MES Will”!!!HI("NilIHIIIUHIHIHIWINILJITTTHI .5. a

310691 1666 ix
Mm Sid L3
University

 

 

 

 

 

 

 

 

 

      

This is to certify that the

thesis entitled
EVALUATION OF AEDES HENDERSONI AND

 

AEDES TRISERIATUS AS POTENTIAL

VECTORS 0F DIROFILARIA IMMITIS
'p""re'sen' fe'a' 5“y "—

JAMES S. ROGERS

has been accepted towards fulfillment
of the requirements for

M. S - degree in ENTOMOLOGY

 

 

AAMJZM

Major professor

 

Date March 24, 1930

0-7639 ‘

  
  

OVERDUE FINES:
25¢ per day per item
RETUMING LIBRARY MATERIAL§:

Place in book return to new
charge from circulation ream

 
    

..i.
1“ mam .'
Jéna; --
‘5 ~£-.'-g1:,e.t!’_ -

'-

r .

   
      

$763767» ’

188 0143
06123135
1V;~~t§: “

1 11

 

 

EVALUATION OF AEDES HENDERSONI AND AEDES TRISERIATUS AS POTENTIAL
VECTORS OF DIROFILARIA IMMITIS

BY

James Speed Rogers

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Entomology

1980

ABSTRACT

EVALUATION OF AEDES HENDERSONI AND AEDES TRISERIATUS AS POTENTIAL
VECTORS OF DIROFILARIA IMMITIS

By

James Speed Rogers

 

The tree-hole breeding mosquitoes Ag. hendersoni and Ag. Agi-
seriatus were separately evaluated as potential vectors of 2. Ag:
migig (dog heartworm) in Michigan. Significantly fewer 2. immitis
developed to the infective stage in Ag. triseriatus than Ag, Agg-
dersoni. Infection with 2, immitis caused significantly greater
mortality in Ag. hendersoni than Ag. triseriatus. Paired verti-
cal ovitrapping in a 10 acre beech-maple woodlot indicated that

Ag. hendersoni slightly outnumbered Ag. triseriatus. {Ag. hender-

 

soni and Ag. triseriatus were respectively the fourth and fifth
most abundant species caught in the dog-baited traps. Paired ver-

tical ovitrapping indicated that both Ag. hendersoni and Ag. tri-

 

seriatus are primarily ground-level feeders. Ag. hendersoni and

Ag. triseriatus are reviewed in relation to breeding habitat, flight

 

range, relative abundance, feeding habits, longevity, and suscep-
tibility to infection. It is concluded that both mosquitoes are

potential vectors of Q. immitis in wooded areas of Michigan.

ACKNOWLEDGMENTS

I would like to express deep gratitude to Dr. H. D. Newson for
his kindness and patience; and for providing the intellectual, moral,
and financial support necessary for this degree. The help of the oth-
er members of my guidance committee, Drs. G. W. Bird, R. L. Fischer,
and J. F. Williams, is gratefully acknowledged. My friend Dr. Mori
Zaim deserves special thanks for making me feel welcome as a new grad-
uate student, and for enthusiastically teaching me so much about field

and laboratory research techniques.

ii

TABLE OF CONTENTS

LIST OF TABLES ...o..................................o.ooo.......oo V
LIST OF FIGURES ...............................o...........o.....o. Vi
INTRODUCTION cocococo.oooooooo0.00000000000000000000000000000.0000. 1
LITERATURE REVIEW ooooooo00000000000000coooooooo00000000000000.0000 2

Taxonomy and Morphology ...................................... 2

Geographical and Ecological Distribution ..................... 3

Epizootiology ................................................ 4
Pathogenesis ................................................. 7
Abnormal Hosts ............................................... 8
Vectors ...................................................... 9

Ag. hendersoni and Ag. triseriatus as Vectors ................ 10
MATERIALS AND METHODS ............................................. 14
Colony Development and Maintenance ........................... 14
Laboratory Infection of Mosquitoes ........................... 18
Susceptibility to 2. immitis ................................. 19
Longevity .................................................... 20
Relative Abundance ........................................... 20
Feeding Habits ............................................... 21
RESULTS ........................................................... 25

susceptibility to 23 immitis oooooooooooo00.000000000000000... 25

LongeVi-ty 0.00......OOOOOOOOOOOOOOOOO0.0..OOOOOOOOOOOOOOOOOOOO 29

Relative Abundance coo000000000000oooooooooooocoooooocoooooooo 33

iii

iv

Feeding Habits .0.0...0..00....0.0..O...OOOOOOOOOOOOOOOOOOCOO

DISCUSSION

SUWARY AND CONCLUSIONS O0.00..0..O...0.0.0....OOOOOOOOOOOOOOOOOCO

APPENDIX A.

APPENDIX B.

APPENDIX C.

APPENDIX D.
APPENDIX E.
APPENDIX F.

REFERENCES

2, immitis-infected Ag. hendersoni dissections ......
Dissections of mosquito heads .......................

Survival of Q. immitis-infected and uninfected

A20 hendersoni ooooooooooooooooo0.0000000000000000...
Ovitrapping reSUItS oooooooooooooo00000000000000.0000
Rat-baited mosquito trapping ........................

Dog-baited mosquito trapping ........................

33

39

46

48

56

6O

61

65

67

71

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table 10.

Table 11.
Table 12.

Table 13.

1.
2.

3.

4.
5.
6.
7.
8.

9.

LIST OF TABLES

Development of D, immitis in Ag. hendersoni ..............
Third-stage Q, immitis in mosquito heads .................

Effect of oviposition on survival of Q, immitis-
infeCted Ago hendersoni one...cooooooooooooooooooooooooooo

Survival rate of Q. immitis infected mosquitoes ..........
Paired vertical ovitrapping summary ......................
Rat-baited trapping summary ..............................
Dog-baited trapping summary ..............................
2. immitis-infected Ag, hendersoni dissections ...........
Dissections of mosquito heads ............................

Survival of Q, immitis-infected and uninfected Ag.

hendersoni 0.00......0..O0.0...0.00000000000000000000000CO
OVitrappi-ng reSUIts C...000......0....OOOOCOOOOOOOOOOOCOO.
Rat-baited moquito trapping O...I...OOOOOOOOOOOOOOOOOOOOO

Dog-baited moquito trapping 0.0.0.0....OOOOOOOOOOOOOOOOOO

48

56

6O

61

65

67

LIST OF FIGURES

Figure 1. Suction forceps for mosquito forced copulation ......... 16
Figure 2. Distribution of mosquito traps in Hudson's Woods ....... 22

Figure 3. Mortality of Q, immitis-infected and uninfected
£2. hendersoni OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 3]-

vi

INTRODUCTION

In recent years dog heartworm, Dirofilaria immitis (Leidy), has
become a serious veterinary problem in Michigan and other parts of

the midwest (Lewandowski, 1977; Otto, 1969). Aedes triseriatus (Say),

 

a tree-hole breeding mosquito, has been incriminated as a potential
vector of 2. immitis in Michigan (Lewandowski, 1977), Massachusetts
(Phillips, 1939), Texas (Keegan ggflgl., 1968), and Mississippi (In-
termill, 1973). These last four studies failed to distinguish between

Ag. triseriatus and a closely related species, Ag. hendersoni Cocker-

 

e11, which is the most widely distributed tree-hole breeding mosquito
in North America (Zavortink, 1972). Ag. hendersoni has never been
specifically evaluated as a potential vector of Q, immitis, although

it is known that Ag. triseriatus and Ag. hendersoni have very differ-

 

ent vector potentials for another disease, California encephalitis
(Watts gg‘gl., 1975). The purpose of performing the field and labor-
atory studies reported here was to separately consider Ag. hendersoni
and Ag. triseriatus in relation to the following factors which are
relevant to Q, immitis vector determination (Ludlam g£.gl., 1970):
flight range; breeding habitat; relative population density; feeding

habits; longevity; and susceptibility to infection.

LITERATURE REVIEW

Taxonomy and Morphology

Ag. triseriatus var. hendersoni was described by Cockerell in
1918. Dyar (1919) placed hendersoni in synonymy with triseriatus,
and Breland (1960) resurrected hendersoni to full specific rank on
the basis of morphological and chromosomal studies. Breland's action
has been fully substantiated by subsequent reports of morphological
and ecological differences between Ag. hendersoni and Ag. triseria-
ggg. According to Zavortink (1972), Ag. hendersoni and Ag. triser-
igggg are sibling species (closely related species which are repro-
ductively isolated but morphologically identical or nearly so; Mayr,
1963).

Morphological differences between Ag. hendersoni and Ag. triser-
igggg are subtle; however, Zavortink (1972) listed five characteris-
tics of adults, six of the male genitalia, six of the larvae, and
two of the pupae, which may be used to differentiate between the Spe-
cies. In addition, Zaim _gflgl. (1977) described differences in the
egg morphology of Ag. hendersoni and Ag. triseriatus, using light and
electron microscopy.

Apparently, both behavioral and morphological differences between

Ag. hendersoni and Ag. triseriatus ensure reproductive isolation. In

small laboratory cages, Ag. triseriatus mates freely, while Ag.

hendersoni will not. Laboratory colonies of Ag. hendersoni must be
propagated by the forced c0pulation technique of McDaniel and Horsfall
(1957). Truman and Craig (1968) used this technique to produce hybrids
of Ag. hendersoni and Ag. triseriatus, and found the larval and adult
hybrids to be morphologically intermediate between the two species.

The F1 hybrid males produced by mating male Ag. hendersoni to female
Ag. triseriatus, however, had deformed genitalia. Grimstad _£_gl.

(1974) reported that less than .5% of field-collected larvae were hy-

brids.

Geographical and Ecological Distribution

Ag. hendersoni is the most wideSpread tree-hole breeding mosqui-
to in North America. Ag. triseriatus is nearly as widespread, and is
sympatric with Ag. hendersoni in all states and provinces east of the
Great Plains (Zavortink, 1972). According to Lunt and Peters (1976),
the range of Ag. hendersoni extends further west because Ag. hender-
gggg is more tolerant of arid conditions. Hanson and Hanson (1970)
described how past land management has inadvertently increased the
number of tree-holes in the midwest.

Although water-filled rot holes in trees are the usual larval
habitat of Ag. hendersoni and Ag. triseriatus, these mosquitoes have
been collected from artificial containers such as tin cans and dis-
carded tires. Ovitraps made of jars or cans are attractive to gravid
females and make useful field samplers for Ag. hendersoni and Ag. £51-
seriatus (Furlow and Young, 1970). The utility of ovitrapping was
greatly enhanced by the report by Zaim._£.gl. (1977) that the eggs of

Ag. hendersoni and Ag. triseriatus could easily be distinguished

morphologically.

The use of ovitraps has permitted study of ovipositioning in the
canopy of woods, and differences between the oviposition sites of Ag.
hendersoni and Ag. triseriatus have been described. Scholl and De-
Foliart (1977) in Wisconsin, and Sinsko and Grimstad (1977) in Indi-

ana, reported that Ag. triseriatus oviposits primarily in ground-level

 

ovitraps, while Ag. hendersoni oviposits primarily in elevated ovi-

 

traps. However, in a special problems project designed to replicate
these ovitrapping studies, Rogers (1978) in Michigan found that Ag.

hendersoni preferred elevated ovitraps, but Ag. triseriatus utilized

 

ovitraps at both levels equally.

