly between third and fourth
instars over a diel period.

Ingested food sources were compared over time. The percentage of total
bacteria, algae and detritus particles found within larval guts was signiﬁcantly
greater at midnight than at noon (Figure 1). Treatment comparisons of larval
dietary sources indicate that larvae consumed more rod bacteria in the
vegegation area (control and algal clump) than the open water zone (control
and algal clump) (Figure 2). In addition, larvae consumed signiﬁcantly more

IPP in the open water zone (control and algal clump) than vegetated areas

89

Table 1. Mean number of microbes found within a lml sample (N = 6 per sample) dissected from third and fourth
instar An. madﬁmamﬂatns larval guts at noon and midnight.a

 

 

 

Instar Time WWW
Rods Cocci Total Bacteria Algae IPP Detritus
(SE) (SE) (SE) (SE) (SE) (SE)
Third Noon 1.01 x 106* 1.19 x 105 1.13 x 106 5.25 x 105 7.42x 104 1.44 x 106*
(33795) (9945) (37835) (33535) (13288) (77677)
Midnight 1.19 x 106* 1.77 x 105 1.83 x 106 9.15 x 105 1.154 x 105 1.46 x 106‘
(54352) (21875) (454300) (196700) (18557) (102000)
Fourth Noon 1.03 x 106* 1.74 x 105 1.20 x 106 6.3 x 105 8.03 x 104 7.88 x 105*
(27223) (22777) (39561) (15159) (19321) (64051)
Midnight 1.06 x 106 * 2.15 x 105 1.27 x 106 6.3 x 105 2.00 x 105 1.33 x 106*
(34882) (27257) (49479) (43659) (44464) (107500)

 

a Data were log transformed for analysis

* Signiﬁcantly different at P < 0.05, ANOVA

thofParﬁekshnl

 

15 0990

lOOOOOO-zc

500000-

 

<5

 

 

 

 

 

 

 

 

Midnight
Time

Figure 1. Mean number of particles found in Q. u '

41W
guts over a diel (24h) period. Error bars represent SEM. * signiﬁcant

@E

Rods
Cocci
Algae
IPP

Detritus

larval

difference between midnight vs. noon, P < 0.05, N = 40 per time period.

91

a R d
Veg.-AC I ° 3
Cocci
Veg.-control I gae
I IPP
D t t
Open- AC I e r1*us

Open-control

 

0 25 50 75 100 125

Percentage of Particles

Figure 2. Percentage of particles/ml of gut content sample from Q.
c larvae 1n four treatments. * signiﬁcant difference
between treatments, P < 0. 05, (N: 24 per treatment).

92

(control and algal clump). The time X treatment interaction of larval detritus
consumption differs signiﬁcantly, i.e. larvae consumed more detritus at
midnight across all treatments. No other signiﬁcant interaction differences
were observed. The available food resources inside versus outside of the
microcosms was tested (Table 2). There were no signiﬁcant differences in rod,
cocci, total bacteria, algae, IPP and detritus concentrations between
treatments.
Larval dietary diﬁ‘erences on microinvertebrates

Preliminary experiments in 1994 showed that Q. gaadrimagalataa
larvae consume an array of microinvertebrates, e.g water mites (Eylaidae:
Eﬂa'g sp.) (Figure 3), cladocerans (I.D. unknown), and Ceratopogonidae larvae.
The cladocerans observed in the guts of third and fourth instar Q.
gaadﬁmagllatas were in very poor condition to be identiﬁed down to species
level, but appear to be Daphaia spp. Although no signiﬁcant differences were
observed over a diel period in 1994, this new and undocumented dietary
discovery provided fodder for future experiments in 1995 (Figure 4). There were
two trials of the same diel feeding experiment conducted in 1995 (July and
August). In order to determine if 24 h was sufﬁcient to void larval guts of mites,
cladocerans and rotifers, a laboratory starvation experiment was performed.
The results ﬁ'om the starvation assay showed that the mean number of mites,
cladocerans and rotifers (Lecanidae: ngana sp. and Brachionidae: W2
sp.) were signiﬁcantly less in the post starvation treatment (Figure 5).

In the ﬁrst trial (July), fourth instar Q. gaadrimagalataa consumed
signiﬁcantly more mites and cladocerans than third instars (Table 3). Although
not identiﬁed in the preliminary study in 1994, rotifers appeared to be a

signiﬁcant contribution to the larval diets. However, in the second trial

93

Table 2. Mean concentration/ml of microbial particles observed in water samples collected from the surface microlayer
inside vs. outside of the ﬁeld microcosms, (SEM). No signiﬁcant differences were observed.a

 

Microbial Concentration (Mean # particles/ml)

 

 

Treatment N Rods Cocci Total Bacteria Algae IPP Detritus
(SE) (SE) (SE) (SE) (SE) (SE)
Inside 12 1.30 x 106 5.35 x 105 1.84 x 106 5.95 x 105 5.35 x 104 1.32 x 106
(134000) (32819) (137000) (279750) (16753) (148100)
Outside 12 1.31 x 106 5.95 x 105 1.96 x 106 5.97 x 105 3.88 x 104 1.29 x 106
(108100) (51893) (139541) (151700) (8552) (113100)

 

a Data were log transformed for analysis.

94

 

Figure 3. Photograph of water mite (size: 25 um) (Eylaidae: Eylaiasp. )
with larval head of Q. W.

ertebrates

V

microin

Mean# of

 

0.5
Mites

Cladocerans

E ceratopogonidae

Log value

._. '
...”

  

 

 

 

 

 

............... k
:I;I:3;2:I:I:Z: 4
D:I:I:o:o:o:o:g “=22“
.:::::::;::I:I_... E51" .5- £7
M1. dm' ght

Time

Figure 4. Preliminary experiment on diel feeding by Q. MW
larvae onmicroinvertebrates, samples collected at noon and midnight. Error
bars representSEM. No signiﬁcant differences were observed, N = 12 per
time period.

invertebrates

micro

per gut (Log value)

Mean # of

 

 

Mites
Cladocerans
Rotifers

 

 

 

 

 

 

Pre Post

Treatment

Figure 5. Mean number of microinvertebrates (N: 25 guts) dissected
from Q. Wm larval guts, pre (before starving) and post
(aﬁer starving) treatments. Error bars represent SEM. * Signiﬁcantly
different from Pre treatment, P < 0.05.

97

Table 3. Mean number of microinvertebrates dissected from third and fourth instar Q. W
at noon and midnight, ﬁrst trial, July, 1995. (Third instars: N=48, 24 noon, 24 midnight; Fourth instars:
N :48, 24 noon, 24 midnight. 1

 

 

 

Instar Time Mites (SE) Cladocerans (SE) Rotifers (SE) Ceratopogonidae(SE)

Third Noon 0.050 (0.023) 0.025 (0.017) 0.404 (0.096) 0.00 (0.00)
Midnight 0.013 (0.013) 0.077 (0.033) 0.877 (0.053) 0.00 (0.00)

Fourth Noon 0.163 (0.046) 0.038 (0.021) 0.535 (0.098) 0.048 (0.037)
Midnight 0.245 (0.043) 0.416 (0.05) 0.409 (0.113) 0.042 (0.042)

 

1 Data are log transformed.

98
(August), there was a shift in microinvertebrate consumption by third and
fourth instars (Table 4). Fourth instars consumed signiﬁcantly more mites and
rotifers than cladocerans. There were no ceratopogonid larvae found within the
guts dissected in the second trial.

Larval microinvertebrate consumption over a diel period was compared
at noon versus midnight (Figure 6). For both trials, larvae consumed
signiﬁcantly more cladocerans at midnight than at noon. Mite, rotifer and
ceratopogonid consumption did not differ during this period. Various larval
habitat treatments were compared for microinvertebrate consumption (Figure
7). Larvae consumed signiﬁcantly more mites in the open water area during the
ﬁrst trial. Mite and rotifer consumption was signiﬁcantly greater in open water
areas during the second trial. There was a signiﬁcant Instar X Time interaction
in the ﬁrst trial, i.e. fourth instars consumed more cladocerans but less rotifers
than third instars. In the second trial, there was a signiﬁcant Time X
Treatment interaction, i.e. more rotifers were consumed at noon in the open
water area than in the vegetated zone.

