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This is to certify that the

dissertation entitled

THE INFLUENCE OF NITROGEN
ON THE FEEDING STRATEGIES OF
HERBIVORES AND DETRITIVORES

presented by

Daniel L. Lawson

has been accepted towards fulfillment
of the requirements for

Ph-D- degree in mm

 

4:2 {6 fig /{/ ”@2%

Major professor

 

Date 2/25/83

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

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RETURNING MATERIALS:
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THE INFLUENCE OF NITROGEN ON THE
FEEDING STRATEGIES OF

HERBIVORES AND DETRITIVORES

by

Daniel Lee Lawson

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Entomology

I983

ABSTRACT

THE INFLUENCE OF NITROGEN ON THE
FEEDING STRATEGIES OF
HERBIVORES AND DETRITIVORES
by

Daniel L. Lawson

The feeding strategies of the herbivores Alsophila @metaria (Harr) (Geo-

 

metridae) and Anisota senatoria (J. E. Smith) (Citheroniidae) and the detritivores

 

Tipula abdominalis (Say) (Tipulidae) and Aedes triseriatus (Say) (Culicidae) were

 

 

contrasted in relation to dietary nitrogen concentration. Larval _A_. pometaria
and Ag. senatoria (J. E. Smith) (Citheroniidae) were reared on oak leaf species
which differed in tannin and nitrogen concentrations. Larvae were unaffected by
the tannin concentration of the diet but were most influenced by leaf nitrogen
concentration. A. Emetaria consumed each species of oak foliage at similar
rates but ECI of high nitrogen leaves were greater. This resulted in growth rates
that were directly related to leaf nitrogen concentration. Nitrogen consumption
and accumulation were also related to leaf nitrogen content. _A_rl. senatoria
grew at the same rate on most leaf species. This was a result of high
consumption and reduced ECI on low nitrogen leaves, and promoted uniform
rates of nitrogen consumption, utilization and accumulation on most leaf species.
A. Emetaria grew faster rate than £3. senatoria as a result of greater

consumption rate rather than enhanced digestive efficiency. It is suggested that

Daniel L. Lawson

the appearance ot early season herbivores is in response to the presence of the
high nitrogen concentration of immature foliage and not the avoidance of
tannins.

Detritivore studies examined the contribution of microbially derived nitro-

gen to insect growth. Larval _'_I'_. abdominalis and _A_. triseriatus were reared on lab

 

conditioned leaves which were inoculated with pure fungal strains and cultured in

15 N incorporated by

the presence of 15N lablelled nitrate. The percentage of
the insect was used to estimate the importance of microbially derived nitrogen
for detritivore growth. Results indicated that from 2.443.296 of the nitrogen of

conditioned leaf litter was of microbial origin. 1. abdominalis assimilated

 

61.2% of the microbial nitrogen which was not significantly different from leaf
NUE or total NUE. It was estimated that microbial nitrogen contributed 15.7%

of I. abdominalis and 1-596 of A. triseriatus nitrogen gains. Microbial biomass

 

was not a major component of leaf litter and contributed little to the nitrogen
nutrition of the detritivores examined.

Herbivores and detritivores utilized a variety of mechanisms to obtain
nitrogen for growth. Digestively, they maintain high gut pH to extract nitrogen
formerly bound with tannins. Consuming leaves with the highest nitrogen
content or nutritional quality and efficiently utilizing the nitrogen present in

foliage or leaf litter are all adaptive strategies to acquire dietary nitrogen.

ACKNOWLEDGMENTS

I would like to thank J. Martin, University of Michigan, for the tannin
assays, and C. Klug, R. Snider and G. Walker for fungal cultures and nitrogen
analyses. I would also like to thank Drs. E. Grafius and J. Miller for guidance
throughout my graduate program. Special thanks are extended to Drs. R. W.
Merritt and M. J. Klug for the excellent research opportunity and the physical

and moral support.

TABLE OF CONTENTS

List of Tables ...................................................... iv
List of Figures ..................................................... v
Introduction ....................................................... I

Chapter 1 The influence of nitrogen and tannin on the
feeding ecology of two larval lepidopterans,
Alsophila pometaria (Geometridae) and Anisota
senatoria (Citheroniidae) ................................. 3

 

Chapter 2 The contribution of microbial nitrogen to the
growth of the detritivores, Tipula abdominalis
(Tipulidae) and Aedes triseriatus (Culicidae) ................. 37

 

Synopsis .......................................................... 59

Appendix The utilization of late season foliage by the
orange striped oakworm, Anisota senatoria (J.
E. Smith) (Citheroniidae) ................................. 6i}

 

List of References ................................................. ‘ 79

Table 1.1.

Table 1.2.

Table 1.3.

Table 1.11.

Table 2.1.

Table A.l.

Table A.2.

LIST OF TABLES

Leaf chemical characteristics among early (May 27)
and late (September 10) season foliage. ---------------------

Indices of A. pometaria larval growth on different
oak leaf species ooooooooooooooooooooooooooooooooooooooooo

Indices of An. senatoria larval growth on different
oak leaf species .........................................

Correlation coefficients between nitrogen and indices
of larval growth of A. mmetaria and A_n. senatoria. ----------

Growth statistics for I. abdominalis and A.

 

triseriatus fed 9. glabra leaves. -_ --------------------------

Indices of [31. senatoria larval growth on different
oak leaf species. ........................................

Leaf chemical characteristics of six oak leaf species. ........

IO

l2

I3

Ill

’46

70
71

Figure 1.1.

Figure 1.2.

Figure 1.3.

Figure 1.1!.

Figure 1.5.

Figure 1.6.

Figure 1.7.

Figure 1.8.

Figure 2.1.

Figure 2.2.

LIST OF FIGURES

Relationship between leaf nitrogen concentration and
the relative growth rate (RGR) of A. Emetaria and
An. senatoria. ..........................................

Relationship between leaf nitrogen concentration and
the relative consumption rate (RCR) of A. pometaria
and A2. senatoria. .......................................

Relationship between leaf nitrogen concentration and
the efficiency of conversion of ingested food (ECI) of
A. pometaria and An. senatoria. ...........................

Relationship between leaf nitrogen concentration and
the approximate digestibility (AD) of A. pometaria
and _A_n. senatoria, .......................................

Relationship between leaf nitrogen concentration and
the efficiency of conversion of ingested food (ECD)
of A. pometaria and AA. senatoria. .........................

Relationship between leaf nitrogen concentration and
the relative nitrogen consumption rate (RNCR) of A.
pometaria and _A_n. senatoria. .............................

Relationship between leaf nitrogen concentration and
the nitrogen utilization efficiency (N UE) of A.
Rometaria and Ag. senatoria. ..............................

Relationship between leaf nitrogen concentration and
the relative nitrogen accumulation rate (RNAR) of A.
Bometaria and Ag, senatoria, ..............................

Schematic representation of the associations between
stream microorganisms and detritivores, and pools of
organic nitrogen (ie., coarse particulate organic
material) and inorganic nitrogen. --------------------------

General methods employed in feeding experiment
which include leaf culture, isotope incorporation in
microbial biomass and partitioning the contribution
of microbial and leaf nitrogen to animal growth. .............

15

I7

19

2l

23

25

27

29

lIl

lI7

LIST OF FIGURES, continued

Figure 2.3. Comparisons of I. abdominalis leaf nitrogen utiliza-
tion efficiency, microbial nitrogen utilization effi-
ciency and total nitrogen utilization efficiency. .............