Epigootiology

According to Otto (1972), Q. immitis was originally enzootic in
areas within 83-125 km of the Atlantic coast, from New Jersey to Tex-
as, and this situation prevailed as recently as 30 years ago. Since
then, there have been reports of 2. immitis infections in several in-
land states and provinces, both north and west of the original enzo-
otic foci. In the North, 2. immitis has been known to be enzootic in
Minnesota for the last two decades (Otto, 1972), and Slocombe (1978)
reported enzootic Q. immitis infections in Ontario and Manitoba. In
the West, 2. immitis is an increasing problem in California (Weinmann
and Garcia, 1974) and Oklahoma (Kocan and Laubach, 1976).

It is of particular interest that the incidence of Q. immitis is
increasing in many, but not all, parts of the midwest (Otto, 1972).

In Ohio, Streitel t AA. (1977) found 11 of 500 (2.2%) dogs necrop-

sied in a Columbus humane shelter to be infected with Q. immitis.

Groves and Kutz (1964) found 7 of 380 (1.8%) necropsied dogs in Ohio to
be infected with Q. immitis. In the 13 year period between those stud-
ies, the incidence of D. immitis had not significantly increased, and
Streitel _g filo (1977) concluded that a highly enzootic area had not
developed in Ohio. Kazacos (1978) reported that 17 of 112 (15%) dogs
necropsied in Lafayette, Indiana were infected with 2. immitis. Mar-
quardt and Fabian (1966) reported that 2. immitis infection rates in
southern, central, and northern Illinois were 35%, 22%, and 10%, re-
spectively.

D. immitis has received considerable attention in Michigan. Like
most other midwestern states, Michigan has experienced an increase in
the prevalence of D. immitis. Leash and Hanson (1961) reported that 4

of 192 (2.1%) blood smears seen at the Michigan State University Vet-

t al. (1970)

erinary Clinic were positive for Q. immitis. Zydeck
found 4 of 248 (1.6%) blood smears taken from dogs in the Detroit dog
pound positive for D. immitis. Worley (1964) found 6 of 123 (5.4%) dogs
from southeastern Michigan dog pounds positive for Q. immitis when nec-
ropsied. Prouty (1972) reported that 195 of 880 (22%) Belleville dogs,
22 of 399 (6%) Detroit dogs, and 41 of 698 (6%) Farmington dogs had D.
immitis microfilariae. Prouty found that older dogs and dogs kenneled
outdoors had a higher probability of being infected with Q. immitis.

The most comprehensive data on the epizootiology of 2. immitis in
Michigan has been collected by Dr. H. D. Newson, who sent out question-
naires to veterinarians throughout Michigan. The data of Dr. Newson
show that there were at least 14,525 confirmed cases of 2. immitis in

54 counties of Michigan from pre-l951 to May, 1972. Since 1972, Q.

6

immitis infections have been reported from six additional counties in
the state. The majority of Q. immitis infections have been reported
from urban counties in the lower peninsula.

The reported distribution of Q. immitis in the midwest is not uni-
form (Otto, 1972), and the different methods of making 2. immitis sur-
veys probably accentuate this apparent uneven distribution. Streitel
‘_g‘gl. (1977) reported that 13 of 24 dogs with Q. immitis in their
hearts did not have a microfilaremia, illustrating a bias in surveys
based on microfilarial findings. Further, the various methods of ex-
amining blood for microfilariae that were used in reported studies are
not equally sensitive. Dogs screened at a veterinary clinic would pre-
sumably show a higher infection rate than dogs randomly chosen at a dog
pound, because infected dogs often present symptoms that would elicit
medical attention. Also, increasing awareness of 2. immitis among
veterinarians in recent years has resulted in more efficient diagnosis.
An additional complication in comparing reported data on Q. immitis
incidence is that microfilarial surveys conducted before 1956 must be
questioned. In that year Newton and Wright described a new species of
microfilariae in dogs, Dipetalonema reconditum (Grassi), that is mor-
phologically very close to 2. immitis. It is probable that earlier
surveys would have mistaken Q. reconditum microfilariae for those of
Q. immitis. Q. reconditum is transmitted by fleas, and produces no
pathology in the dog.

In Michigan, there are two reports of 2. reconditum infections.
Leash and Hanson (1961) reported that 8 of 192 (4.2%) dogs examined at
the Michigan State University Veterinary Clinic were positive for 2.

t AA. (1970) reported that 7 of 248 (2.8%) dogs

reconditum. Zydeck

from Detroit were positive for D. reconditum.

Pathoggnesis

According to McGreevy ££.él- (1974), the third-stage D. immitis
larvae in the labium of an infective mosquito are stimulated to escape
from the tip of the labium when it is bent in the feeding process. A
tiny droplet, probably mosquito haemolymph, is deposited on the skin
along with the larvae. This fluid protects the larvae from desiccation,
and allows them to penetrate the skin at the fasicle puncture site.

Next, the larvae enter the bloodstream and migrate to submuscular
membranes and subcutaneous tissues (Kume and Itagaki, 1955), where they
molt at 9-12 days and 60-70 days after inoculation. After 3-4 months,
the larvae migrate to the heart via the veins. Once in the heart, males
and females attain adult lengths of 15-18 cm and 25-30 cm, respectively.
Sexual maturation is reached about 8 months after inoculation, as indi-
cated by the release of microfilariae into the bloodstream.

According to Garlick (1975), each female 2. immitis releases about
30,000 microfilariae daily into the bloodstream, and the microfilariae
live up to 2 years. The microfilariae of Q. immitis exhibit both dai-
ly and seasonal periodicity. According to Otto (1969), in a 24 hour
period the maximum microfilaremia is from 5 to 50 times the minimum
microfilaremia. Church _g_gl. (1976) described Q. immitis microfilar-
ial fluctuations as "subperiodic" and could not even characterize the

t _a_l_. (1954), Kume

fluctuations as diurnal or nocturnal. Eyles
(1974), and Sawyer (1974) reported that the microfilaremia is lower in
colder months. According to James and Harwood (1969), circadian per-

iodicity is a response of the parasite to its vectors, the maximum

microfilaremia coinciding with the chief biting period of the vector.
According to Ansari (1970), dUring the peak microfilaremia, the micro-
filariae are evenly distributed throughout the circulation, and at low
microfilaremia, the microfilariae are sequestered in the lungs. Hawk-
ing (1967) reported that oxygen tension in the blood is the factor
which determines the distribution of the microfilariae.

A dog infected with D. immitis may be symptomless except for the
presence of circulating microfilariae (Soulsby, 1968). Commonly, dogs
infected with D. immitis have a persistent cough and lack stamina. In
severe cases, there are a variety of other symptoms which can be fatal.
According to Soulsby (1968), the severity of the symptoms is proportion-
al to the number of adult worms in the heart. Fowler _g_gl. (1973),

however, found no correlation between the microfilaremia and the num-

ber of adult worms in the heart.

Abnormal Hosts

Although Q. immitis adults have been found in many types of wild
animals, Otto (1972) reported that there is no evidence of any reser-
voir host. Lewandowdki (1977) listed the following animals as hosts
of adult 2. immitis; fox, beaver, coyote, wolf, dingo, eat, seal, gib-
bon, tiger, jaguar, and sea lion. Goble (1942) reported the muskrat,
Williams and Dade (1976) the wolverine, and Johnson (1975) the black
bear, as having 2. immitis infections.

Human dirofilariasis has been reported with increasing frequency
in the southern and eastern United States and Canada (Blecka, 1978).
According to Schlotthauer gg'gl. (1969), most cases of human dirofil-

ariasis are not caused by Q. immitis, but by Dirofilaria tenuis

Chandler, a raccoon parasite. Schlotthauer _g AA. (1969), however, at-
tributed 14 cases of human dirofilariasis to Q. immitis. Twelve of
these cases produced "coin" lesions of the lung. The lesions may be de-
tected by routine chest X-rays, or X-rays may be prompted by cough and
chest pain (Schlotthauer ggngl., 1969). The chief importance of human

dirofilariasis is its diagnostic interpretation, because the lesions

produced by D. immitis may be confused with a malignancy.

Vectors

Mosquitoes are the only known vectors of 2. immitis (Ludlam gg
AA., 1970), and become infected by ingesting circulating microfilar-
iae along with the blood of the dog. Kershaw gg_gl. (1955) found that
fewer microfilariae are ingested by the mosquito than would be expected
on the basis of the microfilaremia and volume of blood ingested.

Taylor (1960) reported that at 24.40 C, the microfilariae leave
the midgut of Ag. aegypti (Linnaeus) after 24 hours and enter the cells
of the malpighian tubules. Once in the cells of the malpighian tubules,
the microfilariae become less motile. On the sixth or seventh day, the
larvae enter the lumen of the malpighian tubules, and by the tenth day
molt to the second-stage. The 2nd-stage larvae, which are senentary,
molt to become the highly motile 3rd-stage beginning on the 13th day
after ingestion. On the 15th day after ingestion, 3rd-stage larvae
leave the malpighian tubules, and migrate to the mouthparts of the mos-
quito via the haemocoel.

Kutz and Dobson (1974) described how temperature affects the rate

of development of 2. immitis in the mosquito, and how the geographical

range of Q. immitis is influenced by climate. Ho t El. (1974)

10

described how third-stage 2. immitis larvae in mosquito mouthparts may
spontaneously escape. According to Bemrick and Bemrick (1969), third-
stage 2. immitis larvae do not escape from the mouthparts when the mos-
quito takes a blood meal.

Laboratory studies have shown that different mosquito Species
(and strains) vary greatly in their susceptibility to Q. immitis in-
fection (Hu, 1931; Kartman, 1953a). In refractory hosts, blood clot-
ting in the midgut may mechanically impede the microfilariae, or they
may be passed to the hindgut and excreted (Kartman, 1953a, 1953b).

Some hosts resist infection by forming a chitinous capsule around the
developing larvae, and in others, larval development does not procede
even in the absence of encapsulation (Kartman, 1953a, 1953b).

Death of the vector is also a physiological response to Q. immitis
infection, and according to Hamilton and Bradley (1979), is the most
salient problem attending experimental attempts to transmit dirofilar-
ias through laboratory mosquitoes. These authors found that early
death of Q. immitis-infected mosquitoes was due to active, living mi-
crofilariae, and not to dead, intact, or homogenized larvae, whole mos-
quito bodies, or body fractions. Although Intermill (1973) reported
that Q. immitis larvae in Ag. triseriatus did not cause apparent his-
tological damage to the gut or malpighian tubules, Hamilton and Brad-
ley (1979) judged that the malpighian tubules could be damaged without
visible changes.

Ludlam _£.£l- (1970) listed 63 species of mosquitoes in which
complete larval development of Q. immitis has been reported. Despite
this long list, these authors wrote that "the principal mosquito vec-

tors of Q. immitis have not been identified in any area of the world."