In order to determine if Q. W larvae selected for a
particular invertebrate, the surface microlayer from open water and vegetated
areas was sampled at noon and midnight. Surface microlayer water samples
show that over a diel time period, water mites, rotifers and copepods do not
differ signiﬁcantly (Figure 8). However, cladoceran numbers were signiﬁcantly
greater at midnight than noon. Water mites and copepods were found to be
more abundant in open water zones than vegetated areas (Figure 9).
Cladoceran and rotifer abundance did not differ between habitats. There were

no signiﬁcant interactions between time and treatment.

99

Table 4. Mean number of microinvertebrates dissected from third and fourth instar Q. quadrimamlama
at noon and midnight, second trial, August, 1995. (Third instars: N=53, 25 noon, 28 midnight; Fourth instars:

N=57, 29 noon, 28 midnight. 1

 

 

 

Instar Time Mites (SE) Cladocerans (SE) Rotifers (SE) Ceratopogonidae(SE)

Third Noon 0.048 (0.023) 0.000 (0.000) 0.230 (0.050) 0.00 (0.00)
Midnight 0.011 (0.011) 0.076 (0.029) 0258 (0.052) 0.00 (0.00)

Fourth Noon 0.161 (0.051) 0.021 (0.014) 0.488 (0.054) 0.000 (0.000)
Midnight 0.131 (0.032) 0.150 (0.04) 0.418 (0.079) 0.000 (0.000)

 

1 Data are log transformed.

microinvertebrates
Logvalue

Mean# of

rtebrates

microinve
Logvalue

Mean # of

100

 

 

 

 

Mites
Cladocerans
Rotifers

 

0.5 SECOND TRIAL I Ceratopogonids
0.4 - T T

0.3 -

0.2 - *

 

 

 

 

 

 

Noon

Figure 6. Mean number of microinvertebrates dissected from Q.

larval guts at noon (N=24) and midnight (N=24), ﬁrst and
second trials. Error bars represent SEM. * Indicates signiﬁcant difference
from Noon, P< 0.05.

 

101

First Trial

    
 

 

 

vertebrates

microin

Logvalue

 

 

Mean# of

Open-control Open-AC Veg.-control Veg.-AC

Second Trial

 

0.6

 

‘-

* T E Mites
Cladocerans
I Rotifers

0.5 -

0.4 -

 

 

  

 

0.3 -

0.2 -

0.1-

 

 

Mean # of microinvertebrates
Log value

Open-control Open-AC Veg -control Veg-AC

Figure 7. Mean number of microinvertebrates dissected from Q.

larval guts (N= 12 per treatment), comparison between
treatments, ﬁrst and second trials, July and August, 1995. Error bars
represent SEM. * Signiﬁcantly different from vegetated habitat, P < 0.05.

102

 

ertebrates

microinv

per sample (Log value)

 

 

Mean # of

 

Noon Midnight

Tlme Mites l

   
  
   

Cladocerans
H Rotifers
I Copepods

 

Figure 8. Mean number of microinvertebrates found within the surface
microlayer of open and vegetated habitats at noon (N= 10) and midnight

(N: 10).Error bars represent SEM. * Signiﬁcantly different from noon
treatment, P < 0.05.

invertebrates

micro

per sample Log value

Mean # of

103

 

 
    
   
 

 

 

  
  

 

  

 

 

 

Mites 1*I'
0.75 _ Cladocerans
I Rotifers
0.5 - I Copepods
0.25 -
T ‘. f
0 — . W ,,
OPen Water Vegetated Areas
Habitat

Figure 9. Mean number of microinvertebrates found within the surface
microlayer over a diel (24h) period from an open water zone (N: 48) and
a vegetated area (N= 48). Error bars represent SEM. *Signiﬁcantly
different from other treatment, P < 0.05.

104

In 1996, an experiment to determine the frequency of mites, cladocerans
and rotifers in Q. m larval diet over a 24 h period was
conducted. Larval consumption of cladocerans showed a bimodal peak from
9:00 pm. to 9:00 a.m. in open water and vegetation habitats (Figure 10).
Greatest number of cladocerans (18) found in larval guts was at 10:00 pm. in
the open water area, while mite consumption was lowest during this period in
both habitats. Larvae consumed more mites than cladocerans during the hours
of 10:00 a.m. to 4:00 pm. in open water and vegetated habitats. Rotifer
consumption was variable throughout the 24 h period in both habitats (Figure
11). Larvae consumed the greatest number of rotifers (34) in the open water
habitat.

From previous experiments, it was determined that Q._gaadnmagalat1§
larvae were able to consume water mites. It was unknown if mites were being
consumed alive or dead. Therefore, in a laboratory experiment, larvae were fed
live and dead water mites (Eylaidae: Ma sp.). Larvae did not prefer live to

dead mites, or vice versa (Figure 12).

DISCUSSION
To date, there are no data on the quantitative dietary sources for Q.
gaadﬁmagalataa over a diel period. The use of the ﬂuorochromatic stain,
DAPI, has been a recent methodological advance in the quantitative study of
the food sources found within mosquito gut contents (Walker et al. 1988) and
was instrumental in the identiﬁcation and enumeration of such food items as
bacteria, algae, invertebrate parts/protozoans and organic detritus from larvae

reared in different habitats over a diel period.

105

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....p... ...» 9 ...... I .
........ ..\.. I I . .. ..... ...0 .. I
9 ...... ¢.. 0.. I .. ......o... .. I ..
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Mean # of Mites

107

 

 

 

 

Log value

 

 

 

 

 

 

 

 

0.4
0.3 -
0.2-
0.1 -
0 :E:E:E:E:E:E:E:E;E:E:E:3:35:33: 33:3:i=2:E:E:E:l£:i:E:E:E:i:i:£:+
Abve Dead
Treatment

Figure 12. Mean number of mites found 1n the guts (N= 9 guts/
treatment) of dissected 4th instar An. Wm. Error
bars represent SEM.

108

The densities of bacteria from direct counts in the gut contents of larval
An. quadrimaculatus were consistent with the densities observed in previous
studies (Walker et al. 1988; Wallace and Merritt 1996). Within third and fourth
instar guts, bacteria were the most abundant food type, followed by detritus,
algae and invertebrate parts/protozoans. Mean densities of rod bacteria and
and detritus were higher in third instar guts. Diel differences across food types
were fairly consistent, i.e. larvae consumed more bacteria, algae and detritus at
midnight than noon.

However, larvae reared in a vegetated area, regardless of treatment,
consumed more rod bacteria than larvae in open water areas. In contrast,
larvae consumed more IPP in open water areas than vegetation areas.
Although vegetated areas may provide a refuge for anopheline larvae from
predation (Orr and Resh 1989; 1992), they provided different food choices for
An. quadrimaculatus larvae as observed in this study. Some algae, mainly
diatoms were found little digested or not at all in larval guts. This result is not
surprising as the presence of particular organisms within the larval guts does
not prove that they are a food resource (Clements 1992). Moreover, Laird (1988)
stated that up to 75% of ingested algae in larval mosquitoes may be unaffected.
The food choices available in different habitats may affect larval growth and
survivorship. In fact, in Chapter 3, I found that larval growth, developmental
rates and survivorship were signiﬁcantly affected by larval habitat.

A potential food source in a single biotope or microhabitat can be highly
complex, and its composition and/or presence may change dramatically with
time (Clements 1992). &. W larvae consumed numerous types
of microinvertebrates e.g., water mites, cladocerans and rotifers over a diel

period. Early studies on anopheline food resources do report these foods

109
(Coggeshall 1926; Senior-White 1927 ; Boyd and Foot 1928; Hinman 1930).
Larvae consumed more cladocerans at midnight than noon and the signiﬁcant
difference in diel periodic feeding activity on cladocerans was due to their
greater densities in the surface microlayer at midnight. Cladocerans are
known to vertically migrate on a diel basis, most likely to obtain food and/or
avoid predation (Bayly 1986). Larval gut analyses as observed on an hourly
basis show that larval consumption of cladocerans follows such a diel vertical
migration. Larvae exhibited a dietary shift around 10 a.m., consuming more
mites than cladocerans and continued such a trend until approximately 6 pm.
Larvae consumed more microinvertebrates at midnight than noon, suggesting
that larvae may prefer microinvertebrate food items at night than during the
day.