Figure 2.4. Changes in percenta e leaf 15 N and percentage
larval A. triseriatus 1 N as a function of days into
the feeding experiment. A. triseriatus 35 day values
are segregated by instar. .................................

 

Figure 2.5. Simulated changes in A. triseriatus 15 N concentra-
tions, if 0-1096 of the insect's dietary nitrogen is
derived from microbial biomass, as a larva grows
from lst instar (ca. 1 u N) to 3rd instar (_c_a. 25 ug
N). A. triseriatus 1 N concentrations from the
feeding experiment are depicted (*) to estimate the
contribution of microbial N to insect growth. ................

Figure A.l. The relationship between leaf nitro en concentration
(96) and approximate digestibility a), efficiency of
conversion of ingested food (b), efficiency of conver-
sion of digested food (c), relative consumption rate
(d), and relative growth rate (e) of A_n. senatoria and

E. rapae. ...............................................

vi

50

52

5h

72

INTRODUCTION

Deciduous leaves represent the principal organic resource for a diverse
assemblage of insect herbivores and detritivores in both terrestrial and aquatic
habitats. Terrestrial herbivores are known to defoliate large forested areas, yet
as a whole consume only a small portion of the seasonal leaf production (Baker
1972). The remaining foliage enters terrestrial and aquatic systems as leaf
litter. Microbial colonization followed by detritivore feeding together serve to
decompose these organic inputs and recycle nutrients. Although invertebrate
herbivores and detritivores consume a small percentage of annual leaf produc-
tion, their primary role is as regulators of energy and nutrients between trophic
levels (Mattson 1977; Cummins and Klug 1979). Despite the importance of
herbivores and detritivores in terrestrial and aquatic ecosystems, little is known
of the factors regulating the feeding of insects, and therefore, the flow of
energy and nutrients.

An insect's diet, whether foliage or decomposing leaf litter, must provide
the proper nutritional requirements for growth. A qualitative survey of larval
dietary requirements shows a close similarity among most insects (House 1969;
Dadd 1973). Carbohydrates form the the major energy source while protein,
essential amino acids and fatty acids, steroids, vitamins and minerals are all
required for basic metabolic processes. However, specific nutritional require-
ments may vary and knowledge of exact proportions are generally lacking (Dadd
1973). Among the dietary needs of insects, the requirement for protein is the
most critical as nitrogen is a major component of insect structural tissues,

required for metabolic functions and genetic replication. Therefore, nitrogen

has received considerable attention in studies of insect feeding and is considered
as the primary limitation to insect growth (McNeill and Southwood 1978; White
1978; Cummins and Klug 1979; Mattson 1980).

Elemental nitrogen comprises 79% of the earth 5 atmosphere, yet the
majority of organisms can only utilize other inorganic and organic forms of
nitrogen and these are in short supply. Deciduous trees utilize inorganic nitrogen
(i.e. nitrate), which when presented in excess, results in increased plant
production (Larcher 1980). The evolution of nitrogen-fixing. microorganisms and
their symbiotic associations with plants further attests to the limited quantity of
available nitrogen (Bonner and Varner 1976). Since nitrogen is potentially
limiting to plants as well as insects, deciduous trees evolved mechanisms to
reduce nitrogen losses, which have significant impact on the acquisition of
organic nitrogen by herbivore and detritivores. Plant secondary compounds (i.e.
tannins), synthesized by deciduous trees, are postulated to complex with leaf
proteins and insect digestive enzymes, thereby reducing herbivore digestion (Van
Sumere et al. 1975; Swain 1979). The quantity of nitrogen also varies seasonally
(Feeny 1970; Mattson 1980). Foliage high in nitrogen is available for a brief
period in the spring, and during the remainder of the season foliage low in
nitrogen predominates. In autumn, leaf nitrogen is translocated from senescent
foliage and retained within the tree (Kramer and Kozlowski 1979). Leaf litter
nitrogen concentrations are thus very low and only after microbial colonization
occurs and leaf litter nitrogen concentration increases, do detritivores begin to
feed (Cummins and Klug 1979). Therefore, it is hypothesized that the feeding of
herbivores and detritivores is directed for the acquisition of dietary nitrogen. It
was the objective of this study to examine and contrast the feeding strategies of

selected herbivores and detritivores in relation to dietary nitrogen.

CHAPTER I

The Influence of Nitrogen and Tannin on the
Feeding Ecology of Two Larval Lepidopterans,

Alsophila pometaria (Geometridae) and

 

Anisota senatoria (Citheroniidae)

 

ABSTRACT

Larval Alsophila pometaria (Harr) (Geometridae) and Anisota senatoria (J.

 

 

E. Smith) (Citheroniidae) were reared on oak leaf species which differed in
tannin and nitrogen concentrations. Larvae were unaffected by the tannin
concentration of the diet but were most influenced by leaf nitrogen
concentration. A. mmetaria consumed each species of oak foliage at similar
rates but ECI of high nitrogen leaves were greater. This resulted in growth rates
that were directly related to leaf nitrogen concentration. Nitrogen consumption
and accumulation were also related to leaf nitrogen content. fl. senatoria grew
at the same rate on most leaf species. This was a result of high consumption and
reduced ECI on low nitrogen leaves, and promoted uniform rates of nitrogen
consumption, utilization and accumulation on most leaf species. A. Emetaria
grew faster rate than _A_n_. senatoria as a result of greater consumption rate
rather than enhanced digestive efficiency. It is suggested that the appearance at
early season spring feeders is in response to the presence of the high nitrogen

concentration of immature foliage and not the avoidance of tannins.

INTRODUCTION

Seasonal variations in the quantity and quality of foliar nitrogen has
received considerable attention in studies of insect herbivore consumption,
utilization and growth (McNeill and Southwood 1978, Mattson 1980). Early
season (spring) deciduous leaves characteristically contain high concentrations of
nitrogen and provide highly assimilable sources of protein (Feeny 1970; Slansky
and Feeny 1976; Scriber and Feeny 1979). A subsequent reduction in leaf
nitrogen concentration in summer and fall, coupled with the seasonal increase in
plant secondary compounds (i.e., tannins) further reduces the availability of
nitrogen as the summer progresses (Feeny 1969, 1970; Bate-Smith 1971). Early
season feeders are therefore thought to benefit from the greater quantity and
quality of foliar nitrogen, resulting in higher rates of assimilation and growth
than late season foliage feeders (Feeny 1970, Slansky and Feeny 1976). Despite
the widespread acceptance, studies have yet to validate this view by comparing
early and late season herbivores on the same tree Species. The purpose of the
present study was to constrast the consumption, utilization and growth of an
early season herbivore, Alsophila Lometaria, and a late season feeder, Anisota
senatoria, fed on a variety of oak species containing different concentrations of

leaf nitrogen and tannin.