11

According to these authors, the weakness of most reports on potential

2. immitis vectors is that they go no further than reporting develop-
ment of Q. immitis to the infective third-stage under laboratory con-
ditions. Ludlam _g AA. (1970) judged that the ability of microfilariae
to develop to the infective stage in the laboratory does not indicate
that the mosquito in question is an efficient vector in nature. These
authors noted that data on field-collected mosquitoes harboring infec-
tive D. immitis larvae are an important step in vector determination,
but are found in only a few reports. Christensen (1977) pointed out
that dog-to-dog transmission of Q. immitis has been reported for only
three Species of North American mosquitoes. Ludlam _£,_A. (1970) al-

so mentioned other factors which are relevant to vector determination,
but which are often ignored: breeding habitat; flight range; relative
population density; feeding habits; longevity; and genetics of different
strains which affect susceptibility to Q. immitis infection. Michigan
is one of the few regions where a detailed study, giving due consider-
ation to the above factors has been made. Lewandowski (1977) concluded
that Ag. vexans (Meigen), Ag. quadrimaculatus Say, and Ag. walkeri Theo-
bald are D. immitis vectors of primary importance in Michigan, and that

Ag. canadensis (Theobald), Ag. cinereus Meigen, and Ag. triseriatus are

vectors of secondary importance.
Ae. hendersoni and Ae. triseriatus as Vectors

There is some relevant information on the Q. immitis vector poten-
tial of Ag. triseriatus, but until this study there was none on Ag. hen-
dersoni. Benach t 1. (1971) in the laboratory, and Wright and DeFol-

iart (1970) in the field, found that Ag. triseriatus is a general

12

feeder, feeding on man, other mammals, birds and turtles. According to
Loor and DeFoliart (1970), Ag. triseriatus is a daytime feeder, with
chief biting activity in the late afternoon and evening. Morris and De-
Foliart (1971) found Ag. triseriatus to have the highest parous rate
(32-45%) of any Wisconsin woodland mosquito. This finding enhances the
vector potential of Ag. triseriatus (for any disease) because it indi-
cates that this species lives a long time and takes repeated blood
meals.

Phillips (1939) in Massachusetts, was the first to study Ag.‘gg;-
seriatus as a vector of 2. immitis. (The study of Phillips was made
before Ag. hendersoni was elevated to specific rank.) Phillips found
that Ag. triseriatus fed avidly on dogs in the field and laboratory,
and that 2. immitis readily developed to the infective stage in Ag.,£§i-
seriatus in the laboratory. Keegan _g‘_A. (1968) in Texas also found
that Q. immitis developed to the infective stage in Ag. triseriatus in
the laboratory. No mention is made in their study of Ag. hendersoni,
and it is not possible to know if proper care was taken to differentiate
Ag. hendersoni from Ag. triseriatus. Intermill (1973) made the most
detailed study on the ability of Q. immitis to develop in Ag. triseri-
gggg, and judged Ag. triseriatus to be an efficient vector. He noted
that host mortality was low, and that a high proportion of the mosqui-
toes which ingested Q. immitis developed infective larvae, although
some encapsulation of developing larvae took place. Intermill collect-
ed larvae for his study from tree-holes in Mississippi, and notably,
made no mention of any effort to differentiate between Ag. hendersoni

and Ag. triseriatus, although both species occur in Mississippi.

13

Lewandowski (1977) made field studies on the Q. immitis vector
potential of Ag. triseriatus in Michigan. He found that Ag. triser-
igggg.was locally abundant (i.e., occurring in woodlots), and was
attracted to dogs in dog-baited mosquito traps. He judged that Ag.

triseriatus was an excellent host and potential vector of Q. immitis,

 

but due to its local distribution, may have only secondary importance
in the natural maintenance of this disease. Lewandowski made no at-
tempt to differentiate between Ag. hendersoni and Ag. triseriatus, and
in view of the known mixed populations of these two species that are
present in his study area, the "Ag. triseriatus" in his study can
reasonably be considered a mixture of Ag. hendersoni and Ag. triseria-

tus.

MATERIALS AND METHODS

Colony Development and Maintenance

 

The insectary, where all stages of the mosquitoes were maintained,
had a 16 hour photoperiod, including 5 hour crepuscular periods of di-
minished light. The temperature in the insectary ranged from 21.1-
26.70 C and the relative humidity from 80-100%. Larvae were reared in
white enamel pans, and each pan was fed daily 3 pinch of TetraminR
fish food. Cages of adult mosquitoes were provided with a 7% sucrose
solution in a flask from which a cotton wick protruded. Cages were
placed on corks in petri dishes filled with mineral oil to protect
the mosquitoes from predacious ants. Eggs were stored in the insec-
tary for at least one week before hatching in water deoxygenated with

autolyzed yeast (Difco Laboratories, Detroit).

The colonies of both Ag. hendersoni and Ag. triseriatus were

 

 

founded by eggs taken in ovitraps in the summer of 1977. Larvae were
not added to their respective colonies until all the egg shells on the
tongue blade of an ovitrap were checked under a light microscope by
the method of Zaim _£.él° (1977), and found to belong to one species.
If a tongue blade had eggs of both species, all larvae which hatched
from that tongue blade were discarded. Despite this check, several

months after being colonized, the Ag. hendersoni colony was found to

contain Ag. hendersoni-Ag. triseriatus hybrids, as determined by the

 

l4

15

criteria of Grimstad gg EA- (1974). The hybrids were excluded from
the colony by individually segregating gravid adult female Ag. hender-
soni and collecting all the eggs produced by each one. For this, each

gravid female Ag. hendersoni was placed in a 472 ml paper cup, which

 

was covered with mosquito netting and contained a sucrose source and
an oviposition beaker made from a 118 ml paper cup lined on the in-
side with paper toWeling. The entire egg batch produced by each fe-
male, after maturation, was hatched in an enamel larval pan, and the
larvae reared to the fourth-stage. At least ten fourth-stage larvae

of each egg batch were examined and determined to be Ag. hendersoni,

 

Ag. triseriatus, or hybrids, according to the criteria of Grimstad gg

 

21- (1974). If any hybrid or Ag. triseriatus larvae were found, the

 

entire egg batch was discarded. The Ag. hendersoni obtained by this

 

method were placed in a cage, and after one generation, a pure colony
was obtained, as determined by subsequent larval spot checks.

The Ag. triseriatus mated freely in laboratory cages, but the Ag.

 

hendersoni had to be propagated by a forced copulation technique Simi-

 

lar to that of McDaniel and Horsfall (1957). The thorax of the male
was firmly pinched by a pair of forceps. This served as a handle,
and also severed any connection to the brain (which inhibits copula-
tion), eliminating the need to decapitate the male. The female then
was held by "suction forceps" powered by a water-driven aspirator
(Carolina Biological Supply Co.) (Figure 1). The glass tip of the
"suction forceps" was made by drawing out a fine glass tube in a bun-
sen burner.

The mosquitoes were anesthetized for forced copulation by carbon

16

Figure l. Suction forceps for mosquito forced copulation.
a. Threads for faucet attachment.
b. Water exhaust.
c. Rubber tubing (1 m long).
d. Thumb hole, used to make and break vacume.

e. Glass tip, which contacts female mesoscutum.

l7

. _.... .\. r
,»,..(.L/u...

Gull}; L411}

 

 

em

 

      

.v 3.4. .

 

l8

dioxide evolved from dry ice. It was convenient to anesthetize the
mosquitoes in the aspirator which was used to transfer them from the
holding cage. Males were exposed to carbon dioxide until they col-
lapsed (about ten seconds), and were immediately pinched by the for-
ceps. Females were exposed for about 20 seconds, because they had to
remain anesthetized for at least 1 minute for the procedure to be suc-
cessful. (Females which revived during copulation would kick away
their partner.) The forced copulation technique was very time con-
suming, and even after a year of trial and error variations, the suc-
cess rate never rose above 20%. The highest rate of insemination was
achieved when the abdomens of the copulating pair formed an angle of
90-120 degrees, and males were 1-3 weeks old.

Dr. G. B. Craig (personal communication) suggested that if given
a large enough cage, the Ag. hendersoni might swarm and mate freely,
obviating the forced copulation technique. A 2.3 m3 cage was built
and several thousand Ag. hendersoni put into it. The cage had a 16
hour photoperiod, including crepuscular periods created with a rheo-
stat controlling light intensity. Unfortunately, the Ag. hendersoni
did not mate freely, and the forced copulation technique had to be

used.

Laboratory Infection of Mosquitoes

In the laboratory experiments, a dog served as the infective blood
source for Q. immitis. All dogs used in these studies were obtained
through the Michigan State University Laboratory Animal Care Service.

Originally, attempts were made to use the canvas dog-restraining

l9

harness described by Lewandowski (1977), to allow the mosquitoes to feed
on the dog. In later experiments, this apparatus was discarded in favor
of making the dog lie quietly on its side, with a shaved paw extended
into the mosquito cage. The dog tolerated the mosquito-feeding very
well, and no tranquilizer or restraining apparatus was needed. For ex-
perimental purposes, mosquitoes were 4-7 days old at the time of blood
feeding. The order in which the two treatment groups in each replicate
were fed (since they could not be fed simultaneously) was determined by
flipping a coin.

For microfilaremia determination, blood samples were obtained at
9:00 a. m., the time at which each infective feeding replicate was be-
gun. Blood was taken from the cephalic vein of the infected dog in a
syringe containing a pinch of ethylenediaminetetraacetic acid (EDTA)

as an anticoagulant, and 20 mm3

of the sample was transferred from the
syringe and divided between 2 slides. A 20 X 60 mm coverslip was then

placed on each slide, and the microfilariae were counted under a com-

pound microscope.

Susceptibility to D. immitis

Mosquito susceptibility to 2. immitis infection was studied in
two ways. In study 1, several thousand Ag. hendersoni were fed on
the infected dog after determining its microfilaremia. For 15 days
after the infective blood meal, from 11 to 26 mosquitoes were dissected
daily, and the number of larvae, their location, and observations on
their developmental stage recorded. The dissection technique was that
of Jones (1967), in which the entire gut and ovaries were drawn out in-

to a drop of saline with the use of instruments made of minuten pins

20

embedded in applicator sticks.

In study 2, 300 Ag. hendersoni and 300 Ag. triseriatus were fed on
the infected dog, in each of 10 replicates. Sixteen days after the in-
fective blood meal, 12 heads of each species were dissected under a mi-

croscope, and the number of Q. immitis larvae present was recorded.

Longevity

Three studies addressed the subject of mosquito longevity. In
study 3, 100 infected Ag. hendersoni were randomly assigned to a cage
lacking an oviposition beaker and 100 infected Ag. hendersoni were
likewise assigned to a cage with a beaker, in each of ten replicates.
The number of surviving mosquitoes in each cage was recorded 16 days
after feeding on the infected dog.

To begin study 4, several hundred Ag. hendersoni were allowed to
engorge on the infected dog immediately after its microfilaremia had
been determined. Several hundred other Ag. hendersoni were allowed
to engorge simultaneously on the uninfected dog. Immediately after
feeding, 70 engorged females within each treatment group were randomly
assigned to each of six cages. For 17 days, the number of survivors
in each cage was recorded daily.

The mosquitoes used in study 5 were the same as those in study 2.
Sixteen days after the infective blood meal, the number of survivors

of each species was recorded.

Relative Abundance

Study 6 concerned the relative abundance of Ag. hendersoni and

Ag. triseriatus in a ten acre beech-maple woodlot (Hudson's Woods)

21

on the campus of Michigan State University. This woodlot was chosen
because it was within bicycling distance, barking dogs would not dis-
turb people there, it was inaccessible to the public, and because pre-
season reconnoitering indicated that it was well endowed with tree-
holes.