Although most studies suggest that mosquito larvae are not very
discriminatory in the types of food they ingest, laboratory studies indicate that
limited sizes of particles are ingested (Dadd 1971; Pucat 1965; Wallace and
Merritt 1980). In fact, it has been suggested that food particle selection may be
related to morphological and behavioral adaptations (Harbach 1977; Merritt et
al. 1990; Rashed and Mulla 1990).

Clearly, this study has shown through laboratory experiments that
fourth instar An. W are capable of ingesting live and dead water
mites, with no preference for either. Water mites and cladocerans were
estimated to be approximately 20 - 30 um in size and were usually found either
whole or in pieces. This size range falls within the ingested particle size range
for collecting-ﬁltering insects such as An. W (Wallace and
Merritt 1980).

 

110

Third instars consumed signiﬁcantly fewer microinvertebrates than
fourth instars and this was probably a function of microinvertebrate size and
larval mouthpart morphology (Rashed and Mulla 1989). It was not documented
in this study if larvae consume live or dead cladocerans or rotifers.
Interestingly, some cladocerans are capable of preying on rotifers (Kgmtgﬂa
sp.) (Thorp and Covich 1991). Kemtglla rotifers were observed in signiﬁcant
numbers in larval guts. If Q. MW larvae are capable of
consuming cladocerans that have ingested microbes e.g., bacteria and algae or
microinvertebrates, e.g., rotifers then this ﬁnding documents a new pathway
for traphic interactions.

Habitat had a signiﬁcant effect on microinvertebrate consumption by .2.
WM larvae in both feeding trials. In the ﬁrst trial, larvae
consumed more water mites in the open water areas than vegetated zones, a
reﬂection of the higher density of water mites inhabiting the surface microlayer
within this habitat and consistent with other marshes (Campeau et al. 1994).
The second trial showed that more water mites and rotifers (Brachionidae:
Keratella sp) were consumed in the open water area than the vegetated zone. A
greater rotifer abundance difference between the ﬁrst and second trial may be
due to natural population ﬂuctuations caused by microhabitat changes and
trophic status of the pond in August versus July (Edmondson and Litt 1982).
The surface microlayer analysis revealed that water mites were more abundant
in open water areas than vegetated zones and conversely, copepod abundance
was greater in vegetated zones than open water areas. Therefore, this study
shows that &. quadrimaculatus larvae do not preferentially feed on
microinvertebrates on a diel basis. However, larvae may feed selectively on

rotifers in open water habitats.

111

Several of the more recent mosquito biological control agents, such as
bacteria [ﬁagjllus thggingiensis var. melensis (Bti) and ﬁagillus Mg]
and fungi (e.g. Lagenidium giganteum) must be ingested by larvae to be
effective (Chapman 1985; de Barjac and Sutherland 1990). Knowledge of the
"feeding area": or where the larvae forage and optimal particle sizes and food
types by larval mosquitoes in the natural habitats will enhance the success of
Bti and other bacilli as particulate larvicides (Aly 1983; Dahl 1988). Moreover,
the potential to utilize larger particles in the form of microinvertebrate dietary
sources has not been investigated and may be an effective approach to control.
It is clear that research on larval mosquito feeding ecology and perhaps more
speciﬁcally, the nature of their diet should be given increased signiﬁcance in

future years.

112
LITERATURE CITED

Aly, C. 1983. Feeding behavior of Aedes mans larvae (Diptera: Culicidae) and
its inﬂuence on the effectiveness of Bacillus h ' ' n i var.
iaraelensia. Bull. Soc. Vector Ecology. 8294-100.

Bayly, I.A.F. 1986. Aspects of diel vertical migration in zooplankton, and its
enigma variations. Pp. 349-368 in: P. De Deckker and W.D. Williams,
(eds.), Limnology in Australia. CSIRO, Melbourne, Junk, Dordrecht,

Netherlands.
Bekker,E.E. 1938. On the mechanism of feeding in larvae of Whales. Zool.
Zh.,17: 741-62.

Boyd, M.F. and H. Foot. 1928. Studies on the bionomics of American
anophelines: the alimentation of anopheline larvae and its relatio to
their distribution in nature. J. Prev. Med., 2: 219-242

Campeau, 8., HR. Murkin, R.D. Titman. 1994. Relative importance of algae
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Chapman, H.C. ed. 1985. Biological control of mosquitooes. Am. Mosq.
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Carpenter, SJ. and W. J. LaCasse. 1955. Mosquitoes of North America (north
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Coggeshall, LT. 1926. Relationships of plankton to anopheline larvae. Am. J.
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Clements, A.N. 1992. The biology of mosquitoes. Chapman & Hall Publishers,
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Dadd, RH. 1971. Effects of size and concentration of particles on rates of
ingestion of latex particulates by mosquito larvae. Ann. Entomol. Soc.
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Dahl, C. 1988. Control potentials in feeding mechanisms of mosquito larvae.
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de Barjac, H. and DJ. Sutherland. eds. 1990. Bacterial control of the
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BM thmiugiensia var. Malaysia and Bacillus W.
Rutgers Univerity Press. New Brunswick, N .J . 349 pp.

Edmondson, WT. and AH. Litt. 1982. Qaphnia in Lake Washington.
Limnology and Oceanography. 30: 180-188.

Harbach, RE. 1977. Comparative and functional morphology of the mandibles
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Hinman, EH. 1930. A study of the food of mosquito larvae (Culicidae). Am. J.
Hyg., 12: 238-270.

Merritt, R.W., E.J. Olds and ED. Walker. 1990. Natural food and feeding
behavior of WW larvae. J. Am. Mosq. Control
Assoc., 6: 35-42.

Merritt, R.W., R.H. Dadd and ED. Walker. 1992a. Feeding behavior, natural
food and nutritional relationships of larval mosquitoes. Ann. Rev.
Entomol., 37 : 349-376. Technical Bulletin 202, M.S.U. East Lansing,MI.
50pp.

Merritt, R. W., D.A. Craig, E.D. Walker, H.A. Vanderploeg and RS. Wotton.
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114

Angpheles quadrimaculatus (Diptera: Culicidae). J. Insect Behav., 5 (6): 741-
761.

Norkrans, B. 1980. Surface microlayers in aquatic environments. Adv.
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Orr, B.K. and V.H. Resh. 1989. Experimental test of the inﬂuence of aquatic
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— . 1992. Inﬂuence of Myriophyllum aquaticum cover on

 

Anopheles mosquito abundance, oviposition, and larval microhabitat.
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Porter, KG. and Y.S. Feig. 1980. The use of DAPI for identifying and counting
aquatic microﬂora. Limnol. Oceanogr., 25(2): 943-948.

Prescott, G.W. 197 8. How to know the freshwater algae.Wm. C. Brown
Publishers, Dubuque, IA 293 pp.

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mosquito larvae. Queast. Entomol. 1: 41-86.

Rashad, SS. and MS. Mulla. 1990. Comparative functional morphology of the
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bacteria in a marsh habitat of Angphelss W larvae
(Diptera: Culicidae). J. Am. Mosq. Control, In Press.

115

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American Freshwater Invertebrates. Academic Press, San Diego, CA 911
pp.911.

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(Diptera: Culicidae) using DAPI stain and epiﬂourescence microscopy.

J. Med. Entomol., 25: 551-544.

Walker, E.D., R.W. Merritt and RS. Wotton. 1988(b). Analysis of the
distribution and abundance of Anophslss quadrimaculatus (Diptera:
Culicidae) larvae in a marsh. Environ. Entomol., 17 : 992-949.

Walker, ED. and R.W. Merritt. 1993. Bacterial enrichment in the surface
microlayer of an Ms W (Diptera: Culicidae) larval
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insects. Annu. Rev. Entomol., 25: 103-32.

Wallace, J. R. and R.W. Merritt. 1995. Preliminary investigations on the
growth and natural history of Ms quadrimaculatus in Michigan
ponds. Vector Control Bulletin of the North Central States 4: 66-70.

Wallace, J .R. and R.W. Merritt. 1996. Natural food, growth and microhabitat
partitioning of Angphglﬂ in Michigan marshes. Vector Control Bulletin
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Wotton, R.S. (ed.) 1990. The biology of particles in aquatic systems. CRC Press,

116
Boca Raton, FL.
Zar, J .H. 1984. Biostatistical analysis, second edition. Prentice-Hall, Inc.,
Englewood Cliffs, New Jersey. 718 pp.