METHODS AND MATERIALS

Eggs of the fall cankerworm, A. Emetariaz and second instars of the
orange striped oakworm, fl. senatoria, were collected from the field on 6 May
1980 and 30 August 1979, respectively, and reared at 24C under a 15/9 and 12/12

photophase/scotophase, respectively. Larvae were fed red oak (Quercus rubra

 

L.) until the penultimate instar; whereupon, larvae were randomly divided into
six groups and fed one of the following six species of leaves: white oak (Q. alba

L.), swamp-white oak (Q. bicolor Willd.), bur oak (Q. macrocarpa Michx.), pin oak

 

(Q. palustris Huenchh.), red oak (9. 21.1232): black oak (Q. velutina Lami).
However, due to an undetermined nutrient defficiency in spring pin oak, only five
spring oak species were examined. Ten freshly molted #th instar A. wmetaria
and twenty freshly molted 5th instar An. senatoria from each leaf species were
transferred individually to 0A7 l waxed paper cups with a hole punched in the
bottom. The petiole of a freshly harvested leaf was placed through the hole and
the cup placed within another cup filled with 20 ml distilled water to provide a
continuous water supply to the leaf. Larval and leaf fresh weights were
determined and then converted to dry weight (Waldbauer 1968). Fresh leaves

were added and feces removed at 24 hr intervals.

Chemical Assays

Sample Preparation: After excision of the midrib, leaves were lyophilized
and ground to 250 um in a Wiley mill prior to assay.

Tannins: Approximately 6 mg of leaf was extracted in 2.0 ml of 50%
methanol at 80 C for 10 min. Following centrifugation (12,000 x g, ‘1 C, 20 min),
the supernatant was collected. The pellet was resuspended in 10 ml of 50%
methanol, centrifuged as above, the supernatant collected and combined with the
original extract. Aliquats of this extract were used in the following assays.

Total phenols were measured using the Folin-Denis procedure (Swain and
Hillis 1959; Ribereau-Gayon 1972) with Tannic acid (Sigma) serving as a

reference standard. Proanthocyanidins were assayed using the method of Hillis

and Swain (1959) with an 8096 butanol-hydrochloric acid reagent containing
15.1496 (w/v) ferrous sulfate (Govandarajan and Mathew 1965). Protein precipita-
tion was estimated by the bovine serum albumin (GSA) precipitation assay
(Martin and Martin 1982).

Total N: Total nitrogen was determined on subsamples of each leaf species
and larvae in each treatment at the onset of the experiment, and on each larvae
at the conclusion of the experiment. Larvae and leaves were prepared as above,
weighed and analyzed for nitrogen on a model 1102 Carlo-Erba elemental

analyzer.

Growth Indices

The following indices of consumption, utilization and growth were calcu-
lated utilizing the procedures detailed by Waldbauer (1968), and are reported as
dry wt. Mean larval wt. is defined as the average of the sum of larval initial and
final dry weights.
Relative Consumption Rate:

RCR (mg/(mg/day)) = food ingested per unit mean larval biomass per day

Relative Growth Rate:

RGR (mg/(mg/day)) = biomass gained per unit mean larval biomass per day

= (RC R)(ECI)

Approximate Digestibility:

AD (96) = 100 (food ingested-feces)/food ingested

Efficiency of Conversion of Digested Food:

ECD (96) = 100 (biomass gained)/(food ingested-feces)

Efficiency of Conversion of Ingested Food:

ECI (96) = 100 (biomass gained)/food ingested = (AD)(ECD)/ 100

Relative Nitrogen Consumption Rate:
RNCR (ug/(mg/day)) = biomass nitrogen ingested per unit mean larval

biomass per day

Relative Nitrogen Accumulation Rate:
RNAR (ug/(mg/day)) =biomass nitrogen gained per unit mean larval

biomass per day

Nitrogen Utilization Efficiency:

NUE (96) = 100 (biomass nitrogen gained)/nitrogen ingested

Statistics

A Pearson Correlation procedure related the above indices to concentra-
tions of leaf nitrogen, total phenol, proanthocyandin and BSA activity to
investigate the effects of leaf chemistry on A. pometaria and A11. senatoria
consumption, utilization and growth. Mean differences were derived from

standard ANOVA procedures and a posteriori contrasts (Gill 1978).
RESULTS

The Folin-Denis assay showed that spring and fall foliage of each species
had comparable total phenol concentrations except for spring bur and black oak,
which had higher phenolic content than late season leaves (Table 1.1). In

contrast, proanthocyanidin assays of condensed tannins revealed higher concen-

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trations in most late season oak leaves (Table 1.1). Recent studies by Martin and
Martin (1982) questioned the use of the above assays in light of the development
of more meaningful measures of tannin concentration i.e. protein binding
potential (Goldstein and Swain 1965; Schultz et a1. 1981; Hagerman and Butler
1978). Therefore, leaf extracts from identical leaves utilized in the Folin-Denis
and proanthocyanidin assays in this study were measured for their capacity to
precipitate bovine serum albumin (BSA) (Table 1.1). Since leaf samples were
stored for an extensive period prior to assay (Spring, 7 months; Fall 8 months)
absolute values of BSA are unreliable. However, results provided an indication of
the protein binding potential of the ingested leaves (Martin and Martin 1982). In
all cases spring and fall oak foliage had similar protein binding potential which
indicated that leaves were equally defended by tannins.

Results of the feeding experiments for A. pometaria and An. senatoria are
presented in Tables 1.2 and 1.3, respectively. The influence of leaf nitrogen
concentration on the aforementioned indices are presented in Figures 1.1 to 1.8
and summarized as correlation coefficients in Table 1.1!.

A. pometaria larvae grew at a rate (RGR) that was positively correlated
with leaf nitrogen concentration. Larvae consumed leaves (RCR) at approxi-
mately the same rate, but the high nitrogen leaves were more efficiently
assimilated (ECI) and promoted greater growth. This is reflected in the positive
correlation of leaf nitrogen content with ECI; although leaf nitrogen was not
correlated with AD or ECD. An. senatoria consumption rates (RCR) were
negatively correlated with leaf nitrogen concentration as larvae consumed low
nitrogen leaves at a faster rate than high nitrogen leaves. However, larval ECI
and AD were positively correlated with leaf nitrogen concentration. This

resulted in equal growth rates on most leaf species.

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Table 1J5. Correlation coefficients between nitrogen and indices
of larval growth of A. ametaria and fl. senatoria.

 

 

A. pometaria fl. senatoria

n=5 n=6
RCR -0.30 -0.92**
RGR 0.82* 0.51
ECI 0.85* 0.92**
ECD -0.05 0.30
AD 0.50 0.72*
RNCR 0.81 * 0.60
RNAR 0.91 * 0A9
NUE 0.30 0.37

 

*P < 0.05.
**P < 0.01.

15

Figure 1.1 Relationship between leaf nitrogen concentration and the re-
lative growth rate (RGR) of A: pometaria and Ag: senatoria.

 

RGR

0.25

0.40

0.35

ALLLIllLllPLLLLlLlllllLll

0.30

0.20

 

16

 

N
O
CD

2.5

T f I

I r T r

sin
% NITROGEN

Y

17

Figure 1.2 Relationship between leaf nitrogen concentration and the
relative consumption rate (RCR) of 5: pometaria and 52:
senatoria.

RCR

3.5

2.5 3.0
l L L l i 1 L L L L 1 L L L l l L l L |

2-0

 

l8

EDAmaenQchig +
3KA122msn1smg

'8-
£-
@-

 

T 1 l 1 U r

ale
% NITROGEN

19

Figure 1.3. Relationship between leaf nitrogen concentration and the
efficiency of conversion of ingested food (ECI) of 5: P07
metaria and fig, senatoria.

20

 

 

o...
N .