Twenty-one pairs of ovitraps were widely dispersed within the wood-
lot (Figure 2). The ovitraps were made by removing the tops from 354
ml beer cans and spray painting the cans black. The ovitraps were al-
ways paired, one placed at 6 m, and the other at ground level. The
upper traps were attached to a cord, and could be raised and lowered
for servicing. The cords were placed by using a ladder, and looped
either over a convenient limb or small nail. Each trap had a wooden
tongue blade wrapped with paper toweling clipped into it with a large
paper clip. At weekly intervals, the old tongue blades were collected
and replaced with new ones, and the traps were refilled with water.
The tongue blades with attached eggs were stored for at least one
week in the insectary to allow them to maturate. Maturated eggs were
hatched by placing the tongue blades containing the eggs in a beaker
of water, adding a pinch of autolyzed yeast, and leaving them over-
night. Hatched egg cases were cleared by the technique of Zaim _g__A.
(1977) and examined under a light microscope. A hand counter was used

to tally the number of Ag. hendersoni and Ag. triseriatus eggs.

FeedingiHabits

The 10 rat-baited mosquito traps used in study 7 were the same as
those used by Shaw (1976) as chicken-baited traps. These traps were

paired, one at ground level and the other at 10 m, and were widely

22

Figure 2. Distribution of mosquito traps in Hudson's Woods

I dog-baited trap

pair of vertical ovitraps

A = pair of rat-baited traps

 

 

23

N \ Figure 2.

 

 

100 m

 

 

J

r 9.
T.o’.
1

LA

 

 

 

 

 

 

Bennett Rd.

 

 

24

dispersed in Hudson's Woods (Figure 2). The captured mosquitoes were
killed in the evening with chloroform.

The three dog-baited mosquito traps used in study 8 were widely
dispersed in Hudson's Woods (Figure 2). The traps were described by
Lewandowski (1977). The dogs remained in the traps 24 hours per day.
The louvered side panels containing mosquitoes were emptied twice
daily, at approximately 10 a. m. and 7 p. m., by placing the panels in
a large plastic bag and killing the mosquitoes with chloroform.

Adult Ag. hendersoni and Ag. triserigtus collected in the animal-
baited traps were identified on the basis of their mesoscutal scale
patterns (Grimstad ggflgA., 1974), and their tarsal claws (Harmston,
1969). The possibility of encountering hybrids was judged to be neg-
ligible, since Zaim (1978) in the same locality in Michigan found 400
Ag. hendersoni and 1,100 Ag. triseriatus, and no hybrids among 4th-

stage larvae derived from eggs caught in ovitraps.

RESULTS

Susceptibility_to D. immitis

The microfilaremia of the dog used in study 1 was 25,000 per cm3.

Developing D. immitis larvae were recovered from the malpighian tubules
of every mosquito dissected during the extrinsic incubation period (Ap-
pendix A). The lst 3rd-stage Q. immitis reached the head and mouth-
parts of Ag. hendersoni on the 16th day after the blood meal (Table 1).
No encapsulation or other resistance to the development of Q. immitis
was observed. The development of Q. immitis in Ag. hendersoni (Ta-
ble 1) was similar to that reported by Intermill (1973) in Ag. triseri-
gggg. The decreasing mean and standard deviation (Table 1) of the num-
ber of larvae found in the dissections on successive days suggests that
the mosquitoes with a heavier parasite load tended to die earlier,
leaving as survivors those mosquitoes with fewer parasites. (Although
several thousand mosquitoes fed on the infected dog, none were alive 18
days later.)

Study 2 addressed the relative susceptibility of Ag. hendersoni and
Ag. triseriatus to Q. immitis by comparing the number of third-stage
larvae in the heads and mouthparts of these mosquitoes after the extrin-
sic incubation period (16 days). Dissection results are shown in Appen-
dix B, and the computed mean number of larvae for each treatment within

each replicate are in Table 2. The ten replicates in this experiment

25

26

mcofiuoommfiv .0:

k.

 

.coom umuwm om>um~ HH mwmum o
o
.zuan m“ name“: 0
.oHHuoE you one uaw
sows ecu aw omwumfiwmouows mafiafiwEom o
o
o
o
o
o

.monsu smenwfiafiwe ecu aw mum omfiumfi
tamouowe umoE .wcwvoom umuwm : NH u< 1

 

 

mcowum>uomno mounqnusoe d can: aw
mauoz .0: new:

~.mH
o.mH
©.o~
H.h~
m.o~
¢.Nm
H.mm
H.oH

m.o¢

 

cowumw>oc
cumcamum

AONV
AHNV
AONV
ASNV
AHNV
AONV
Aoav
ASNV

«ASNV

w.o¢
w.mm
o.H¢
m.o¢
o.o¢
o.mm
H.¢m
w.Hm

~.No

o

 

 

moaannu Gownmfiawwa Home coca
aw meuos .0: new:

A

noumm when

 

 

 

.ficomumccwc qMfl aw mflufiEEw mm mo uaoeaofio>on .H oHnm

B

27

 

.o>«~w who mooufiacwoe oz

.HHH owmum one om>um~ umoz
.cmo: ecu CH

comm umuwm ohm om>umH HHH owmum
.moflsnau swanwwaame o5»

cfinuwa mum om>um~ HHH owmum ~H<

.comm umuwm one mm>um~ HHH owmum

.HH owmum mum om>pmH Ham xHumoz

 

mcowum>pomco

AHHV S.SH

ANHV m.o

o

 

muummnusoe w new: CH
mayo: .oc one:

¢.NH
n.m~
o.~H

o.o~

 

cowumfi>ow
ouvaMum

AHHV e.m~
AONV o.em
AONV H.mm

AHNV H.5m

 

ma

ma

0H

ma

«a

Ma

NH

HH

0H

 

moasnau ammnwwaame Home cooHn
:« mahoa .0: new: known when

 

.Av.u:oov

H ofinwe

28

.oH xmv coHuoomaHnumoa co mmouHsvmoE comoso >HEovnmu NH mo mcoHuoomch
CH vasoH om>HmH mHuHEEH am owmumucanu mo pecan: some on» mH >Huco some

 

k.

 

 

m; m... SS m... a... AS A... 0.... is a... 633823.64.

 

¢.m o.mH m.w ~.oH H.w n.m ¢.w m.m N.q *m.o Hcomuocaoa tMfl

 

 

 

0H 0 w m c m a m N H moHoon
mumoHfimmm

 

 

.mvmm: cuHsvmoE CH mHuHEEH am owmumuquze .N mHnme

29
were performed over a 15 week period, during which the microfilaremia

of the dog varied from 13,000 to 20,000 per cm3. Ag. hendersoni sup-

 

ported the development of a significantly greater number of Q. immitis

to the third-stage than Ag. triseriatus (p( .01).

Longevity

Study 3 was preliminary, designed to determine if ovipositing by
Ag. hendersoni infected with D. immitis affected longevity. The results
of this study had practical Significance, since if ovipositing did af-
fect longevity, oviposition beakers would have to be supplied to cages
in subsequent studies. (Because presumably mosquitoes have the oppor-
tunity to oviposit in the field.) The number of surviving mosquitoes
in each cage was recorded 16 days after feeding on the infected dog
(Table 3). There was no significant difference between the longevity
of those mosquitoes which oviposited and those which did not (p) .2).
Therefore, oviposition beakers were not used in the other studies.

In study 4, cummulative longevity curves were constructed for Ag.
hendersoni which had fed on an uninfected dog, and on a dog which had
a 2. immitis microfilaremia of 15,000 per cm3. For 17 days after the
blood meal, the number of survivors in each treatment group was recorded
daily (Appendix C). The mortality of the uninfected mosquitoes was
small and gradual (Figure 3), with 89% of the mosquitoes surviving 17
days after feeding. The mortality of the infected mosquitoes was much
larger (Figure 3), with only 18% of the original mosquitoes surviving
17 days after the blood meal.

Study 5 compared the longevity of Ag. hendersoni and Ag.

30

 

onHQasm uo:
mm muoxmmn coHuHmoaH>o

ooHHamam
mm muoxmon :oHuHmoaH>o

 

 

H uaoeumwue

 

m 0 mm om 0N «H mm mm

NH w Hm mm as «H m on

o w n o m a m N
oumoHHomm

 

coHuomwcHuumom mzmn oH
ARV Haemuovcoc .Mfl wcH>H>u=m

 

 

 

.Hcomuwuaon

 

.Mfl wouomwcHumHuHEEH am we Hm>H>uam co

:oHuHmoaH>o mo uoommm .m mHnt

31

Mortality of 2. immitis-infected and uninfected
Ag. hendersoni. (Vertical bars indicate 95%
confidence intervals.)

Figure 3.

 

32

Hams vOOHH Hmumm m%mo

 

2 8 3 2 2 w a e N
vmuummafl
.+...+. +
+ * + + + + f:..+....+....+....+:..+
.32....

m oustm

 

NH

wH

«N

on

on

we

no

qm

co

00

N5

 

aarte ruosxapuaq 'av 'ou ueew

33

triseriatus which were infected with Q. immitis. Sixteen days after
feeding, the number of survivors in each replicate was recorded (Ta-

ble 4). Ag. triseriatus had significantly greater longevity than Ag.

 

hendersoni when both were infected with l_)_. immitis (p( .05).

Relative Abundance

Study 6 was relevant to the relative abundance of Ag. hendersoni
and Ag. triseriatus, and incidentally, to the vertical ovipositioning
preferences of these mosquitoes. From June 13 to August 22 (1979) all
eggs collected in the ovitraps were identified to species (Appendix D),
and weekly results summarized (Table 5). Apparently, Ag. hendersoni
slightly outnumbered Ag. triseriatus in Hudson's Woods during the trap-
ping period because 6,653 total Ag. hendersoni eggs and 5,136 Ag. 5;;-
seriatus eggs were captured. Ag. hendersoni, as expected, displayed a
strong preference for the upper traps (Table 5). Ag. triseriatus, how-

ever, utilized traps at both levels equally (Table 5).

FeedingiHabits

Paired vertical rat-baited mosquito traps were used in study 7 to
determine whether Ag. hendersoni and Ag. triseriatus fed at different
heights. Both Ag. hendersoni and Ag. triseriatus fed at 0 and 10 m,
but greater numbers of both species were caught in the traps at O m,
(Table 6). The number of mosquitoes of all species caught in the rat-
baited traps was small (Appendix E); either white rats do not make good
bait, or the traps were poorly designed.

Dog-baited mosquito traps were used in study 8 to determine the

extent to which Ag. hendersoni and Ag. triseriatus feed on dogs. From

34

OH xmn :oHuoomcHuumom so oouczoo ohms muo>H>H3m¥

 

 

 

 

 

 

Hm SHH «ma mm SN as SH LN mm on magmanmmnuu .m«

6H ms 6N km mH NS NS SS 6H 1mm acompmeeo: .m«

OH 6 w A e m s m N S mmaomam
mumoHHmwm

 

moOuHmmmoE wouoomcH oom\muo>H>u=m

 

.mmOuHscmoE wouowmcHumHuHEEH .n mo bump Hm>H>u=m .q mHnmP

35

Table 5. Paired vertical ovitrapping summary.

 

 

 

 

 

 

 

 

Ag. hendersoni Ag. triseriatus
End of trap-
ping week 0 m 61h _ggg_ 6 m
June 20 0* 161 o 0
27 60 459 0 33
July 4 64 964 106 166
11 120 566 547 1083
18 153 559 721 525
25 105 543 673 549
Aug. 1 195 528 106 245
8 815 734 141 34
15 O 400 77 62
22 .22 .1332. ___6§_ __2
Total: 1532 5121 2439 2697

 

3!.