CHAPTER 3
POPULATION DYNAMICS OF AN OBI-_IELES W:
THE EFFECTS OF HABITAT, NUTRITION AND PREDATION ON
LARVAL GROWTH AND SURVIVORSHIP

ABSTRACT

The ﬁeld and lab components to this study were designed to examine
the inﬂuence of temperature (as measured by two distinct habitat treatments,
open water and vegetated zones), nutrition (as measured by two different
microhabitat treatments (with/without algal clumps) and predation on An.
MW larval growth and developmental rates, survivorship and
adult size at emergence. Recently, the need for ecological management of
mosquitoes sparked a new interest in their larval ecology as an important
component in malaria control programmes.

A ﬁeld manipulation study was conducted to determine the effects of
habitat (temperature), microhabitat and predation on relative growth and
developmental rates and survivorship as well as adult dry mass (mg). A second
ﬁeld assay was conducted in open water and vegetation habitats to determine
the effects of habitat on individual relative growth rates for the different larval
stages. The laboratory study was conducted to determine the eﬂ'ects of
temperature, food quantity on larval E. W growth,
development and mortality. Previous studies have shown that An.
WM larvae selectively consume some microinvertebrates over
others during a diel period. A microinvertebrate dietary supplement feeding
experiment was conducted to measure the effects on larval development and

117

1 18
survivorship.

Surface microlayer temperatures between these habitats were
marginally to highly signiﬁcantly different (Range: Open water maximum =
28°C; minimum = 21°C, Vegetation maximum = 297°C; minimum = 18.5°C.
Relative growth rates developmental rates, adult size and survivorship were
affected by treatment and marginally by habitat. In addition, survivorship was
signiﬁcantly affected by temperature, food type and amount in the laboratory.
Field and lab studies provide support for a multi-hypothsis explanation to

predict An. W larval distribution and success.

119
INTRODUCTION

The mosquito, Angphslss gum (Say), is distributed
throughout the eastern half of North America and was the principal vector of
human malaria in this region before eradication of the disease (Carpenter &
LaCasse 1955). This mosquito remains an important pest and disease vector,
particularly in more southerly regions of its distribution (Horsfall 1955).
Immature stages of mphﬂss have been known to be associated with ﬂoating
or emergent vegetation, or ﬂoating debris (Rozenbloom and Hess 1944; Aitken
1945). In fact, larval An. mm aggregate at air-water interfaces
(surface microlayer) of plant stems and algal mats (Orr and Resh 1989; 1992)
and feed on food particles from the surface microlayer by using the collecting-
ﬁltering feeding mode (Merritt et al. 1992a). Recently, the need for ecological
management of mosquitoes sparked a new interest in their larval ecology as an
important component in malaria control programs (Hess 1984; Laird 1988).

Vector competence (i.e. the internal physiological factors that govern the
infection of human pathogens in a mosquito, e.g. malarial agent Elasmgdimn)
(DeFoliart et al. 1987) varies with the quality of the larval environment
(Merritt et al. 1992). For some mosquito species, adverse effects on larval
growth and development rates, survival, adult size and adult ﬁtness appear to
be primarily caused by larval stress due to temperature and food limitations
within habitats. Some principal environmental factors that affect rates of
larval growth and development are temperature (Huffaker 1944; Meyer et al.
1990; Clements 1992; Hu et al. 1993; Atkinson 1994), nutrition (Merritt et al.
1992), and predation (Orr and Resh 1989, 1992).

Temperature is a particularly important and widespread correlate of

differences in size, and rates of growth and development (Atkinson 1994).

120
Temperature effects on bioenergetic factors such as growth rates of Anopheles
larvae may be in part due to the sensitivity of the microorganism community
within the surface microlayer to temperature changes. The duration of the
mosquito larval period is an important life-history parameter because it is a
critical component of most life-table and production estimates for aquatic
insects (Sweeney 1984).

Most studies that have investigated habitat effects on mosquitoes and
other aquatic invertebrate presence/absence have focused on the predation-
refuge hypothesis (See Gotceitas and Colgan 1989; Orr and Resh 1989, 1991 ).
The predation-refuge hypothesis indicates that predator efﬁciency decreases as
habitat complem'ty (i.e. plant density or structural complexity) increases, and is
supported by several ﬁeld experiments on anopheline mosquitoes (Collins et al
1988; Orr and Resh 1989, 1991). In addition, the mosquito oviposition site
preference hypothesis is supported by several ﬁeld studies (Russell and Rao
1942; Orr and Resh 1992; Rejmankova et al. 1991, 1993, 1996). These studies
suggest that anophelines perfer to oviposit in or among vegetation or algal
mats. As suggested by Orr and Resh (1989), the effects that potentially
alternative hypotheses (not necessarily mutually exclusive), e.g. enhanced food
resources (Walker and Merritt 1993; Wallace and Merritt 1995; Smith et al.
1997) and favorable microclimates (Collins et al. 1985) have on Angpheles
larval growth, development and survivorship has been little studied.

The ﬁeld component to this study was designed to examine the
inﬂuence of microclimates (as measured by two distinct habitat treatments,
open water and vegetated zones), nutrition (as measured by two different
microhabitat treatments (with/without algal clumps) and predation on An.
MW larval growth and developmental rates, survivorship and

121
adult size at emergence. The lab experiments address the effects of varying
temperatures, surface microlayer food quality, quantity and microinvertebrate

dietary supplements on larval growth, development and survivorship.

MATERIALS AND METHODS
Si a i c

This study was conducted in a permanent pond, located approximately 8
km south of Michigan State University at the Collins Road Entomology Field
Laboratory(42°42'00" N Latitude; 84°30'20" W Longitude), Ingham County,
Michigan, U.S.A. The pond is a permanent pond and is approximately 2.25 ha
in size. It is bordered with cattails Challis latifolja.) and emergent grasses e.g.
foxtail (m spp.).

Larval gr. quadrimaculatus growth and development were compared
between two habitats, an open water zone and a vegetated area. The open
water zone consisted of an area of the pond that was without ﬂoating or
emergent vegetation within 4 m of the larval microcosms. The vegetated area
provided ample emergent vegetation, e.g. grasses (Setaria spp.) and cattails
(limbs laﬂfglia) around which microcosms were placed. These two habitats
were selected based upon available food resources and temperature differences.
Surface water temperatures were monitored with maximum/minimum
thermometers during the 1995 and 1996 seasons.

A_n. quadrimaculatus larvae were maintained in ﬁeld microcosms for the
ﬁeld growth experiment. The microcosms were constructed of a 4 liter clear,
plastic, round container (20 cm diam X 25 cm length). Microcosms were kept in

a speciﬁc location with a monoﬁliment line tied to a garden stake (Figure 2,

122
Chapter 1) for sketch). Microcosms were placed randomly approximately 2 m
apart in open water and vegetated areas in order to simulate areas where
anopheline larvae are present and absent.
WW
Microhabitat / predator manipulation ﬁeld study

A ﬁeld manipulation study was conducted to determine the effects of
habitat (temperature), microhabitat and predation on relative growth and
developmental rates and survivorship as well as adult dry mass. A 2 X 3
factorial design was used in this study. Second instar An. W (N
= 40) were collected with a 250 m1 dipper and pipeted into ﬁeld microcosms.
Microcosms were placed, prior to larval introductions, in two habitats (open
water zones and vegetated areas) and treatments were 1) with algal clump, 2)
with algal clump/predator, 3) with predator only. Control microcosms contained
Q. guadrg’ aculatus larvae only. The predator species used in this experiment
were early instar backswimmers (N otonectidae: NW spp.). A single
notonectid was added to each predator treatment in both habitats. Surface
microlayer temperatures from open water and vegetation habitats were
measured with a ﬂoating max/min thermometer during the experimental
period.

Microcosms were checked every other day until larvae began to pupate.
Pupae were pipeted from microcosms and held inside 15 ml collection tubes
ﬁlled with pond water. Collection tubes were maintained inside ﬂoating
containers until adults emerged. Adults were aspirated into a second collection
tube and transported to the laboratory where they were killed, dried and

weighed for analysis.