ED Amss’mismg
4 *6 AW
.4
E)“
l 41

m it +

2* i l 1
. ll!
.1
J

I T T r I r T T V T 1 1'— r r FT I Ij
2.0 2.5 3.0 3.5 4.0

% NITROGEN

21

Figure 1.h. Relationship between leaf nitrogen concentration and the ap-
proximate digestibility (AD) of A: pometaria and 52: sen-
atoria.

 

22

 

n
T
T
lxl .
1
Iii r
r
T
fl
um: T
x... m
T
I
O I
mam. m f
mm r
m; m .
“AJdddddelddlqlqlqddldel-ldd‘l1d

 

om cm 0. on on
o<

% NITROGEN

23

Figure 1.5. Relationship between leaf nitrogen concentration and the ef-
ficiency of conversion of ingested food (ECD) of L pometaria
and All; senatoria.

211

S

hm.a 1v.
m*+

1

.eji~r .
3.0
% NITROGEN

T

r T T T
2.5

T

17

 

«JJJ Jddld deJ 4+ld1- *de
_ a A a 4

8 8 S on ow
00m

 

2.0

25

Figure 1.6. Relationship between leaf nitrogen concentration and the re-
lative nitrogen consumption rate (RNCR) of A. pometaria and
fig. senatoria.

26

 

1

r

r

.

lxll. .

T

+ .

a

.

laT T

.m .

llllllll m r

. r

fl.

.

. .

“m1“ mun .

EX m r
1....Jllq.u..ll.44..

 

ow“ no“ on em
mozm

73 NITROGEN

27

Figure 1.7. Relationship between leaf nitrogen concentration and the
nitrogen utilization efficiency (NUE) of A. pometaria and
51. senatoria.

 

28

 

 

% NITROGEN

u)—
(D
4m .ggngg°
1*22m201fi
3.3.?
«l
.1
cl
'3'? ‘I’¢ lIJ
J
. Ill
wi'l’ {-
j 'I
.. ,-.r.r.fi. r. .j
2.0 2.5 3.0 3.5 4.0

29

Figure 1.8. Relationship between leaf nitrogen concentration and the re-
lative nitrogen accumulation rate (RNAR) of A. pometaria and
An. senatoria.

 

30

4-0

 

 

T

IT: T
5
TI 0
3

+ r

r
1.... TO.
3

m T

+ '

m T

T
T5
2

T

MA .m. T

.m: .m T

EX T
nw

—TI 4 4 Id 4 J I! I4 I—

S on 8 2

73 NITROGEN

31

Uniform consumption rates by A. pometaria on leaves which varied in
nitrogen concentration (Table 1.1), resulted in nitrogen consumption rates
(RNCR) which were directly related to the nitrogen concentration of the diet.
Nitrogen utilization efficiency (NUE) reflected no major differences among leaf
species, and combined with RNCR resulted in nitrogen accumulaltion rates
(RNAR) that were positively correlated with leaf nitrogen content. In contrast,
_Fm. senatoria nitrogen indices were not correlated with leaf nitrogen concentra-
tion. Larval consumption rate (RCR) which compensated for leaves low in
nitrogen, resulted in uniform rates of nitrogen consumption (RNCR), utilization
(N U13) and accumulation (RNAR) on most leaf species.

_f_\_. pometaria and fl. senatoria dry weight and nitrogen indices were
contrasted utilizing covariance analysis with leaf nitrogen concentration as a
cofactor. In all cases, leaf nitrogen explained significant (p < 0.05) variation in
the dependent variable and was employed throughout the analysis. Results
revealed that A. wmetaria consumption (RCR), assimilation (AD, ECI) and
growth (RGR) were all significantly greater (p < 0.05) than fl. senatoria.
However ECI's differed by less than 0.0896. Only the ECD of Ag. senatoria was
signficantly higher (p . 0.05) than A. pometaria. Nitrogen indices demonstrated
a similar trend. A. pometaria nitrogen consumption (RNCR) and accumulation
(RNAR) were signficantly greater (p < 0.05) than _A_n_. senatoria. However, 53.

senatoria NUE was significantly greater (p < 0.05) than that of _5. pometaria.

DISCUSSION

Leaf Tannins as Defensive Compounds

Leaf tannins are postulated to deter herbivory by complexing with leaf
proteins (Goldstein and Swain 1965; Van Sumere et a1. 1975) particularly in
mature foliage which is thought to contain the highest seasonal concentration
(Feeny 1970). In this study, total phenol and condensed tannins demonstrated
seasonal distributions similar to those described by Feeny (1970). Spring leaves
had high phenolic concentration while mature leaves were higher in condensed
tannins. In Feeny's study, leaves collected only 12 days apart elicted two-fold
differences in winter-moth pupal weight. This was attributed to the presence of
condensed tannins in more mature leaves (Feeny 1970). Martin and Martin (1982)
found that total phenol and proanthocyanidin assays do not adequately quantify
the capacity of leaves to bind with protein. Spring foliage in this study had
lower condensed tannin than mature leaves but significant protein binding was
evident. This would indicate that early season oak leaves are as well protected
chemically as more mature leaves in these oak species. These results add one
more example to a growing list of plant species in which young leaves have high
levels of tannins (Bate-Smith 1973; Rhoades and Cates 1976; Fox and Macauley
I977; Waterman et al. 1980; Becker and Martin 1982).

Leaf tannins have been described as quantitative secondary defense com-
pounds which act as dose-response inhibitors of herbivore digestion (Feeny 1976).
Among the indices of insect assimilation, larval NUE is the most sensitive
parameter of the inhibition of protein digestion. Yet, in this study there was no

significant correlation between a leaf's tannin concentration, as measured by

33

FoIin-Denis, proanthocyanidin, or BSA and the resulting NUE's of A. pgmetaria
or A_n. senatoria. As these insects were exposed to similar foliar tannin
concentrations, they were equally adapted to the protein binding capacities of
their diets. These data conflict with Feeny (1970, 1976), but conform to an
emerging view of tannins as potential, but not insurmountable, barriers to
efficient protein digestion (Fox and Macauley 1977; Bernays 1981; Lawson et al.

1982).

Leaf Nitrogen and Insect Herbivory

Leaf nitrogen concentration is often recognized as a primary nutritional
factor in insect feeding studies (McNeill and Southwood 1978; Mattson 1980). In
this investigation, leaf nitrogen concentration also had a major impact on larval
dry weight and nitrogen indices; although the influence of nitrogen was mediated
by larval consumption rate. A. Emetaria consumed all foliage at consistent
rates, regardless of nitrogen concentration. In contrast, An. senatoria adjusted
consumption rate in relation to foliar nitrogen concentration, as larvae presum-
ably compensated for the low nitrogen content of the leaves by increasing
consumption rate (McGinnis and Kasting 1967; Slansky and Feeny 1977; Lawson
et a1. 1982). Larval assimilation (ECI) of both insect species was greater on high
nitrogen foliage. Therefore, the different patterns of growth displayed by A.
pometaria and Ag. senatoria were due to disparities in consumption rate rather
than utilization. A. pometaria consumed foliage at a constant rate and grew at a
rate directly related to leaf nitrogen concentration. In the same way, uniform
consumption promoted greater nitrogen consumption rates and nitrogen accumu-

lation rates on high nitrogen leaves. M. senatoria utilized compensatory

31+

consumption to grow at equal rates on most leaf species. This enabled larvae to
stabilize rates of nitrogen consumption, utilization and accumulation. The
stabilization of nitrogen accumultion rates has been shown to be an effective
mechanism by which larvae obtain sufficient nitrogen for growth, independent of

leaf nitrogen concentration (Slansky and Feeny 1977).