O

no. eggs

36

Table 6. Rat-baited trapping summary.

 

Trap height

 

 

Species 0 m 9 m
Ag. hendersoni 9* 4
Ag. triseriatus 15 l

 

 

*no. mosquitoes

37

June 19 to August 2 (1979), 1,537 mosquitoes were taken in the 3 dog-
baited traps. The traps were emptied twice daily on 33 days during

this period, and all mosquitoes identified to species, or species com-
plex (Appendix F). ‘Ag. hendersoni and Ag. triseriatus were caught in
nearly equal numbers, being respectively the fourth and fifth most abun-

dant species taken in the dog-baited traps (Table 7).

38

Table 7. Dog-baited trapping summary.*

 

 

 

 

 

 

 

Species Number
9. restuans-pipiens 565
Ag. vexans 500
Ag. stimulans 174
Ag. hendersoni 115
Ag. triseriatus 110
Ag. trivitattus 43
Ag. quadrimaculatus 18
Q. tarsalis 6
Ag. sticticus 4
Ag. cinereus 2

 

*mosquitoes were trapped from June 19
to August 2.

DISCUSSION

Barnett (1960) lists four criteria which should be met to in-
criminate an arthropod as a vector:

1. demonstration of feeding or other effective con-

tact with the host under natural conditions,

2. demonstration of a convincing biological associ-
ation in time and/or space of the suspected ar-
thropod Species and occurence of clinical or sub-
clinical infection in the host.

3. repeated demonstrations that the arthropod, un-
der natural conditions, harbors the infective stage,

4. and, transmission of the agent under controlled con-
ditions.

In the case of Q. immitis, these four criteria are hard to meet,
and in fact, no mosquito has ever met all four of them. Strictly
speaking, then, every suspected mosquito species should be referred
to as a "potential" vector of Q. immitis. Of course, "potential" mos-
quito vectors do transmit Q. immitis, because there is no recent re-
port suggesting that other insects or arthropods transmit D. immitis.

Only a few attempts have been made to meet the third criterion of
Barnett, and none have been successful. Christensen (1977) isolated
third-stage nematodes from Ag. trivitattus in Iowa, but it is possible

that these nematodes were not Q. immitis. Lewandowski (1977)

39

40

encountered similar difficulties in Michigan. He also isolated third-
stage nematodes from several mosquito species (but not Ag. triseriatus)
and could not identify the nematodes to species. Until a method is de-
vised for positively identifying third-stage Q. immitis larvae, further
attempts to meet Barnett's third criterion cannot be conclusive. Other
difficulties also prevent application of this criterion to Ag. hender-
gggg and Ag. triseriatus. First, effective trapping methods for Ag.
hendersoni and Ag. triseriatus do not exist (Zaim, 1978). Since, even
in an important vector, 2. immitis infection rates would be very low
(Dr. H. D. Newson, personal communication), many thousands of a mosquito
Species, from several locations, must be examined to make reasonable
inferences. Presently, it is not possible to collect such large num-
bers of Ag. triseriatus or Ag. hendersoni. Secondly, even if it were
possible to collect large numbers of these two species, it would not be
possible to differentiate between them on a large scale. Differentia-

tion of Ag. hendersoni and Ag. triseriatus is very time consuming, and

 

is particularly difficult for old and flight-worn individuals (which
are the only individuals which would harbor infective third-stage lar-
vae). Q. immitis isolation attempts require fast handling of large
numbers of mosquitoes, and in such attempts, Ag. hendersoni and Ag. gg_-
seriatus would have to be pooled, defeating the purpose of the study.
Only three North American mosquitoes have met the fourth criter-
ion of Barnett. Newton (1957) transmitted Q. immitis from one dog to
another in the laboratory with Ag. quadrimaculatus, Bickley g£,gA.
(1977) transmitted Q. immitis with Ag. canadensis, and Christensen
(1977) accomplished this with Ag. trivitattus. According to Dr. J.

F. Williams (personal communication), 2. immitis transmission attempts

41
using only one or a few dogs are not meaningful, and I did not even at-
tempt to meet the fourth criterion of Barnett with Ag. hendersoni.

The first criterion of Barnett is relatively easy to satisfy, and
this has been accomplished for the Ag. triseriatus-Ag. hendersoni com-
plex by Phillips (1939) and Lewandowski (1977). (Neither author dis-
tinguished between Ag. hendersoni and Ag. triseriatus.) In study 8 I
have explicitly satisfied the first criterion of Barnett for both Ag.

hendersoni and Ag. triseriatus.

 

Even though it is not feasible to meet all of the criteria of
Barnett, it is both reasonable and necessary to follow other lines of
evidence in order to make inferences about potential vectors of 2. Ag:
gigig. Ludlam _g__g. (1970) discussed the difficulties involved with
meeting the third and fourth criteria of Barnett, and listed complemen-
tary factors which are relevant to Q. immitis vector determination:

l) breeding habitat, 2) flight range, 3) relative population density,
4) feeding habits, 5) longevity, and 6) genetics of different strains
which affect susceptibility to Q. immitis infection.

I could not improve on the studies of Lewandowski (1977), Phillips
(1939), Intermill (1973), or Keegan _gugl. (1968) in the sense of sat-
isfying more of the criteria of Barnett. However, unlike the authors
of these previous "Ag. triseriatus" vector potential studies, I did con-
sider the sixth Q. immitis vector determination factor of Ludlam ggflgl.,
by recognizing the genetic difference between Ag. hendersoni and Ag.
triseriatus. The fact that Ag. hendersoni and Ag. triseriatus are actu-

ally different Species (instead of strains of "Ag. triseriatus") makes

it even more important to consider these mosquitoes separately in

42

relation to Q. immitis. Of course, potential species differences be-
tween Ag. hendersoni and Ag. triseriatus need to be considered not on-
ly for the Sixth D. immitis vector determination factor of Ludlam gg
gl., but for the first five as well.

In relation to the first and second 2. immitis vector determina-
tion factors of Ludlam t al. (breeding habitat and flight range, re-

spectively), Ag. hendersoni and Ag. triseriatus are similar; both mos-

 

quitoes could play a role in the transmission of Q. immitis in wooded
areas. Tree-holes, the breeding habitat of Ag. hendersoni and Ag. g1;-
seriatus, occur in significant numbers only in wooded areas. The dif-
ferent oviposition altitude preferences shown by Ag. hendersoni and

'Ag. triseriatus (study 6), while ecologically interesting, have no ob-

 

vious bearing on the Q. immitis vector potential of these mosquitoes.
The short flight range of Ag. hendersoni and Ag. triseriatus confines
these mosquitoes to the woodlot of their origin. During three summers
of field work, I never encountered either Ag. hendersoni or Ag. triser-
Agggg outside of a woodlot. Nonetheless, both Ag. hendersoni and Ag.

triseriatus could be important 2. immitis vectors, because dogs often

 

frequent wooded regions such as campgrounds, parks, suburban subdivi-
sions, and farm woodlots.

Data on the third vector determination factor of Ludlam gg_gi.,
relative population density, supports the conclusion that both Ag.
hendersoni and Ag. triseriatus are important woodland potential vec-
tors of Q. immitis. Gorton (1973) sampled mosquito populations in an
Owosso, Michigan woodlot by means of human biting collections. He
found that "Ag. triseriatus" was the most common mosquito in the wood-

lot. Gorton made no mention of efforts to differentiate between Ag.

43

hendersoni and Ag. triseriatus, and it is reasonable to regard the

VAg. triseriatus" in his study as a mixture of Ag. hendersoni and Ag.

 

triseriatus. As measured by dog-baited mosquito trapping during the
summer of 1979 (study 8), Ag. hendersoni and Ag. triseriatus were re-
spectively the fourth and fifth most abundant mosquitoes in Hudson's
Woods. Morris and DeFoliart (1971) noted that Ag. hendersoni and Ag.
triseriatus are reluctant (compared to other mosquitoes) to enter an-
imal-baited traps, and will be underestimated in studies using such
traps. In a special problems project (Rogers, 1978) eggs of both Ag.
hendersoni and Ag. triseriatus were captured in all seven East Lansing,
Michigan woodlots in which paired vertical ovitraps were placed. It
is concluded that Ag. hendersoni and Ag. triseriatus are common Mich-
igan woodland mosquitoes. Ecological factors which would favor one
species over the other are not known.

Feeding habits, the fourth Q. immitis vector determination factor
of Ludlam gg.gl., was addressed by using rat- and dog-baited mosquito
traps (studies 7 and 8, respectively). The results of the dog-baited

mosquito trapping are consistent with the conclusion that both Ag. tri-

 

seriatus and Ag. hendersoni are potential vectors of Q. immitis. Both
Species were attracted to dogs in the field, with the total catch of
Ag. hendersoni slightly exceeding that of Ag. triseriatus (115 and 110,
respectively). In view of the fact that the concurrent paired verti-
cal ovitrapping (study 6) indicated that Ag. hendersoni slightly out-
numbered Ag. triseriatus in Hudson's Woods, it appears that these mos-
quitoes are equally attracted to dogs in the field. Consistent with
this conclusion are the results of the vertical rat-baited trapping,

which found that neither Ag. hendersgni nor Ag. triseriatus are

44

primarily canopy feeders.

The fifth D. immitis vector determination factor of Ludlam gg_gl.,
longevity, was investigated for the Ag. triseriatus-Ag. hendersoni com-
plex in Wisconsin by Morris and DeFoliart (1971). They found that mos-
quitoes of this complex had the highest parous rate of any Wisconsin
woodland mosquitoes. This finding supports the conclusion that Ag.

hendersoni and Ag. triseriatus are potential vectors of Q. immitis, be-

 

cause it indicates that these mosquitoes live a long time and take re-
peated blood meals. Study 5 demonstrated that relative to Ag. triseri-

atus, Ag. hendersoni suffers more mortality when infected with Q. immi-

 

gig. The great mortality experienced by 2. immitis-infected Ag. hender-
gggg_in study 4 does not necessarily indicate that infected Ag. hender-
gggg_would suffer similar mortality in the field. Hamilton and Bradley
(1979) judged that the high mosquito mortality which has occurred in
most laboratory infections with 2. immitis was an artifact.

The sixth vector determination factor of Ludlam gg_gl., genetics
of different strains which affect susceptibility to Q. immitis infec-

tion, was addressed by studies 1 and 5. The daily dissections of Ag.

hendersoni during the extrinsic incubation period of Q. immitis demon-

 

strated that Ag. hendersoni is an excellent intermediate host of Q.Ԥg-

 

giggg in the laboratory. In all cases, mosquitoes which lived long
enough (17 days) supported the develOpment of 2. immitis to the infec-
tive stage. No encapsulation or any other refractory influence was
encountered in the development of Q. immitis. Intermill (1973) on

the basis of a similar daily dissection study of Q. $mmitis-infected

Ag. triseriatus concluded that Ag. triseriatus was an excellent inter-

 

mediate host of Q. immitis. The results of Intermill on E.

45

triseriatus differed from the results of study 1 on Ag. hendersoni
mainly in that he encountered some encapsulation of Q. immitis. This
discrepency suggested that Ag. hendersoni has greater susceptibility

to D. immitis than Ag. triseriatus. Accordingly, study 2 tested this
hypothesis, by comparing the number of third-stage Q. immitis larvae in
the heads and mouthparts of Ag. hendersoni and Ag. triseriatus after
the extrinsic incubation period. A greater number of Q. immitis lar-
vae reached the heads and mouthparts in Ag. hendersoni than in Ag.

triseriatus, so Ag. hendersoni should be considered the more suscepti-

 

 

ble Species. (It should be kept in mind, however, that both Ag. hender-
gggi and Ag. triseriatus supported the development of Q. immitis to the
infective stage.)