123

Individual instar growth rate study

A second ﬁeld assay was conducted in open water and vegetation
habitats to determine the effects of habitat on individual relative growth rates
for the different larval stages. Five microcosms in each habitat were stocked
with 35 ﬁrst instar m. gaadm'maem. A subsample of ﬁrst instars was
collected for dry weights. Larvae were subsampled by removal periodically as
they molted to the next instar, killed in boiling water, dried, and weighed to
the nearest milligram. The growth rate of an average individual in each
microcosm population was expressed as a relative growth rate (RGR).

= (mean ﬁnal mass -- meaa initial mass)
mediaa mass

time interval

where median mass is (initial mass + ﬁnal mass) / 2. The resulting expression
multiplied by 100 is growth as mg% body weight per day and is essentially
equal to instantaneous growth rate for short intervals, especially during
periods of rapid growth (Cummins et a1 1973).

Lab study

A laboratory study was conducted to determine the effects of
temperature, food quantity and quantity on larval Aa. W
growth, development and mortality. A 2 X 3 X 3 factorial experimental design
was used in this study. Two food types of surface microlayer water samples
representing a vegetated and Open water habitats were collected with a
modiﬁed Harvey-Burzell technique (Norkrans 1980) and stored in sterilized
jars and returned to the lab. Sterile 10 m1 pipets were used to pipet food

124

samples of each habitat type for larval container inoculations. Larvae were
maintained in circular plastic rearing containers (21.0 cm diameter X 7.5 cm
depth). Three food amounts (surface microlayer samples: 10, 50 and 100 ml)
were added to each larval container. There were six replicates of each
treatment placed inside Percivel® Growth chambers at 18°, 23° and 28°C.
Larval containers were stocked with 20 ﬁrst instars collected as mentioned in
earlier chapters. These temperatures were selected because they represent the
maximum and minimum range of temperatures E. W larvae
are exposed to at highest population densities (August-September) in the ﬁeld
in southern Michigan. Microcosms were checked daily to record mortality,
time to maturity and adult emergence. Adults were collected, frozen, dried
and weighed.
Larval diet manipulation experiment

Previous studies (Wallace & Merritt 1996, 1997) have shown that ﬂ}.
gaadﬁmalemtas larvae selectively consume some microinvertebrates over
others during a diel period. A microinvertebrate dietary supplement feeding
experiment was conducted to measure the effects on larval development and
survivorship. Due to collecting and handling logistics, cladocerans (Daphnia sp)
were used to supplement fourth instar An. W diets. There were
two food treatments (cladocerans, no cladocerans) and ﬁve
replicates/treatment. Fourth instar m. gaadmimaematas (N=250) were
collected in the ﬁeld, maintained in sieved pond water (sieve size = 125 um)
and transported to the laboratory. Pond water containing numerous
cladocerans was also collected and transported to the laboratory. Pond water
was sieved with mesh X 10 pm to exclude most microinvertebrates typically

found in larval guts.

125

In the laboratory, larvae (N= 25) were placed into 1 L plastic containers
ﬁlled with 250 ml of sieved pond water. Twenty-ﬁve cladocerans were added to
each food treatment container. To control for any particulate organic or
inorganic food item in the sieved pond water that was added with the
cladocerans, 5 ml of sieved pond water was added to the no-food (no
cladocerans) treatment. Plastic containers were maintained inside climate
controlled growth chambers, 16:8, L:D and 23°C. Daily additions of 25
cladocerans per food treatment and sieved pond water were performed until all
larvae had pupated. Pupae were separated and allowed to emerge as adults
inside 50 ml centrifuge tubes. Adults were sexed and killed by freezing, dried
at 47°C for 48 h and weighed with a Cahn Electrobalance to the nearest
hundredth of a milligram. Adult dry weights, larval development rates and

survivorship parameters were compared between the two dietary treatments.

III Dat Anal sis
The following parameters will elucidate g1. gaadm'magﬂajms

performance in the open water vegetated habitats: relative growth rate,
development rate, adult size and survivorship. A three factor analysis of
variance was used to measure the effects that habitat, treatment and sex had
on these parameters (P < 0.05 is signiﬁcant) (Super Anova 1989). The effects of
habitat on individual instar relative growth rates was examined with a one-
way analysis of variance.

In the lab growth experiment, the effects of temperature, food quality
and quality, sex and their interaction on adult size, development rate and
survivorship were analyzed with an three-way analysis of variance. Signiﬁcant
F-ratios were further analyzed with Fisher's PLSD specialized ttest of the

means. A life table was constructed for each treatment in order to determine

126
which life stage was most sensitive to temperature, food type and amount as
measured in terms of mortality. A Single Species Key Factor Analysis (Manly
1994) was used to estimate the variation in mortality rates in different larval
stages from all temperature and food treatments in order to gain
understanding of which sources of variation are particularly important for A14.
ggmdﬁmamlatas population dynamics. Regression coefﬁcients, calculated by
regressing k-values on their sum K from the key factor analysis were compared
with an analysis of variance and MANOVA. This multivariate analysis of
variance technique performs an AN OVA on more than one factor (e.g.,
temperature, food type and food amount) and takes into account the correlation
among the dependent values (instar regression coefficients). For the larval
dietary manipulation using microinvertebrates experiment, a two sample
ttest was used to compare mean adult dry weights, development rates and

larval survivorship (Zar 1984).

RESULTS

1. Eield mierehabitat/predator manipulation assay

Surface microlayer temperatures between these habitats were
marginally to highly signiﬁcantly different (Range: Open water daily maximum
= 28°C; daily minimum = 21°C, Vegetation daily maximum = 297°C; daily
minimum = 185°C; ). Daily minimum surface temperatures in the vegetated
habitats were signiﬁcantly cooler than open water habitats. Vegetation areas
also provide higher maximum surface temperatures (not signiﬁcantly different
(Figure 1).

Relative growth rates calculated as described above were signiﬁcantly

different between treatments (Figure 2). Larvae reared in the predator

40

 

 

Max. temp
Min. temp

 

 

 

Temperature °C

 

 

 

 

 

 

Open Water Vegetation

Habitat

Figure 1. Temperature (°C) differences between open water (N: 12) and
vegetation (N: 9) habitats. Error bars represent SEM. * indicates
signiﬁcant difference from open water habitat.

 

 

 

 

 

 

 

 

 

 

 

 

 

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Control AC AC/P P

Figure 2. Treatment effects on relative growth rates for A_n.
quagmimamlgmm in ﬁeld growth experiment. Error bars represent

SEM. * indicates signiﬁcant difference from other treatments. AC = algal
clump (N=49), AC/P = algal clump/predator (N =19), P = predator (N=11).

 

129
treatment grew more per day than in any other treatment. Female relative
growth rates were signiﬁcantly higher than males' (Table 1). Although, habitat
effects were not signiﬁcant, results show that marginal signiﬁcance was
observed (F-ratio = 3.841, P < 0.0525). In addition, there was a signiﬁcant
difference in the habitat X treatment effect on RGR (Figure 3). There were no
other signiﬁcant interaction differences observed.

Larvae reared in algal clump and algal clump predator treatments
developed faster than the predator and control treatments, regardless of
habitat (Figure 4). Although habitat effects on development rates was not
signiﬁcant, results indicate that habitat had some effect on development (F-
ratio = 3.416, P = 0.0672).

Adult mosquitoes reared from algal clump and algal clump/predator
treatments weighed signiﬁcantly more than those adults from the predator and
control treatments (Figure 5). As expected, females were signiﬁcantly larger
than males. Algal clump and predator treatments yielded signiﬁcantly longer
female wing lengths as compared to adults reared in the control and algal
clump/predator treatments (Figure 6). There was a signiﬁcant habitat X
treatment effect, i.e. females reared in algal clump and algal clump/predator
treatments in the open water habitat had longer wing lengths than females in
the vegetated area (F-ratio = 2.762, P < 0.05). Interestingly, a single female
reared in the predator treatment in the vegetation habitat had the longest
wing length (Table 2).

Survivorship in the algal clump/predator and predator treatments was
signiﬁcantly lower than in the algal clump and control treatments (Figure 7).
Although not signiﬁcantly different, habitat effects may be marginally

130

Table 1. Mean RGR (% mg/mg/day) a and dry weight (mg) b measurements for
An. gaadmmaematas male and female adults in the ﬁeld growth study.