Insect Herbivory and Foliar Seasonality

The differences in growth rate between early and late season feeders has
been ascribed to the low tannin contents and high nitrogen concentrations of
spring foliage (Feeny 1970; McNeill and Southwood 1978) However as previously
discussed, tannin content of spring foliage approximates late summer leaves and
thus represent equally defended diets. Foliar nitrogen concentration had a
greater influence on larval growth as high nitrogen spring foliage supported
higher growth (RGR) than low nitrogen, late season leaves.

A reduction in larval assimilation efficiency is associated with low leaf
nitrogen concentration (Mattson 1980; Lawson et a1 1982) and is thus thought to
contribute to the lower RGR's of late season herbivores which feed on nitrogen
poor diets (Feeny 1970, 1976; Rhoades and Cates 1976). Results from this study
do not conform to this hypothesis. The assimilation efficiencies (ECI) of A.
Emetaria and Ag. senatoria differed by less than 0.0896, and indicated that late
season leaves were as efficiently utilized (ECI) as early season foliage. This
result was consistent with Lawson et al (1982) when Ag. sentoria was contrasted
with a non-tannin feeder, but contrary to conventional views of late season

herbivory and digestive efficiency (Feeny 1970, 1976; Scriber and Feeny 1977).

35

A. pometaria consumed oak foliage at a higher rate than Ag. senatoria.
The higher consumption combined with nearly equal ECI, as discussed, resulted in
the differences in growth found between A. mmetaria and An, senatoria. In
constrast to earlier findings (Feeny 1970; Scriber and Feeny 1979), the primary
contribution to higher growth rates of an early season feeder in this study was
the high consumption rate rather than enhanced digestive efficiency. High
consumption rate has been shown to be a characteristic feeding strategy of
spring feeders (Slansky and Feeny 1977; Scriber and Feeny 1977) but not without
possible rate limitations imposed by reduced digestive efficiency.

The major disparities in consumption rate between early and late season
feeders also had a major influence on nitrogen consumption, utilization and
accumulation. A. Emetaria had the highest consumption rate, and fed on spring
foliage with the seasonally highest nitrogen concentration. This enabled A.
pgmetaria to ingest nitrogen at a consistently higher rate than An, senatoria.
The minor differences in nitrogen utilization were compensated by the greater
nitrogen consumption by A. pgmetaria which resulted in a higher relative

nitrogen accumulation rate than An. senatoria.

CONCLUSIONS

The process of leaf maturation from budbreak to senescence induce
alterations in leaf chemistry and tannin concentrations which may utimately
affect the feeding strategies of the associated insect fauna. The polyphagous A.
pometaria, which hatch at budbreak, complete larval development by early June
(Mitter et al. 1979), and are exposed to major fluctuations in nitrogen and tannin

(Feeny 1970) within and between leaf species. In this study, all foliage of spring

36

oak species had high protein binding capacitites by late May and therefore it is
reasonable to predict that A. Emetaria is exposed to tannins throughout much of
its developmental period. Thus, it is not suprising to find a spring feeder such as
A. mmetaria or late season feeders adapted to the presence of tannins in their
diets (Berenbaum 1980; Bernays 1981; Lawson et a1. 1982).

A. pomentaria does not utilize or possess the capacity to adjust consump-

 

tion rate to compensate for low nitrogen diets. Larvae consume foliage at equal
rates and grow at a rate directly related to leaf nitrogen concentration.
Therefore, the appearance of larvae early in spring is likely an adaptation to
feed on high nitrogen foliage rather than the avoidance of tannins. Ar_1. senatoria
employs an alternate strategy and actively feeds on low nitrogen foliage in late
summer and early fall (Lawson et a1. 1982). Consumption rates are much lower
than its early season analog, but larvae adjust consumption to compensate for
low leaf nitrogen concentration. The differences in consumption rate may be a
reflection of the greater nutritive value of high nitrogen spring foliage. Early
season feeders can consume foliage at a higher rate, and yet maintain digestive
efficiency. Late season feeders must utilize lower consumption rates (i.e. high
gut residence time) to maximize digestion of low nitrogen foliage. This is
demonstrated by the nearly equal rates of dry weight assimilation with large
disparities in consumption between insects. Early season feeders appear adapted
to grow at maximum rates dependent on leaf nitrogen concentration, while late

season feeders maximize digestive efficiency by regulating rate of food intake.

CHAPTER 2

37

The Contribution of Microbial Nitrogen
to the Growth of the Detritivores,

Tipula abdominalis (Tipulidae) and

 

Aedes triseriatus (Culicidae)

 

ABSTRACT

Larval Tipula abdominalis (Say) (Tipulidae) and Aedes triseriatus (Say)

 

 

(Culicidae) were reared on lab conditioned leaves which were inoculated with

15N labelled nitrate. The

pure fungal strains and cultured in the presence of
percentage of 15N incorporated by the insect was used to estimate the
importance of microbially derived nitrogen for detritivore growth. Results

indicated that from 2.443.296 of the nitrogen of conditioned leaf litter was of

microbial origin. 1. abdominalis assimilated 61.2% of the microbial nitrogen

 

which was not significantly different from leaf NUE or total NUE. It was
estimated that microbial nitrogen contributed 15.7% of I. abdominalis and 1-5%
of A. triseriatus nitrogen gains. Microbial biomass was not a major component
of leaf litter and contributed little to the nitrogen nutrition of the detritivores

examined.

INTRODUCTION

The degradation of autumn-shed leaf litter in stream systems involves a
complex array of abiological and biological events mediated by aquatic insects and
microorganisms (Fig. 2.1). Fresh leaf litter is not readily consumed by detritivores
but requires days to weeks of conditioning before a leaf is acceptable to stream
insects (Kaushik and Hynes 1971). The initial stages of conditioning involve the
leaching of soluble organics and inorganics which amount to a 5-30% loss in initial
leaf dry wt. (Kaushik and Hynes 1971; Cummins et al. 1973; Peterson and Cummins
1974). Subsequent microbial colonization results in a physically softened and
chemically modified leaf which is more readily consumed and more efficiently
digested than less colonized leaves (Triska 1970; Barlocher and Kendrick l973a,
l973b, I979; Cummins et al. 1973; Peterson and Cummins 1974). However, it is not
yet known whether the feeding preferences demonstrated for microbially colonized
leaves are based on the nutritive value of the leaf or the associated microbial
biomass.

Detritivores are unable to digest the major structural carbohydrates of leaf
litter (Bjarnov I972; Nielson I962, 1963; Martin et al. 1980, 1981a, 1981b). Stream
insects are therefore thought to benefit nutritionally from the microbial biomass
associated with the leaf, particularly as a source of available nitrogen which is
limited in aquatic systems (Newell I965; Fenchel I970; Levington and Lopez 1977;
Ward and Cummins 1979; Cammen 1980, Rice 1982). However, microbial biomass is
generally considered to be a minor component of the diet (Iverson 1973; Baker and
Bradnam 1976; Cummins and Klug 1979), and was recently demonstrated to be a
minor source of nitrogen in a marine detritivore (Findlay and Tenore 1982). No

studies have yet directly estimated the contribution of microbial nitrogen to

140

Figure 2.1.