Whether the greater 2. immitis-susceptibility of Ag. hendersoni
is outweighed by the greater longevity of Ag. triseriatus is impos-
sible to judge. The resolution of vector potential studies based on
the six 2. immitis vector determination factors of Ludlam _g.gl. is
not keen enough to allow precise rating of vector potential. Vector
potential is an elusive quality because it is based on a variety of
uncontrollable factors which have complex interactions. After review-
ing Ag. hendersoni and Ag. triseriatus in relation to the six vector
determination factors of Ludlam ggugl., it is concluded that both Ag.

hendersoni and Ag. triseriatus should be considered important poten-

tial vectors of Q. immitis in wooded areas of Michigan.

SUMMARY AND CONCLUSIONS

In the laboratory, 2. immitis readily develops to the infective

third-Stage in Ag. hendersoni (study 1).

 

Ovipositing by Ag. hendersoni infected with D. immitis has no

 

influence on mosquito mortality (study 3).

Heavy infection with Q. immitis greatly reduces the longevity
of Ag. hendersoni in the laboratory (study 4).

In the laboratory, Ag. hendersoni supports the develOpment of
more 2. immitis in the heads and mouthparts than Ag. triseri-
gAgg (study 2).

Greater numbers of both Ag. hendersoni and Ag. triseriatus were

 

caught in rat-baited traps at 0 m than 10 m (study 7), suggest-
ing that neither species is primarily a canopy feeder.

Ag. hendersoni has a strong preference for elevated ovitraps

 

but Ag. triseriatus uses both basal and elevated ovitraps
equally (study 6).

As indicated by paired vertical ovitrapping (study 6), Ag. Agg-
dersoni Slightly outnumbered Ag. triseriatus in Hudson's Woods.

Slightly greater numbers of Ag. hendersoni were caught in the

 

dog-baited trapping (study 8) than Ag. triseriatus. Ag. hender-
soni and Ag. triseriatus appear to be equally attracted to dogs

in the field.

46

47

On the basis of the studies reported in this dissertation, and

studies in the literature, Ag. hendersoni and Ag. triseriatus

 

were reviewed in relation to the six 2. immitis vector deter-

mination factors of Ludlam gg. l. (1970) (breeding habitat,

flight range, longevity, relative population density, feeding
habits, and genetics of different strains which affect suscep-
tibility to D. immitis). It is concluded that both Ag. hender-

soni and Ag. triseriatus are important potential vectors of Q.

immitis in wooded areas of Michigan.

APPENDICES

APPENDIX A

Table 8. ‘2. immitis-infected Ag. hendersoni dissections.

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number malpighian tubules and mouthparts

l l 65 0
2 76 0

3 32 0

4 133 0

5 142 0

6 l3 0

7 22 O

8 107 O

9 132 0

10 164 0

11 41 0

12 27 0

13 30 0

14 9 O

15 ll 0

16 101 0

17 68 0

18 69 0

19 76 0

20 21 0

21 ll 0

22 50 0

23 29 0

24 63 0

2 l 27 0
2 59 0

3 21 0

4 66 0

5 57 O

6 32 O

7 50 0

8 35 0

9 100 o

48

49

Table 8 (cont'd).

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number mglpighian tubules and mouthparts

2 10 49 0
ll 48 0

12 40 0

13 32 O

14 39 O

15 78 0

16 58 0

17 36 0

18 36 0

19 61 0

20 6O 0

21 79 O

22 65 O

23 71 O

24 39 0

3 1 l9 0
2 86 0

3 36 O

4 132 0

5 43 0

6 33 O

7 36 0

8 60 0

9 19 0

10 19 0

ll 73 0

12 101 0

13 71 0

14 120 O

15 89 0

16 22 0

17 4O 0

18 72 0

19 17 O

4 1 34 0
2 100 0

3 31 O

4 35 O

5 4 0

6 90 0

7 58 0

8 65 0

9 50 0

50

Table 8 (cont'd).

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number malpighian tubules and mouthparts
4 10 31 0
ll 34 0
12 115 0
13 24 0
14 52 O
15 112 0
16 49 0
17 106 0
18 46 0
19 24 0
20 51 O

5 1 85 0
2 39 0
3 22 0
4 41 0
5 39 0
6 47 0
7 55 0
8 57 0
9 53 0
10 49 0
11 100 0
12 27 0
l3 9 0
14 53 0
15 29 0
16 59 0
17 29 0
18 36 O
19 59 0
20 40 0
21 39 0
6 1 22 0
2 78 O
3 64 O
4 87 0
5 83 O
6 43 0
7 12 0
g 22 0
9 69 O
10 64 0

51

Table 8 (cont'd).

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number malpighian tubules and mouthparts

6 ll 7 O
12 75 0

13 13 O

14 20 O

15 75 0

16 54 0

17 59 0

18 53 0

l9 13 O

20 26 0

21 74 O

22 10 0

23 83 O

24 73 O

25 54 O

26 55 0

7 l 60 0
2 ll 0

3 68 O

4 68 0

5 31 O

6 61 0

7 4 O

8 53 0

9 17 0

10 95 0

ll 47 0

12 19 0

13 22 0

14 64 0

15 36 0

16 81 0

l7 7 O

18 20 0

19 18 0

20 47 0

8 1 12 O
2 35 0

3 36 O

4 13 0

5 35 0

6 34 O

7 48 O

Table 8 (cont'd).

52

 

Days after
blood meal

8

10

Mosquito

number

8

9
10
ll
12
13
14
15
16
17
l8
19
20
21

\OQVO‘UIDWNH

No. larvae in
malpighian tubules

21
19
29
30
41
82
21
38
47

8
65
36
45
15

22
55
23
35
23
45
46
31
18
53
65
26
40
30
43
49
68
53
3O
61

41
56
41
62
32
25
15

No. larvae in head
and mouthparts

OOOOOOOOOOOOOO

OOOOOOOOOOOOOOOOOOOO

OOOOOOO

53

Table 8 (cont'd).

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number malpighian tubules and mouthparts
10 8 15 0
9 l3 0
10 81 0
ll 28 0
12 23 0
13 84 0
l4 l6 0
15 34 0
16 32 0
17 36 0
18 19 0
19 48 0
20 19 0
21 6O 0
ll 1 52 O
2 7 0
3 66 0
4 35 0
5 29 0
6 46 0
7 29 0
8 26 0
9 20 O
10 24 O
11 42 0
12 25 0
13 21 0
14 30 0
15 26 0
16 40 0
17 39 0
18 41 O
19 28 0
20 36 0
12 l 33 O
2 28 0
3 59 0
4 7 0
5 62 0
6 34 0
7 l8 0
8 48 0

54

Table 8 (cont'd).

 

 

 

 

Days after Mosquito No. larvae in No. larvae in head
blood meal number malpighian tubules and mouthparts
12 9 39 O
10 7 0
ll 36 0
12 14 0
13 43 0
14 53 0
15 24 0
16 33 0
17 34 0
18 52 0
19 33 0
20 28 0
13 l 42 0
2 43 0
3 41 0
4 15 0
5 13 O
6 14 0
7 19 0
8 24 0
9 28 0
10 10 0
ll 30 0
l6 1 undetermined l7
2 " 23
3 " 23
4 " 10
5 " 0
6 " l4
7 " ll
8 " 0
9 " 3
10 " 3
ll " 0
12 " 10
17 undetermined 14

II 19
n 14

n 28
H 30

\lO‘UiJ-‘UONt-I
0"

55

Table 8 (cont'd).

 

 

 

Days after Mosquito No. larvae in
blood meal number malpighian tubules
17 8 undetermined
9 fl
1 O M

11 H

No. larvae in head
and mouthparts

 

APPENDIX B

Table 9. Dissections of mosquito heads and mouthparts.

 

Replicate l
Mosquito No. Ae. hendersoni Ae. triseriatus

*
1 6 1
2 1 2
3 6 0
4 l6 5
5 O O
6 4 6
7 11 12
8 3 3
9 l 1
10 3 1
11 3 13
12 27 9

Replicate 2
Mosquito No. Ae. hendersoni Ae. triseriatus

1 8 18
2 10 7
3 6 l
4 ll 7
5 0 0
6 5 13
7 2 O
8 0 7
9 3 ll
10 3 O
11 O 13
12 8 O

 

* No. Q. immitis larvae

56

57

Table 9 (cont'd).

 

Replicate 3
Mosguito No. Ae. hendersoni Ae. triseriatus

1 3 4
2 7 2
3 0 0
4 0 5
5 8 1
6 2 1
7 0 9
8 7 1
9 8 3
10 3 2
ll 1 8
12 5 0

Replicate 4
Mosquito No. Ae. hendersoni Ae. triseriatus

 

 

12
14
10
7
3
6
5
14
7
10 16
ll 5
12 2

Hp—a

\OOO\IO\U11>L.JN1—i
OwNONHH-L‘ONON

H

Replicate 5
Mosquito No.7 Ae. hendersoni Ae. triseriatus

1 1 6
2 l 3
3 6 7
4 3 2
5 1 2
6 1 O
7 0 2
8 0 1
9 3 1
10 3 l
11 10 6
12 15 1

58

Table 9 (cont'd).

 

Replicate 6
Mosggito No. Ae. hendersoni Ae. triseriatus

 

l 1 8
2 2 4
3 2 7
4 ll 10
5 4 O
6 7 O
7 17 9
8 34 4
9 8 O
10 4 6
11 5 5
12 3 6

Replicate 7
Mosquito No. Ae. hendersoni Ae. triseriatus

 

8
ll
14

5

5

3
23

7
ll
10 12
11 14
12 9

\OCDVOLn-L‘ri-d
L‘HHNNomNLflt—nwo

Replicate 8
Mosquito No. Ae. hendersoni Ae. triseriatus

l 14 3
2 13 20
3 10 3
4 7 2
5 4 6
6 13 4
7 3 7
8 12 7
9 8 8
10 8 O
11 5 7
12 5 6

59

Table 9 (cont'd).