 

 

 

 

Si N RGR (SE) Dry Weightjmg)
Male 45 1.29 (0.064) 0.154 (0.006)
Female 83 1.46 (0.057)* 0.179 (0.05)*

 

a RGR data were arcsin transformed

b Dry weight data were log transformed
* indicates signiﬁcant different from Male

 

131

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Control AC AC/P

Figure 4. Treatment effects on development rate (days) for Am.
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clump/predator (N: 19), P: predator (N: 11). Error bars are present but not
visible and represent SEM. * indicates signiﬁcant differences from control.

133

 

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Control AC AC/P P

Figure 5. Treatment effects on adult dry weights for An.
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(N: 49), AC/P = algal clump and predator (N: 19), .P = predator (N: 11),
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signiﬁcant differences from control and predator treatments.

134

 

 

 

 

 

 

 

 

 

 

 

 

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Q) 0 ‘ ‘ ‘ ‘I “““““ F‘“ l I

F“ Control AC ACIP P

Figure 6. Treatment effects on adult female wing length (mm) for
An. gaadriamealatas. Control (N: 33), AC: algal clump (N: 30),
AC/P: algal clump/predator (N: 11), P: predator (N: 7) Error bars
are present but not visible and represent SEM. * indicates signiﬁcant
differences from control.

135

Table 2. Mean wing length for female &. gaamjmamlajms mosquitoes reared in
two habitats (open water and vegetation) and three treatments: algal clump,

algal clump/predator and predator.a

 

 

 

Habitat Treatment Wing Length (mm) (SE) N
Open Water
algal clump 0.739 (0.007) 16
algal clump/predator 0.752 (0.007 ) 4
predator 0.735 (0.009) 6
control 0.715 (0.005) 22
Vegetation
algal clump 0.721 (0.006) 14
algal clump/predator 0.723 (0.008) 7
predator 0.778 (0.000) 1
control 0.720 (0.009) 11

 

a data were log transformed

136

 

10

Survivorship (%)

 

 

 

 

 

 

 

 

 

Control AC

Figure 7. Treatment effects on larval survivorship for An.

lat .Control (N: 10), AC: algal clump (N: 10), AC/P- =
algal clump/predator (N: 10), P: predator (N: 10). Error bars represent
SEM. * indicates signiﬁcantly different from control.

137
inﬂuential in larval survivorship, i.e. open water habitats yielded a higher
survivorship than vegetated habitats (F-ratio = 3.799, P = 0.06).
2, Individaal instar growth rate assay

Habitat effects on individual instar growth rates were not signiﬁcantly
different, but indicate greater growth per day occured for the l'V-Pupa stage in
the open water habitat and III-IV stage in the vegetation habitat (Figure 8).
Between instar compa1isons show that relative growth rates were signiﬁcantly
higher in the III-IV and IV -Pupa stages (Figure 9). Individual dry weights were
signiﬁcantly different between second, third, fourth instars and pupae (Figure
10).

r e/f 8

There were no signiﬁcant effects of temperature, food type and amount
on adult size and development rates (Table 3). However, larval survivorship
was signiﬁcantly different between temperatures, food types and amounts
(Figure 11). Signiﬁcantly more larvae survived at 18°C than 23°C or 28°C.
More larvae reared on a surface microlayer diet from vegetated habitats
survived than from those reared on a surface microlayer diet from open water
areas. Finally, larval survivorship was the greatest from a diet of 100ml of
surface microlayer.

The effects of temperature, food type and amount on individual instar
mortalities as reﬂected through their regression coefﬁcients is presented in a
life table sumarization (Table 4). Key factor analyses on individual instar
mortality show that ﬁrst and second instar mortality are signiﬁcantly affected
by temperature (Table 5). However, MANOVA analysis of all instar mortalities

collectively show that these variables are correlative and that the overall

RGR % (mg/mg/day)

138

 

  

 

 

0 m . -
Open Water Vegetation

Habitat

Figure 8. Relative growth rates for immature life stages of
ri s reared in two habitats, open water areas

and vegetated zones.

 

RGR % (mg/mg/day)

139

 

 

 

 

 

 

 

 

 

10 ,,,,,,,,,,,,,,,,,,,,,,,,
m4? "338
7.5-
ﬁfzfzlefizfzgfé: I
[-11 II-III III-IV IV -Pupa

Life Stage

Figure 9. Relative growth rates for immature stages of
. Error bars are present but not visible and
represent SEM. * indicates signiﬁcant difference from all other

11 dr'

life stages.

 

140

 

 

Dry Wt. Log value

 

 

 

 

 

 

 

 

0.3
n=1 1
*
0'25' .szszzgaszszs:
0.2-
0.15 -
n=27 éﬁﬁﬁi
01- f.
n=20 4*
0.05- "=27 ”
0
Figure 10. Mean dry weights (mg) for An. '

immature life stages. Error bars represent SEM. * indicates
signiﬁcant differences from 1 instar.

 

141

Table 3. Temperature, food quality, and quantity effects on Aa. gaaddmagalatgm
development rate, adult dry weights and female wing length in the laboratory.ail

 

 

 

Factor Development Rate Dry Weight Wing Length
df F P F P F P
Temperature 2 2.071 0.14 1.068 0.35 0.192 0.82
Food 'IB'pe 0 NV* NV NV NV NV NV
Food Quantity 1 0.042 0.83 2.704 0.10 0.082 0.77

 

a data were log transformed.
* NV = No Value

 

142

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

10
9 . .
'5, 7 5 El % Surv1vorsh1p
0E g 5‘
5
”J 2.5-
6°
0
8
.81 *
'5 6-
‘5 .E
> m
0E g4-
5
U) 2-
8°
0- l
Open Water Vegetation
Food Type
10
.81
"S 7.5-
‘5 .E
>
0; § 5..
In
5 CB
”3 2.5-
6°

 

 

 

 

 

 

10

50 100
Food Quantity (ml)

Figure 11. Temperature (C), food type (open water, vegetation) and
quantity (ml) effects on An. geadaimaemlatas larval survivorship (%).
Data have been arcsin transformed. Error bars represent SEM.*
indicates signiﬁcant difference from other treatments.

143

Table 4. Life table on the effects of temperature, food type and amount on An.
gaadrimaealams mortality through immature life stages. Data are regression
coefﬁcients calculated when k-values are regressed on their sum K. k: mortality
for each instar, K = mortality for entire cohort.

 

 

 

Wm
Temperature (C°) Food Type Food Amount Instar

(ml) 1 2 3 4 Papa

18 Open 10 .57* .33 .38 -.36 -.08
50 .62 .63* .24 -.49 .00

100 .67* .37 .61 -.65 .00

Veg 10 .66* .14 .48 -.21 -.07

50 .43 .44 .49* -.49* .13

100 .15 .21 .52* .16 -.05

23 Open 10 .66* .25 .21 -.12 .00
5O .93* .15 .11 -.18 .00

100 .77* .50 .06 -.33 .00

Veg 10 .62* .29 .48 -.39 .00

50 .47* .15 .45 -.26 .20

100 .66* .02 .29 -.26 .30

28 Open 10 .69* .15 .39 -.22 .00
50 .82* .12 .14 -.08 .00

100 .74* .11 .21 -.07 .00

Veg 10 1.07* .08 .00 -.14 .00

50 .84* .07 .27 -.18 .00

100 .66* .05 .37 . -.20 .12

 

* indicates maximum regression coefﬁcient or key factor in terms of mortality for
each treatment according to Podoler and Rogers, 1975.

144

Table 5. Analysis of variance and MANOVAa on individual instar regression
coefﬁcients calculated from key factor analyses to determine temperature, food
type and amount effects on instar mortality.

 

 

 

Treatment ﬂQVA MANOVA
INSTAR F-value P-value F-value P-value
Temperature 1 4.2 0.03* 1.7 0.16
2 5.5 0.01*
3 3.7 0.05
4 1.4 0.26
Pupa 1.0 0.38
Food Type 1 1.5 0.23 0.9 0.49
2 4.3 0.05
3 2.4 0.14
4 0.4 0.53
Pupa 0.20 0.18
Food Amount 1 0.5 0.56 0.595 0.79
2 0.3 0.73
3 0.2 0.78
4 0.1 0.88
Pupa 0.7 0.48

 

a MANOVA analysis is for all instars combined.