#1

Schematic representation of the associations between stream
microorganisms and detritivores, and pools of organic nitro-
gen (i.e., coarse particulate organic material) and inorganic
nitrogen.

 

 

112

 

 

TERRESTRIAL I
/\_I
ORGANIC
N
POOL STREAM

   

 

 

      
   
   

 

INORGANIC
N
POOL

4‘ LEACHING _>

 

 

 

 

 

 

 

 

 

 

 

 

 

DECOMPOSITION

113

stream detritivore growth. The purpose of this study is to estimate and constrast
the importance of microbially derived nitrogen to the stream shredder, Tipula

abdominalis (Say), and the browsing tree hole mosquito, Aedes triseriatus (Say).

 

 

METHODS

Leaf Culture

Pignut hickory (Carya glabra) leaves were collected in the fall in above-

 

ground traps, air dried, and stored. Leaves were rehydrated and cut into 2 cm leaf
discs with a cork borer. Discs were weighed in 0.5 g groups (ca. 20 discs) and
sterilized by ethylene oxide and in later experiments by gamma radiation. Each
group of discs was added to a 500 ml flask containing 125 m1 of sterilized media

containing 5 mM KHZPO 3 mM NaCl, 2 mM MgSO 0.5 mM CaC122(HZO) and 20

4’ 4’
mM MOPS buffer (Sigma) and adjusted to pH 7.5. Inorganic nitrogen was added in

the form of stable isotope as 10 mM 2.5 atom 96 K15

studies and 1 mM 25 atom96 KUNO

 

NO3 in the I. abdominalis

3 in the A. triseriatus experiment. Flasks were

inoculated with the aquatic hyphomycete Tetracladium marchialianum DeWild, and

 

incubated for 10 days at 18 C in a reciprocating water-bath shaker.
Larval Feeding

‘_I'_. abdominalis: Nineteen lIth instars were individually reared at 10 C on the

 

above leaves for 14 days in plexiglass feeding chambers that insured constant
current flow. Larval and leaf fresh weights were determined and then converted to
dry weights (Waldbauer 1968). Leaves were replaced on the 7th day with freshly
cultured leaves. Leaf and larval total nitrogen was determined in a Carlo-Erba

elemental analyzer, while 15N concentrations were determined by a Micro Mass

model 622 mass spectrometer after reduction to ammonia by micro-Kjeldahl

digestion.

M

A. triseriatus: Twelve groups, six 4th instars each, were reared for 25 days
at 18 C in glass scintillation vials with 250 um mesh in the bottom to preclude
reingestion of feces. Larval and leaf initial dry wt. was estimated as above and
fresh leaves replaced at 5-6 day intervals. These procedures yielded consumption,
utilization and growth data based on total dry wt. and nitrogen. An alternate mass
rearing experiment was required to assess the contribution of labelled nitrogen to
this insect's growth. Approximately 2000 freshly hatched lst instars were reared
as above on 15 N labelled leaves wih subsamples of leaves and larvae collected
periodically. Larvae were reared until ca. 5096 attained the 3rd instar and then
were sorted by instar. These larvae and the above subsamples were assayed for
15N as described previously.

Growth Indices

The following indices of consumption, utilization, and growth were calculated
using the procedures detailed by Waldbauer (1968), and are reported as dry wt.
Mean larval wt. is defined as the average of the sum of larval initial and final dry
weights.

Relative Consumption Rate:

RCR (mg/mg/day) = food ingested per unit mean larval biomass per day
Relative Growth Rate:

RGR (mg/mg/day) = biomass gained per unit mean larval biomass per day
Efficiency of Conversion of Ingested Food:

ECI (96) = 100 (biomass gained)/food ingested
Relative Nitrogen Consumption Rate:

RNCR (ug/mg/day) = biomass nitrogen ingested
per unit mean larval biomass per day

Relative Nitrogen Accumulation Rate:

l+5

RNAR (ug/mg/day) = biomass nitrogen gained
per unit mean larval biomass per day

Nitrogen Utilization Efficiency:
NUE (96) = 100 (biomass nitrogen gained)/nitrogen ingested
Additionally, leaf and mirobial nitrogen utilization was partioned in the I.

abdominalis feeding experiment, and are calculated as follows:

 

Leaf Nitrogen Utilization Efficiency:
Leaf N UE (96) = 100 (leaf nitrogen gained)/(leaf nitrogen ingested)
Microbial Nitrogen Utilization Efficiency:

(15

Microbial NUE (96) = 100 N nitrogen gained)/(15 N nitrogen ingested)

Statistics
Mean differences were derived from standard ANOVA procedures and a
posteriori contrasts (Gill 1978). Signficant differences were determined at the 95%

confidence level.

RESULTS

1. abdominalis and A. triseriatus consumed (RCR) leaf litter at approxmi-

mately the same rate (Table 2.1). However, I. abdominalis grew (RGR) faster due

 

to greater assimilation (ECI) of the ingested diet. The more highly assimilated

leaves of I. abdominalis had slightly higher nitrogen concentration (1.14% i

 

0.015E) than the leaves utilized by A. triseriatus (0.94% i 0.015E). Nitrogen
concentrations may reflect levels of microbial conditioning and therefore, differ-
ences in the nutritional quality of leaves between experiments. The reduced

assimilation (ECI) by A. triseriatus is further reflected in generally lower nitrogen

46

Table 2.1. Growth statistics for I. abdominalis and A. triseriatus fed 9. glabra
leaves. Values are SE (SE).

 

 

 

 

RCR RGR ECI RNCR RNAR NUE
(mg/(mg/day» (96) (ug/(mg/day)) (96)
(ug/(mg/day»
I. abdominalis .268 .020* 8.3* 3.043 1.899 65.7*
(.029) (.002) (0.9) (0.324) (0.232) (6.1)
A. triseriatus .273 .010 3.6 2.573 1.036 39.4
(.027) (.002) (0.5) (0.257) (0.182) (5.1)

 

*Values are significantly different (p < 0.05) (Students T-test).

47

Figure 2.2. General methods employed in feeding experiment which include
leaf culture, isotope incorporation in microbial biomass and

partitioning the contribution of microbial and leaf nitrogen
to animal growth.

48

 

 

 

 

 

 

FUNGI gNfiBrgAN'C
PT (“M
V V

 

LEAF LITTER

 

 

MICROBIAL N LEAF N
I=I-c-‘5NI-I2 R-C-NH2
L J

T

‘XaMICROBIAL N « ANIMAL UPTAKE H ‘Xo LEAF N

l+9

consumption (RNCR), nitrogen utilization (NUE) and nitrogen accumulation rate
(RNAR)

The colonization of leaves by '_l'_. marchialianum in the I. abdominalis

 

 

experiment incorporated 15 N labelled nitrogen within the leaves as microbial
biomass and metabolic by-products (Fig. 2.2). Microbially derived nitrogen
accounted for 13.2% i 0.35E of total leaf nitrogen. The assimilation of microbial
nitrogen into larval tissue (Fig. 2.3) approximated that of leaf derived nitrogen and

indicated that I. abdominalis did not differentially assimilate from either nitrogen

 

source. Leaf and microbial NUE's were thus not significantly different from total

NUE. The conversion of microbial nitrogen to I. abdominalis biomass nitrogen

 

contributed 15.7% i 2.75SE of the nitrogen gained by the animal.