 

Replicate 9
Mosquito No. Ae. hendersoni Ae. triseriatus

 

18
7
5
1

12

17

16
9

17

10 23

ll 24

12 7

sooouombcoww
1—- 1--
t-‘NONJ-‘LDI—‘(DUII—‘OO

H

Replicate 10
Mosquito No. Ae. hendersoni Ae. triseriatus

H

\OCDVO‘U‘IwaI-I

H
walUH—ubxlxlooh-Oo
O‘HHOOOuNUIOwO

60

moOuHsumoE oN can NHHmcHwHuo owmo some

 

 

 

 

 

 

 

 

 

 

«
mm Oe oe Om on «e NH w OH 6 HN nH eH NH
em me Ne on on «e eH O OH m NN nH mH eH
em we we Oe Oe ee mH O HH O MN eH mH mH
em me we Oe we ee «H O HH OH mN wH ON 6H
wm we de Ne qe Ne mH OH NH NH eN NN NN wH
Oe me me Ne qe Ne NH OH NH NH wN qN NN NH
Oe me me Ne qe Ne HH HH NH wH wN qN wN HH
Oe «e we Ne «e we OH mH NH «H on ON Nw OH
He qe me Ne qe we 9 MH NH wH Nm ON «n O
me «e me me «e we w mH NH ON mm ON mm w
we ee me we ee we N wH mH HN on Hm em N
ee qe we de ee we e HN wH HN we mm Nm e
ee me me «e we we m Om eN eN Ne wm me n
ee ee me qe we we q Nw ON wN me He ee q
Ne Ne ee me we me m cm Nw Nm Hm eq Om m
Ne me Ne Ne ON me N Nm em O¢ mm mm mm N
0e ON we we ON oe H mm Nd Hm Ne Om Oe H

e n e m N H HmmE eooHe e n e m N H HmmE OooHn

unease uwwo Houwm Non Mensa: undo Houmm Nan

muo>H>usm muo>H>wsm
wouoomchs .oz wouoomcH .oz
.Hcomwoecoc .Mfl cwuoomcHa: nan emuoomcH1mHuHEEH am no Hm>H>usm .oH oHeme

U XHOzmmm<

61

 

maumHummHuu .o<uu .Hcomuoeamn .Mflmnr

 

 

 

 

 

 

 

 

o SA SS 0 o A S SS S SS S

o o S o o o o o o o o S

o 0 AH SS S o 0 AS 0 o S

SS 0 SH 0 o o o o o o o S

oA SH 0 SS S SA o SA 0 SS S

SSS S SS SS o o o o o o o A

SS 0 o o S SS 0 SS o o S

o o o o o o o o o o o S

S SS SS AS S mm o o o o S

SS HS o o o o o S o o o S

SS SS SS SA SS AS 0 SN o o S

S o o o o o o o o o o S

SS SS 0 o S SS o SN 0 o S

SS. 0 o o o o o o o o o S

mm o o SA 0 SS 0 o o S S

SS 0 SA 0 SA SA o o o o o A

S SS NS NS om SS 0 SH 0 o S

AS 0 SSS S SS 0 o S o o o S

S S S S S S S a S a: Hummmmaq

SS Saga SS SASS S Scan AN Scam SA Scan SSSSSS

S

.0: Soup

 

.muHsmmu wcHaamuuH>o .HH oHeme

D XHOzmmm<

Table 11. (cont'd).

 

Trap no.

 

July 18

 

Julygll

 

July 4

 

June 27

 

June 20

 

height
meters

 

 

 

 

 

107

178

10

54

175

43

10

ll

48

63
4O
81

23

76

12

94

17

l7

13

135

88

22

46 82

135

22

14

62

37

15

16

3O
49

33

105

173

32

55

17

30 24 87

28
49

8O

18

27

33 82

105

19

31

72

40

80

35

20

85

83

24

21

25
31

21

50

65

Table 11 (cont'd).

 

Aug. 22

 

15
1:

Aug.

 

Aug; 8

 

End of trapping week
1
t

Aug.

 

July_25

 

 

Trap no.
&

height

meters

 

 

 

 

 

10

48
55

82

116

ll

22

98

19

71

15
67

55

159

132

76

51

55

30

50

46

85
210

41

126

83

76

34

63

48

O
29

49

15

19
29

139

20

14

99

111

52

40

100

27

37

43

43

33

28

10

Table 11 (cont'd).

 

Aug. 22

 

Aug. 15

 

Aug. 8

 

End of trapping week
1
t

Aug.

 

Julyg25

 

 

Trap no.
&

height

meters)

 

 

 

 

 

ll

10

61

3O
89

23

12

33
52

13

25

10

30

58

14

59

30

15

64

15

30

126

69

105

16

93

17

15

50

22

18

94

34

19

15

30
44

99

20

21

35

28 18

179

10

21

32

123

65

 

S A o S o S S o o o S A o S o S SSSASAS-SSSSSSSS .w

 

 

 

 

 

 

 

S S S o S S o o S o S S S A o A SSSSSSA>ASS .m«
S S S o o o o o S S A A o S S A SSSxS> .SS
0 A o S o S o S S o S A o S S o SSSSASSSASS .m«
S o o S o S S o o A S S o S S S ASSSSSSSSS .mfi
mowumam

S S S S S S S S S o S S S S S S ”ASSSSSSV

SSSASS

SA SASS SA SASS SA SASS AA SASS SA SASS SA SASS SA SASS SA SASS "SASS

 

wcHaamHu oAanmoE emuHmeaumm .NH oHemB

m xHozmmm<

66

 

 

 

 

 

H .w:< Hm NHnfi Om NHSH oN NHDH wN AHHAAH. NN NHDH eN AHHan.

 

mooHaHnumam3umou aw

 

mauuouH>Huu (MO

 

moox6> xMfl

mauwHuomHuu .o<

 

 

Hcomuoeao: .o<

 

moHoonm

"Annouosv

SSSASS

«mama

 

.AS.SSoSS AA SASSS

67

ooHuooHHoo .E.o oouoHn: AGOHuooHHoo .E.m ooANne¥

 

 

 

 

 

 

 

 

 

 

 

 

 

S S S S S S o S S S S S o A S o S SSSSASSSSAASSSS .m...»
S o o S S S S o S S S o o S S o S SAASSSSS ...u.
A S S S S S S A o S S A S A S A A SSSASSSSSSSSSSS .w
S S S S S S S S S o S S S S o o o SSSASSASS .3
S o S o o S S o S S S S S S S o S 29.? .MS.
S S A S S S S S S o o o o o o o S SSSSSSAS .3
S S S S S S S A S S S S AA AA S AA AA SSSASSASS sad
0 o A S o S S S o S A S S o o o o SSSSSSAZES .NH.
O S A S S S A o S S A o A A A o A A8338: .md
A A S A A S S o A S A o S S S S S SSSSAASSAAS .SIAA
SSA SSA SSA SSA SAA SAA SSA SSA SSA SSA SAA SAA SAA SAA SSA SSA SSA SSASSSS
Sense CH mama

 

.wcHaamSu OqucmoE eouHmeuwon .MH oHemB

m xHOzmmm<

68

 

S S S S S S S S S S S S S S S S SSASASSSSASSSSS mmw

 

S S S S S S S S S S S S S S S S SAASSSSA aw

 

m o m «H m NH H 0 Ha N OH OH o H w o mcofimfimumamaummh .U

 

 

 

 

 

 

 

 

 

S S S S S S S S S S S S S S S S SSSAASAAS .m<
AA AS SA S S S S S S S S S S S S S SSSxS>..m«
S S S S S S S S S S S S S S S S 3323 .m«
S A S A S S S A S S A S S S S S SSSASeAAS Sufi
S S S S S S S S S S S S S A S S SSSASSA>ASA .m«.
A S A S S S S S S A S A S A S A ASSSSSSSSS .m«
S S S A S A S S S A S S S A S S SSASASSSASS .mfl
SAA SAA SAA SAA SSA SSA SS SS SS SS SS SS SS SS SS SA SSASSSS

 

.NHSH a“ mama

 

.AS.SSSSV SA mASS

69

 

 

 

 

 

 

 

 

 

 

 

S A S S S A S S S S S S S S S S SSASASSSASASSSSS .3
S S S S A A S S S S S A S A S S SAASSSS .m
S AS AA SA A AA S SS A A S AA A A S SA 23393338 .w
S S S S S S S S S A A A S S S S SSSAASASS .3
S S S A A S S S S AS AA SA SA SAA SA AS 823 .3
S S S S S S S S S S S S S S S S SSSSSAS .3
S S A S S A A S S S S A A S S S SSSASASAAS .3
A A A A S A A S S S A S S S S S 33322» .3
A S S S A A S A S S A S A S S A ASoSSmSSSAA .3
S A S S A S S S A S A S A S S S 3323:: .3
SSA SSA SSA SSA SSA SSA SAA SAA SSA SSA SSA SSA SSA SSA SSA SSA SSASSSS

 

Sash GA mama

 

.Av.ucoov ma mfinme

70

 

A S S S S S A A S A S A S S A A SSSSASSSSASSSSS {m3

 

S A A S A A S A S S S S S S S S SAASSSSS Sm

 

N me m o H ON n ¢ NA #N m 0H mH mm HH 0 maowmfimanMSummu .U

 

 

 

 

 

 

 

 

 

S S S S S S S S S A S S S S S S SSSASSASS .md
AA AA SA AA A S S S AA S A A S S S SA SSSxS> {m«
S S S S S S S S S S S S S S S S SSSSSSAS .m«
S S S S S S S A S A A S S A S A SSSASeASS SSS
S A A S AA S S S A S A S A S S A SSASSSA>ASS SSS
S S S S SA A A A S S S S S A SA S ASSSSSSSSS .mfl
S A S SA S A S A A S S A A S AA S SSASASSSASS .m3
SA SA SAS SAS SSS SSS SSA SSA SSA SSA SSA SAA SSA SSA SSA SSA SSASSSS
unawd< SAah GS mama

 

.AS.SSSSS SA SASSS

REFERENCES

Ansari, J. A. 1970. A review of the mechanisms of microfilarial
periodicity. Zool. Anz. 185:387-392.

Barnett, H. C. 1960. The incrimination of arthropods as vectors of
disease. Proc. 11th Inter. Congr. Entomol. 2:341-345.

Bemrick, W. J., and D. M. Bemrick. 1969. Sugar solution feeding
and its effect on the retention of infective Dirofilaria fig-
mitis larvae in Anopheles quadrimaculatus. J. Med. Entomol.
6:278-279.

Benach, J. L., L. G. Cuiston, and J. Azzone. 1971. Preliminary
studies on the blood meal source and olfactory responses of
Aedes triseriatus with notes on the biology of the species.
Proc. N. J. Mosq. Exter. Soc. 58:126-138.

Bickley, W. E., R. 5. Lawrence, G. M. Ward, and R. B. Shillinger.
1977. Dog-to-dog transmission of heartworm by Aedes canaden-
sis. Mosq. News 37:137-138.

Blecka, L. J. 1978. Human subcutaneous dirofilariasis in Illinois.
J. Amer. Med. Assn. 240:245-246.

Breland, O. P. 1960. Restoration of the name, Aedes hendersoni
Cockerell, and its elevation to full specific rank (Diptera:
Culicidae). Ann. Entomol. Soc. Amer. 53:600-606.

Christensen, B. M. 1977. Laboratory studies on the development and
transmission of Dirofilaria immitis by Aedes trivittatus. Mosq.
News 37:367-371.

Church, E. M., J. R. Georgi, and D. S. Robson. 1976. Analysis of
the microfilarial periodicity of Dirofilaria immitis. Cornell
Vet. 66:333-345.

Cockerell, T. D. A. 1918. The mosquitoes of Colorado. J. Econ. En-
tomol. 11:195-200.

Dyar, G. G. 1919. Westward expansion of the Canadian mosquito fauna
(Diptera: Culicidae). Ins. Ins. Mens. 7:11-39.

71

72

Eyles, D. E., C. L. Gibson, F. E. Jones, and M. E. G. Cunningham.
1954. Prevalence of Dirofilaria immitis in Memphis, Tennes-
see. J. Parasitol. 40:216-227.

Fowler, J. L., J. L. Young, R. T. Sterner, and R. C. Fernau. 1973.
Dirofilaria immitis: lack of correlation between numbers of mi-
crofilariae in peripheral blood and mature heartworms. Amer.
Anim. Hosp. Assn. J. 9:391-394.