* indicates signiﬁcant difference from other instars Within treatment.

145

effects observed in the univariate AN OVA cannot be considered signiﬁcant at
the 0.05 level.
Microinvertebrate dietary addition assay

The mean dry weights for males and females showed no signiﬁcant
difference, however, males reared in diets supplemented with cladocerans were
larger than those reared without cladocerans (Figure 12). Development rates
for males and females did not differ between dietary supplements (Figure 13).
Cladoceran dietary supplements did not have a signiﬁcant effect on overall
percentage survival (Figure 14).

DISCUSSION

Temperature and nutrition are important environmental factors to all
species. Life-history patterns in nature are compromises between a complex of
environmental demands as a result of the interactions between these factors.
Senior-White (1928) noted that the temperature in small hoof-mark pools in
grass might be 5°C cooler than in a large pool six inches away. I contend that
Senior-White's (1928) hoof-mark pool in grass and large pool six inches away
example is analogous to the discrete habitats I compared in this study, i.e. the
open water and vegetated areas. This study shows that range between
maximum/minimum temperatures in vegetated habitats is greater (thus more
variable) than open water habitats which appear to be more constant
comparatively.
Field experiment

Numerous studies have observed high positive correlations between
larval growth rates of natural populations and water temperature (Sweeney

197 8; Mackay 1979; Wallace et al.1992). In this study, the maximum/minimum

Dry Weight (mg)

Log value

146

 

 

0.15
Males
Females
0.1—
0.05-

 

 

 

 

Cladocerans No Cladocerans

Figure 12. Mean dry weights (mg)o for male and female adult
EL. 51W mdiet experiment with two
dietary treatments. (Males: t-value= 1.899; P: 0.06,
Females: t-value= 0.055; P: 0.95). Error bars
represent S.E.No signiﬁcant differences observed.

Development Rate (days)

 

147

 

1.5

 

E3 Males

Females

 

 

 

 

 

 

 

 

 

Cladocerans No Cladocerans

Figure 13. Mean development rates for male and female adult An

from diet experiment with two dietary treatments.
(Males: t-value = 1.019; P: 0.311, Females: t-value: 0.055; P: 0.812).
Error bars are present but not visible and represent SEM. No signiﬁcant
differences observed

 

P

V0

Mean%

148

80

 

 

60—

40-

20-

 

 

 

 

 

 

 

 

Cladocerans No Cladocerans

Figure 14. Mean percentage survivorship for An.

adults from diet experiment with
two dietary treatments. (t—value- — -1. 075; P: 0.31)
Error bars represent S. E. No signiﬁcant differences
observed.

149
temperature range was not as great in the open water habitat as in the
vegetated habitat. Field experiment results demonstrate that larvae reared in
open water habitats grew signiﬁcantly more per day than vegetated habitats.
In fact, fourth instar growth rate was higher in the open water habitats.
Furthermore, habitat affected developmental rates, i.e., larvae developed and
emerged as adults faster in the open water habitats than those in vegetated
habitats. These results support those found by Huffaker's (1944) seminal
laboratory study where developmental rates accelerated when larvae were
exposed to higher temperatures for a shorter time period than over an
extended period. Perhaps, the cooler temperatures experienced by larvae in the
vegetated habitats reduced the relative growth and developmental rate
differences for these larvae.

Temperature effects on bioenergetic factors such as larval growth rates
and adult size at emergence of Anopheles mosquitoes may be in part due to the
sensitivity of the microorganism community within the surface microlayer to
temperature changes. In this study, habitat and treatment (algal clump, algal
clump/predator) signiﬁcantly impacted adult female size, i.e. producing larger
females in the open water habitat. Dommergues et al. (197 8) observed
microorganisms within the surface microlayer to be sensitive to temperature
changes. If temperature changes affect microbial and microinvertebrate
abundance and distribution directly, then An. W growth rates in
addition to adult size measurements will ﬂuctuate according to the indirect
effect of greater maximum/minimum temperature differences on quantity and
quality of food sources in the surface microlayer.

In the previous chapter, larvae reared in open water habitats consumed

signiﬁcantly more microinvertebrates, e. g. water mites, cladocerans and

150
rotifers than larvae from vegetated habitats. To determine if larval growth and
development rates as well as survivorship were affected by microinvertebrate
dietary supplements, a preliminary lab experiment showed that grth and
development rates and survivorship did not differ between larvae fed a
microinvertebrate dietary supplement versus those that were not. Denhnie
spp.were used in this experiment and may have posed several biological and
mechanical problems for fourth instar &. W consumption.
First, no exact identiﬁcation of the previously consumed cladocerans has been
made, therefore a different cladoceran may have been used in this study.
Second, cladocerans selected for dietary supplements were done so by eye and
may have been too large for An. Willa larval consumption. It is
possible that An. quadrimaculatus larvae are passively consuming Dephnie
spp. body parts or exuviae. Since ﬁeld studies indicate that larval growth and
developmental rates as well as survivorship are higher in the open water
habitats, intuitively larvae must be supplementing their late instar diets with
necessary food items possibly in the form of microinvertebrates that inﬂuence
such life history parameters. Further studies are required to determine if a
microinvertebrate does in fact serve as a strategy to improve growth,
development and larval survivorship.

As discussed earlier, ﬂoating and emergent aquatic macrophytes have
been implicated in the predator-refuge hypothesis that has been suggested to
play a major role in both the abundance of immature Anepheles and
recruitment of Anephelee (from oviposition or larval movement) (Orr and Resh
1991, 1992). To test this hypothesis, Backswimmers (HemipterazNotonectidae)
were used because they have been shown to have large impacts on mosquito

populations as well as other components of aquatic communities in various

151
lentic habitats (e.g. Ellis and Borden 1970; Hazelrigg 197 4; Scott and Murdoch
1983; Chesson 1984; Murdoch et al. 1984; Chesson 1989). Indeed, in this study,
a ﬂoating macrophyte treatment (algal clump) did not signiﬁcantly inﬂuence
larval survival. As predicted, larval survivorship within a notonectid predator
treatment was signiﬁcantly less than the control or algal clump treatments.
However, contrary to the predator-refuge hypothesis, survivorship was
signiﬁcantly lower in the algal clump/predator treatment than the control or
algal clump treatments. Thus, in this study, a ﬂoating macrophyte refuge did
not provide adequate plant cover to inhibit notonectid predation on @-
geedrimeeelatus larvae. This evidence does not support the predator-refuge
hypothesis proposed by Orr and Resh (1992).
Lab experiment
Most laboratory work has centered around Aedee gem (L.) after that

species was recognized as a vector of yellow fever in the Caribbean (Finlay
1886; Reed et al. 1900). Despite their tremendous importance as disease
vectors, Anepheles mosquitoes have been neglected in laboratory studies,and
comparatively little is known about the interaction of temperature, food type
and amount on larval growth and survivorship. The diversity and abundance of
food quality and quantity in addition to the broad range of temperatures
present an accurate description of the cause and effect temperature and larval
food sources has on An. Wm performance. The relative
importance of food is not biased nor compromised with the combination of a
broad range of temperatures selected in this experiment.

One of the effects of available limiting foods and/or nutrients may be
optimal larval survivorship in addition to growth and development thus
inﬂuencing adult mosquito size and ﬁtness. Results from this experiment

152

indicate that survivorship was impacted greatly by the interaction of
temperature, food type and food amount. It appears that 18°C provides an
optimal developmental and survival temperature in the laboratory for Q1.
W. This optimal temperature within constrained food amounts
is contrary to the optimal survival temperature proposed by Huffaker's (1944)
study without food limitations. Although open water habitats in nature .
signiﬁcantly inﬂuenced larval growth and development rates as well as -
survivorship, lab studies show that 100 ml of surface microlayer food type from
vegetated habitats provide optimal growth and survivorship. Rejmankova et al.

 

(1996) suggest that several environmental factors, e. g., amount of ﬁlamentous
algae and Na+, K+, Ca+, and PO4-P concentrations served as positive
predictors of An. Menus larval occurrence in Mexico. Since open water
habitats provide signiﬁcantly more microinvertebrate dietary resources for An.
quedzimegﬂetge larvae, it is suspected that the addition of these food items
may increase survivorship within this food type/amount treatments.