Results of the A. triseriatus feeding experiment revealed that colonized
leaves had less l5N label incorporated than the previous experiment. Nitrogen of
microbial origin amounted to 2.4% i: 0.8SE of total leaf nitrogen. This could be
attributed to lower microbial colonization of these leaves, as reflected by reduced

nitrogen values compared to the _‘I_'_. abdominalis experiment. Patterns of A.

 

triseriatus 15 N incorporation over time (Fig. 2.4) demonstrated that larval concen-
trations closely followed leaf concentrations, even when leaf levels rose in the last
2 batches. This indicated that larvae were not selectively utilizing microbially

15

derived nitrogen over nitrogen of leaf origin. If this were not the case larval N

content would change rapidly over time. Figure 2.5 describes simulated changes in

animal 15

N concentration if larvae were to utilize 0 to 10% of their nitrogen gains
from microbially derived nitrogen as a larva grows from lst instar (ca. 1 ug N) to
3rd instar (ca. 25 U3 N). AS shown, larval 15N concentrations would change rapidly

and approach final concentrations within the first 5 ug of nitrogen gained by the

50

.mm H x Ocm mo:_m> .>oco_o_:o c032
I23: comet: 7.30.82: .>oco_o_to coCmNZZJ comet: “F.0— mIOEEOEO .H “To mcomCquOu

 

.m.~ ot:m_u

U1
-4

 

 

 

 

 

 

 

 

 

 

 

 

FTTfiTT

0'08

0'09

Tfi‘Tlejj

0‘0?
(x) 3m

0.

1
OZ

Tfi T—T

 

TOTAL

MICROBIAL

52

 

.Lmumc_ >3 tOummOLmOm 0cm mo:_m> >mn mm mzum_com_cu .<

 

m>mv mo co_uocae m mm 2 maum_LOmmcu am _m>cm_ OmmucooLOa new 2

m_

.ucoe_coaxo m:_v00m OLu oucm

mmo_ OmmucooLoa c_ momcmcu

m_

.s.~ ot:m_u

m><o

 

 

 

mm on mm ow m“ a: m o
—IbbbpbbbpLhPPbI—FFI-PI—BbIIrLbbbbbebbb
T
a
T0
D\8 To
9
E T.
T
a TI
.10
.05 T
#1.
Tm:
En an movo< B T
92 .63 x T
a

 

D'Z

SIN %

Figure 2.5.

54

Simulated changes in A: triseriatus 15N concentrations, if

 

0-10% of the insect's dietary nitrogen is derived from micro-

bial biomass, as a larva grows from lst instar (ca. 1 ug N)

15

to 3rd instar (ca. 25 ug N). A, triseriatus N concentra-

 

tions from the feeding experiment are depicted (*) to esti-

mate the contribution of microbial N to insect growth.

% ANIMAL N15

 

55

 

 

 

 

   

 

 

 

ug ANIMAL NITROGEN

9
m"!
1
I 10%
.1
.1
C:
N
d *
‘ x
J
0
0—1
'I
I 4%
1 0%
Di
0. T—rTTIT'TrTIFlI—Y’rrfirrit 1
0.0 5.0 10.0 15.0 20.0 25.0

56

insect. When experimental A. triseriatus 15 N concentrations are superimposed on
this figure, it can be estimated that larvae assimilated approximately 1-5% of their

nitrogen from microbial sources.

DISCUSSION

Nitrogen is recognized as a limiting nutrient in aquatic systems, but for
detritivores, not only the quantity but the quality of available nitrogen may be
limiting (Suberkropp et a1. 1976; Odum et al. 1979; Rice 1982). Nitrogen
concentrations in leaf litter are low in comparison to fresh foliage (Mattson 1980)
and much of the protein is complexed with polyphenols and lignin (Suberkropp et al.
1976; Odum et a1. 1979). This is reflected in the low assimilation efficiencies and

rates of growth of I. abdominalis and A. triseriatus is this study, and for other

 

detritivores which have been examined (Cummins et al. 1973; Iverson 1974;
Barlocher and Kendrick 1975; Berrie 1976; Cummins and Klug 1979). Iverson (1974)
contrasted leaf species with different nitrogen contents and observed that leaves
higher in nitrogen were more digestible and supported greater growth. Leaf
nitrogen concentration also varies within a leaf species as a function of condition-
ing time (Kaushik and Hynes 1968; Iverson 1973; Triska et al. 1975; Suberkropp et
a1. 1976). The more conditioned, high nitrogen hickory leaves in this study were

more efficiently assimilated and produced greater growth rates in I. abdominalis.

 

However, Suberkropp et a1. (1976) suggested that as leaf decompostion proceeds
beyond an optimal period, leaf nitrogen may become bound to lignin and made less
available to detritivores. This was thought to contribute to reduced nitrogen

utilization, assimilation efficiency and growth of ‘_1'_. abdominalis when fed leaves

 

conditioned for more than 5 weeks (Klug unpublished).

S7

The preference demonstrated by stream detritivores for leaves well colonized
by microorganisms has received considerable attention (Kaushik and Hynes 1968,
1971; Barlocher and Kendrick 1973a, l973b; Cummins et al. 1973; Peterson and
Cummins 1974; Cummins and Klug 1979). However, microbial colonization is
closely correlated with changes in the chemical composition of leaf litter during
decompostion. The relative percentages of leaf cellulose and hemicellulose change
little with time, while lipids decrease and total nitrogen and lignin increase with
decompostion (Suberkropp et al. 1976). Thus, demonstration of cause and effect
relationships between detritivore feeding preferences and/or leaf chemical compo-
sition is indeed difficult (Cummins and Klug 1979).

Microbial biomass is thought to provide a primary source of nutrition for
detritivores (Newell 1965; Fenchel 1970; Hargrave 1976; Levington and Lopez 1977;
Cammen 1980), because microbial biomass is readily assimilated (42-97%) (Har-
grave 1976) and shown to be preferred over leaf litter (Barlocher and Kendrick
1973a, l973b). However, in this study microbial nitrogen accounted for at most
13% of leaf nitrogen and constituted a minor percentage of a detritivore's diet. I.
abdominalis did not discriminate between nitrogen sources as nitrogen of leaf and
microbial origin were equally assimilated (Fig. 2.1). As a result the contribution of

microbial nitrogen to the growth of I. abdominalis and A. triseriatus was related

 

to the level of microbial biomass in the leaf. Leaves with 2.4 and 13.296 microbial
nitrogen contributed respectively, ca. 1-5 and 15.7% of the nitrogen gained by the
insects. Differences in feeding mode between insects had no impact on the results.
A. triseriatus, a browser, was observed to reduce densities of surface colonized
fungi (Fish and Carpenter 1982), and perhaps more exclusively utilize microbial

biomass. The percentage of microbial nitrogen incorporated was similar to the

58

shredder I. abdominalis, considering the levels of microbial nitrogen present in

 

each insects diet.

In conclusion, microorganisms associated with leaf litter apparently do not
provide enough biomass for detritivores to explain the selective feeding demon-
strated for well colonized leaves. Microbial nitrogen is not a major component of
leaf litter and contributes a small percentage to the nitrogen nutriton of stream
detritivores (Iverson 1973; Baker and Bradnam 1976; Cummins and Klug 1979;
Findlay and Tenore 1982). Perhaps microorganisms predigest leaf material in such
a way that protein becomes more digestible with time (Suberkropp and Klug 1980).
The microbes themselves may provide enzymes utilized by detritivores for their
own digestive needs, critical growth factors or nutrtional requirements. Further
studies are required in these areas to elucidate the role of microorganisms to

stream detritivore growth.