Furlow, B. M., and W. W. Young. 1970. Larval surveys compared to
ovitrap surveys for detecting Aedes aegypti and Aedes triseri-
atus. Mosq. News 30:468-470.

Garlick, N. L. 1975. The management of canine dirofilariasis. Ca-
nine Practice 2:22-27

Goble, F. C. 1942. Dog heartworm in the muskrat in New York. J.
Mammal. 23:346.

Gorton, R. J. 1973. An epidemiological study of mosquito-borne
California encephalitis in a southern Michigan subdivision.
M. S. thesis, Michigan State University.

Grimstad, P. R., C. E. Garry, and G. R. DeFoliart. 1974. Aedes heg-
dersoni and Aedes triseriatus (Diptera: Culicidae) in Wisconsin:
characterization of larvae, larval hybrids, and comparisons of
adult and hybrid mesoscutal patterns. Ann. Entomol. Soc. Amer.
67:795-804.

Groves, H. F., and F. R. Kutz. 1964. Survey of microfilariae in
Ohio dogs. J. Amer. Vet. Med. Assn. 144:600-602.

Hamilton, D. R., and R. E. Bradley, Sr. 1979. Observations on early
death by Dirofilaria immitis-infected mosquitoes. J. Med. En-
tomol. 15:305-306.

Hanson, R. P., and M. G. Hanson. 1970. The effect of land use prac-
tices on the vector of California encephalitis (La Crosse) in
north central United States. Mosq. News 30:215-221.

Harmston, F. C. 1969. Separation of the females of Aedes hendersoni
Cockerell and Aedes triseriatus (Say) Diptera: Culicidae by the
tarsal claw. Mosq. News 29:490-491.

Hawking, F. 1967. The 24 hour periodicity of microfilariae: biolog-
ical mechanisms responsible for its production and control. Roy.
Soc. (London) Proc., Ser. B. Biol. Sci. 169:59-76.

Ho. B. C., M. Singh, and E. H. Yap. 1974. The fate and migratory pat-
terns of the infective larvae of Brugia malayi, Dirofilaria immi-
tis, and Breinli sergenti in Aedes togoi denied access to a host.
J. Med. Entomol. 11:622-628.

73

Hu, S. M. K. 1931. Studies on host-parasite relationships of Dirofi-
laria immitis Leidy and its culicine intermediate hosts. Amer. J.
Hyg. 14:614-629.

Intermill, R. W. 1973. Development of Dirofilaria immitis in Aedes
triseriatus. Mosq. News 33:176-181.

James, M. T., and R. F. Harwood. 1969. Herm's Medical Entomology.
Macmillan Co., N. Y. 484 pp.

Johnson, C. A. 1975. Ursus americanus (black bear) a new host for
Dirofilaria immitis. J. Parasitol. 61:940.

Jones, J. C. 1967. Methods for dissection of mosquitoes. Mosq. News
27:76-82.

Kartman, L. 1953a. Factors influencing infection of the mosquito
with Dirofilaria immitis (Leidy). Expt. Parasitol. 2:27-78.

Kartman, L. 1953b. An observation on the loss of microfilariae from
the mosquito host during its infective blood meal. J. Parasitol.
39:571-572.

Kazacos, K. R. 1979. The prevalence of heartworms (Dirofilaria im-
mitis) in dogs from Indiana. J. Parasitol. 64:959-960.

Keegan, H. L., C. M. Fitzgerald, T. L. McCrary, and M. D. Doyle.
1968. Studies on dog heartworm in South Texas. Tex. Rep. Biol.
Med. 26:321-330.

Kershaw, W. E., M. M. F. Lavoipierre, and W. N. Beesley. 1955. Stud-
ies on the intake of microfilariae by their insect vectors. VII.
Further observations of the intake of microfilariae of Dirofilaria
immitis by Aedes aegypti in laboratory conditions: the pattern of
the uptake of a group of flies. Ann. Trop. Med. and Parasitol.
49:203-211.

Kocan, A. A., and H. E. Laubach. 1976. Dirofilar;§_immitis and Dipe-
talonema reconditum infections in Oklahoma dogs. J. Amer. Vet.
Med. Assn. 168:419-420.

Kume, S. 1974. EXperimental observations of seasonal periodicity of
microfilariae. lg: Proceedings of the heartworm symposium '74,
pp. 26-31. H. C. Morgan, G. Otto, R. F. Jackson, and W. F. Jack-
son, eds. V. M. Publishing, Inc., Bonner Springs, Kansas.

Kume, S., and S. Itagaki. 1955. On the life cycle of Dirofilaria imp
mitis in the dog as the final host. Brit. Vet. J. 111:16-24.

74

Kutz, F. R., and R. C. Dobson. 1974. Effects of temperature on the de-
velopment of Dirofilaria immitis (Leidy) in Anopheles quadrimacula-
£u§_Say and on vector mortality resulting from this development.
Ann. Entomol. Soc. Amer. 67:325-331.

Leash, A. M., and N. Hanson. 1961. The incidence of heartworm reported
at the Michigan State University Clinic. Mich. State Univ. Vet. 21:
70.

Lewandowski, H. B., Jr. 1977. Determination of the important poten-
tial vectors of dog heartworm in Michigan. Ph. D. dissertation,
Michigan State University.

Loor, K. A., and G. R. DeFoliart. 1969. An oviposition trap for de-
tecting the presence of Aedes triseriatus (Say). Mosq. News 29:
487 ‘488 o

Ludlam, K. W., L. A. Jachowski, and G. F. Otto. 1970. Potential vec-
tors of Dirofilaria immitis. J. Amer. Vet. Med. Assn. 157:1354-
1359.

Lunt, S. R., and G. E. Peters. 1976. Distribution and ecology of
tree-hole mosquitoes along the Missouri and Platte rivers in
Iowa, Nebraska, Colorado, and Wyoming. Mosq. News 36:80-84.

Marquardt, W. D., and W. E. Fabian. 1966. The distribution of 11-
linois filariids of dogs. J. Parasitol. 52:319-322.

Mayr, E. 1963. Animal species and evolution. Harvard Univ. Press.

McDaniel, I. N., and W. R. Horsfall. 1957. Induced copulation of
Aedine mosquitoes. Science 125:745.

McGreevy, P. B., J. H. Theis, M. M. J. Lavoipierre, and J. Clark.
1974. Studies on filariasis. III. Dirofilaria immitis: emer-
gence of infective larvae from the mouthparts of Aedes aegypti.
J. Helminth. 48:221-228.

Morris, C. D., and G. R. DeFoliart. 1971. Parous rates in Wisconsin
mosquito pOpulations. J. Med. Entomol. 8:209-212.

Newton, W. L. 1957. Experimental transmission of dog heartworm,‘2i-
rofilaria immitis by Anopheles guadrimaculatus. J. Parasitol. 43:
589.

Newton, W. L., and W. H. Wright. 1956. The occurrence of a dog fil-
ariid other than Dirofilaria immitis in the United States. J.
Parasitol. 42:246-258.

Otto, G. F. 1969. Geographical distribution, vectors, and life cycle
of Dirofilaria immitis. J. Amer. Vet. Med. Assn. 154:373.

75

Otto, G. F. 1972. Epizootiology of canine heartworm disease. In
Canine heartworm disease: the current knowledge, pp. 1-14. R.
E. Bradley, ed. Univ. Fla. Press, Gainesville.

Phillips, J. H. 1939. Studies on the transmission of Dirofilaria
immitis in Massachusetts. Amer. J. Hyg. 29:121-129.

 

Prouty, D. L. 1972. Canine heartworm disease in southeastern Mich-
igan. J. Amer. Vet. 161:1675-1676.

Rogers, J. S. 1978. Unpublished data.

Sawyer, T. K. 1974. Seasonal fluctuations of microfilariae in two
dogs naturally infected with Dirofilaria immitis. In: Proceed-
ings of the heartworm symposium '74, pp. 23-25. H. C. Morgan,
G. F. Otto, R. F. Jackson, and W. F. Jackson, eds. V. M. Pub-
lishing, Inc., Bonner Springs, Kansas.

Schlotthauer, J. C., E. 6. Harrison, and J. H. Thompson. 1969. Di-
rofilariasis-an emerging zoonosis? Arch. Environ. Hlth. 19:887-
890.

Scholl, M. J., and G. R. DeFoliart. 1977. Aedes triseriatus and
Aedes hendersoni: vertical and temporal distribution as meas-
ured by oviposition. J. Environ. Entomol. 6:355-358.

Shaw, D., Jr. 1976. The determination of the potential vectors of
eastern equine encephalitis in Michigan. M. S. thesis, Michi-
gan State University.

Sinsko, M. J., and P. R. Grimstad. 1977. Habitat separation by dif-
ferential vertical oviposition of two tree-hole Aedes in Indiana.
J. Environ. Entomol. 6:485-487.

Slocombe, J. O. D. 1978. Heartworm in dogs in Canada. Can. Vet. J.
19:244.

Soulsby, E. J. L. 1968. Helminths, arthropods, and protozoa of do-
mesticated animals. London: Bailliere, Tindall, and Cassel.
824 pp.

Streitel, R. H., P. C. Stromberg, and J. P. Dubey. 1977. Prevalence
of Dirofilaria immitis infections in dogs from a humane shelter
in Ohio. J. Amer. Vet. Med. Assn. 170:720-721.

Taylor, A. E. R. 1960. The development of Dirofilaria immitis in
the mosquito Aedes aegypti. J. Helminth. 34:27-38.

Truman, J. W., and G. B. Craig. 1968. Hybridization between Aedes
hendersoni and Aedes triseriatus. Ann. Entomol. Soc. Amer. 61:
1020-1025

 

76

Watts, D. M., P. R. Grimstad, G. R. DeFoliart, and T. M. Yuill. 1975.
Aedes hendersoni: Failure of laboratory-infected mosquitoes to
transmit La Crosse virus (California encephalitis group). J. Med.
Entomol. 12:451-453.

Weinmann, C. J., and R. Garcia. 1974. Canine heartworm in California,
with observations on Aedes sierrensis as a potential vector. Ca-
lif. Vector Views 21:45-50.

Williams, J. F., and A. W. Dade. 1976. Dirofilaria immitis infection
in a wolverine. J. Parasitol. 62:174-175.

Worley, D. E. 1964. Helminth parasites of dogs in southeastern Mich-
igan. J. Amer. Vet. Med. Assn. 144:42-46.

Wright, R. E., and G. R. DeFoliart. 1970. Association of Wisconsin
mosquitoes and vertebrate hosts. Ann. Entomol. Soc. Amer. 63:777-
786.

Zaim, M. 1978. Impact of wastewater irrigation on the population of
the tree hole breeding mosquito, Aedes triseriatus (Diptera: Cu-
licidae). Ph. D. dissertation, Michigan State University.

Zaim, M., H. B. Lewandowski, H. D. Newson, and G. R. Hooper. 1977.
Differentiation of Aedes triseriatus and Ag. hendersoni (Dip-
tera: Culicidae) based on the surface of the egg shell. J. Med.
Entomol. 14:489-490.

Zavortink, T. J. 1972. Mosquito studies (Diptera: Culicidae) XXVII.
The New World species formerly placed in Aedes (Finlaya). Con-
trib. Amer. Entomol. Inst. 8:1-206.

Zydeck, F. A., I. Chowdkowski, and R. R. Bennett. 1970. Incidence
of microfilariasis in dogs in Detroit, Michigan. J. Amer. Vet.
Med. Assn. 156:890-891.