Because An. W mosquito populations are continuously
breeding in nature, a horizontal life table approach was constructed, i.e. a
stage-speciﬁc analysis of a generation or cohort of individuals, whose numbers
and mortalities were determined over the course of time for each of a series of
stages, (in this experiment stages = instars) (Bellows et al. 1992). Life tables
constructed from temperature, food type and amount information indicated
that the key factors, ﬁrst and second instar An. ggeghjmeeeletge were most
sensitive, i.e. these instars were the key factors to temperature and food
quality/quantity differences. The key factor analysis procedure in this study
utilized Podoler and Rogers (1975) technique of regressing each stage speciﬁc

k-v'alue against the total K-value in order to identify the key factors responsible

153
for population change. It is important to mention that identiﬁcation of a key
factor does not necessarily point to the factor or factors that may regulate the
population density (Southwood 1967; Debach et al. 1976).

In summary, support for a multi-hypothesis explanation to predict Ag.
We larval distribution and success may be in order as a result of
the ﬁeld and laboratory experiments presented in this study. First, open water
habitats are typically characterized by cyanobacterial mats. Several studies
(Rejmankova et al. 1996; Rodriquez 1993) have documented that temperature
and C02 emissions associated with cyanobacterial mats may serve as
ovipositional cues for female AnepheLee mosquitoes. This study has shown that
An- guadrimamﬂatus gTowth and
survivorship are strongly inﬂuenced by the temperature differences between
open water and vegetated habitats. Second, although this study did not support
the predator refuge hypothesis described by Orr and Resh (1992), certainly
there is some support for this hypothesis with other species of Anenhelee (Orr
and Resh 1992). Finally, data presented in previous chapters along with
corroborating data from this study indicates that open water habitats provide
important stage-speciﬁc dietary resources that may enhance larval growth,
development and survivorship. In addition to the predator-refuge hypothesis,
proposed by Orr and Resh (1992), clearly, this study provides convincing
evidence to support an additional hypothesis, i.e., the enhanced food resource
hypothesis. Therefore, the growth, development and survivorship of larval An.
MW mosquitoes and ultimately vector competence may be

inﬂuenced by several hypotheses acting in concert.

154
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Wallace, J .R. and R.W. Merritt. 1997. Population dynamics of Anephelee
MW: inﬂuence of diet and predation. Vector Control
Bulletin of the North Central States, In Press.

Zar, J .H. 1984. Biostatistical analysis, second edition. Prentice-Hall, Inc.,

Englewood Cliffs, New Jersey. 718 pp.

CONCLUSIONS AND RECOMMENDATIONS

Habitat choice, food resources and predation are three factors that play
an important role in the larval feeding ecology of An. Wm and
mm in southcentral Michigan ponds. In both permanent or
temporary ponds, larvae of both species tend to aggregate at air-water
interfaces of either algal mats or cattails in a permanent pond, or ﬂoating
debris and woody vegetation in a vernal/aestival pond. Larvae may move
freely, to some extent, between microhabitats, e.g. open water and vegetated
areas. However, further study on larval movement within habitats is needed to
determine if larvae actively choose their microhabitat based on food resources,
predator avoidance, microclimate or if larval habitat is a function of adult
oviposition selection.

Larval dietary resources, e.g. bacteria, algae, detritus and
microinvertebrates varied Within a spatial and temporal context in the
permanent and temporary pond. In the permanent pond, larval diets differed
spatially with regard to microhabitat and temporally relative to seasonal
differences. Temporal or diel feeding differences in microinvertebrate
consumption existed within the An. Wm larvae inhabiting the
permanent pond. It appears that larvae exhibited a food preference for rotifers
in the open water habitat. Such preferential feeding is a novel concept for
these as well as most types of ﬁlter feeders and warrants further study.
Furthermore, M.We larvae were capable of consuming live and
dead water mites, whether there is a preference for either needs additional
work.

The digestibility of microorganisms, e.g., certain protists, cladocerans,

160

161

water mites and some algae is determined by the resistant properties of their
outer wall and the duration of exposure in the gut. Larval ingestion of water
mites and cladocerans appears to have a gut retention time of less than 24
hours. It could be argued that those algae found in larval guts upon dissection
may represent nonfood items and pass through the gut undigested. However,
the consumption of certain algae may provide certain exudates in the form of
dissolved organic material that can be of high nutritive value to mosquito
larvae or have phagostimulatory effects on larval feeding. Further work is
needed on larval feeding rates on these speciﬁc microorganismal diets in order
to determine if poor growth resulting from one type of food is the result of
deﬁciencies in speciﬁc nutrients and not the effects of underfeeding. Also,
knowledge of the "feeding areas" or microhabitats as well as their available
food resources may enhance the success and development of novel types of
recombinant microorganisms that have the potential to provide effective
control of a wider range of mosquito species for a longer duration than the
naturally occurring bacilli. .

Perhaps due to the ephermeral nature of temporary ponds or because
Anephelee larvae are in low densities, there is little information regarding An.
W115 and mm larval feeding ecology. This study

documents a quantitative evaluation of anopheline food sources in a temporary
pond. Anephelee larval diet differed between years and reﬂected the relative
abundance of the available microbial components within a temporary pond. @.
metipemﬁe larvae in temporary ponds rarely, if ever, consumed water mites,
cladocerans or rotifers. Adult non-wintering spring migratory mosquitoes,

such as An. WM and metipennie leave these pools before the

onset of a dry phase for more permanent aquatic systems. The exploitation of

162
temporary ponds by these species may enhance dispersal and colonization
tendencies in more permanent waters (Figure 1). Typically, An.
W and metipem’e inhabit permanent ponds, whether their
presence in temporary ponds is a potential evolutionary stable strategy in the
regulation of these populations or not requires further study. On the other
hand,there is ample evidence that there is in fact a complex of four species
within the An. eeedijmeeeletls designation. If those individuals found within
the vernal pond are different species from those found in the permanent pond,
then this type of dispersal and colonization phenomenon may be a speciation
event. Further molecular taxonomic analyses would elucidate probable
answers to this question.

It has been hypothesized that Angphelee larvae primarily inhabit
vegetated areas in marshes to avoid predation. Field and lab experiments
suggest that temperature, food quality and quantity (as reﬂected by habitat
differences in the ﬁeld) and to some extent, predation had a signiﬁcant impact
on the larval growth and survivorship in permanent ponds. m1.
geedrimeeeleme larvae grew and developed faster and survived better in open
water habitats. Moreover, experimental results from ﬁeld treatments that
provided algal clump refuges for Anephelee larvae did not support the predator
refuge hypotheis. Interestingly, this study offers support for an alternative
hypothesis, i.e. enhanced food resources, that inﬂuence larval growth and
survivoship.

This study has developed a broader picture of Aeephelee feeding ecology
both from a spatial and temporal perspective. Habitat heterogenity provides
different abiotic and biotic constraints that affect Anephelee grth and

163

    

MAMJJASONDJFMA

\

Figure 1. Diagram depicting the time frame of when non-
wintering spring migrants enter a temporary pool and leave
before the dry phase for more permanent habitats (Modiﬁed from
Wiggins et al. 1980).

164
survivorship. Data indicates that open water habitats provide important stage-
speciﬁc dietary resources that may enhance larval growth, development and
survivorship. Moreover, this study provides support for alternative hypotheses
e.g., an enhanced food resource and favorable larval microclimate explanation
that predicts An. We larval distribution and success. These
hypotheses may be acting in concert with others e.g., predator refuge and
oviposition site preference to inﬂuence Melee population dynamics and life
history strategies.

APPENDICES

APPENDIX A

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No. : 1911-2

Title of thesis or dissertation (or other research projects):

Larval feeding ecology of Anopheles quadrimaculatus (Say) and
ﬁg. punctipennis (Say) (Diptera: Culicidae) in southcentral
Michigan ponds.

 

Mbseum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator's Name (8) (typed)
John R. Wallace

 

 

Date 1 July 1997

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
North America. Bull. Entomol. Soc. Amer. 24:141-42.

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator,
Michigan State University Entomology Museum.

165

APPENDIX B

VOucher Specimen Data

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