SYNOPSIS

Nitrogen exists in inorganic and organic "pools", some of which are in
forms readily utilizable by plants and animals. Microorganisms exploit nearly all
forms of nitrogen, and are largely responsible for the tranformations of nitrogen
between the various pools. For continued growth, plants are dependent on
inorganic nitrate made available from the microbial conversion of other
inorganic forms of nitrogen and the decomposition and recycling of nutrients
from organic material (Bonner and Varner 1976). Insects are incapable of
directly assimilating inorganic N, and exploit the capabilities of plants and
microorganisms to synthesize organic nitrogen (eg. protein, amino acids). All
forms of organic nitrogen are not readily utilizable, but may be complexed with
inhibitors that reduce digestive efficiency (Van Sumere et al. 1975; Swain 1979).
Secondary plant compounds, most notably tannins, are thought to act as
herbivore defenses by binding with leaf protein and insect digestive enzymes at
neutral or acidic pH's. Chemically related lignins and humic acids act in a
similar manner by forming complexes with proteins and carbohydrates during
decompostion (Swain 1979). Therefore, the temporal variations in nitrogen
abundance and concentrations of digestive inhibitors are of particular
significance to herbivore and detritivore strategies to obtain nitrogen.

The concentration Of nitrogen in deciduous foliage varies seasonally as a
function of the growth phase of the tree (Feeny 1970; Kramer and Kozlowski
1979; Mattson 1980). Actively growing spring foliage has the highest nitrogen
concentration and by early summer, the leaves mature and nitrogen

concentrations are reduced (Feeny 1970). Leaf nitrogen concentrations remain

59

60

at a lowered level until autumn when protein is translocated away from
senescent foliage. Freshly fallen leaf litter concentrations are thus 20-40% of
spring nitrogen contents (Whittaker et al. 1979). The concetration of tannins, as
shown in this study, are high by mid to late spring and remain so for the
remainder of the season. In stream systems, it is recognized that leaf litter
nitrogen content increases with time in relation to microbial colonization (Triska
et al. 1975; Suberkropp et al. 1976). Tannins are present in fresh leaf litter, but
Suberkropp et a1. (1976) found a rapid reduction in leaf tannins within the first 2
weeks exposure in the stream. However, the F Olin-Denis assay was used in these
experiments and thus may require further study (Martin and Martin 1982). During
the latter stages of decomposition protein becomes bound to lignin or lignin-like
compounds, potentially reducing nitrogen availability to detritivores in the latter
stages Of decomposition (Suberkropp et al. 1976).

The occurrence of tannins in the diets of herbivores and detritivores
appears to have led to the development of counteradaptations by insects to
exploit pools of "bound" nitrogen. Evidence from this study demonstrated no
significant tannin effect on herbivore nitrogen utilization efficiencies. In search
of possible digestive adaptations, Martin (unpubl. studies) surveyed the digestive
capabilities of the herbivores A. pometaria and Ag. senatoria. A common
feature was the presence of high midgut pH (ca. 9.0-10.5) and a highly active,
alkaline stable proteinase. Tannin protein complexes are known to destabilize
under alkaline conditions and would thus provide these insects a mechanism to

exploit protein-bound with tannin. The detritivores, I. abdominalis and A.

 

triseriatus, and other detritivores examined, possess similar midgut enzymology

and high pH and are thought to utilize identical digestive strategies (Dadd 1975;

61

Martin et al. 1980, 1981a, 1981b). The capacity for detritivores to utilize
protein-lignin or protein-humic complexes appears limited (Swain 1979; Rice
1982) as evidenced by the slow turnover and accumulation of humic substances in
terrestrial soils. The binding of leaf litter protein with lignin and/or lignin-like
substances was thought to contribute to the poor utilization of nitrogen by I.

abdominalis fed leave conditioned for more than 7 weeks in the stream (M. J.

 

Klug unpubl. studies). Therefore, herbivore and detritivores apparently can
utilize high gut pH (Berenbaum 1980) and other digestive mechanims (Bernays
1981) to assimilate nitrogen from protein-tannin complexes, but not from lignin
or humic complexes.

The timing of insect feeding is an important strategy utilized by herbivores
for the acquisition of nitrogen (Feeny 1970; Mattson 1980). The herbivores, A.
pometaria and the closely related winter moth, Operophtera brumata, examined
by Feeny (1970) take advantage of foliage with the highest nitrogen
concentration by hatching at budbreak in the spring. The successful timing of
lst instar hatch with leaf flush is a major determinant of larval survival (Feeny
1970; Mitter 1979). In the case of A. ometaria, larvae consume foliage at a
maximum rate regardless of leaf nitrogen concentration. The assimilation of
leaves with high nitrogen concentration is greater, and larvae grow at a rate
directly related to leaf nitrogen content. As A. Egmetaria is adapted to tannins,
the feeding strategy of this early season feeder, and perhaps others, is to appear
early in the spring, consume foliage maximally and complete development before
the early summer reduction of leaf nitrogen concentration.

The late season herbivore An. senatoria utilizes an alternate strategy Since

leaf nitrogen levels are lower in late season foliage, a premium is placed on the

62

efficient utilization of nitrogen. Larvae compensate for low leaf nitrogen
concentration by consuming more foliage. Even though low nitrogen leaves are
less efficiently assimilated, high consumption compensates for low assimilation,
and larvae grow at the same rate regardless of leaf nitrogen content. In
comparison to an early season feeder, Ag. senatoria consumes foliage at a lower
rate to maximize assimilation as demonstrated by similar dry weight and higher
nitrogen utilization efficiencies than A. pometaria. Thus, late season feeders
grow at a slower rate, but may benefit from an extended feeding season. Foliage
of similar dietary quality is available for ca. 2 months for M. senatoria versus
ca. 1 month for early season feeders. The extended developmental period might
increase risks of parasitism or predation. However, the bright orange and blank
striped coloration and gregarious habits of A_n. senatoria may be a warning of low
palatability, and be a mechanism to reduce predation.

Feeding selectively is an adaptive strategy particularly well developed by
detritivores, which enable larvae to discriminate among leaf litter differing in
food quality (Barlocher and Kendrick l973a, 1973b, 1979; Cummins et al. 1973;
Cummins and Klug 1979). The basis of this selection is unknown, however
evidence by Motyka (unpublished) suggests that random trial and contact
chemoreception are involved. Leaf species which are highest in nitrogen
concentration are more highly consumed and support greater growth than low
nitrogen leaves (Iverson 1974; Anderson and Grafius 1975). Preference is also
demonstrated, within a leaf species, for leaves which are maximally colonized by
microorganisms (Iverson 1973; Triska et al. 1975, Klug unpublished) Yet, as
demonstrated in this study, the preference is not based on a nutritional

dependency for microbially derived nitrogen. The nitrogen of leaves well

63

colonized by microorganisms is more readily assimilated than less colonized
leaves, and is likely a result of enzymatic predigestion of the leaf by microor-
ganisms (Suberkropp and Klug 1980; Klug unpubl. studies).

Therefore, herbivores and detritivores utilize a variety of mechanisms to
obtain nitrogen for growth. Digestively, they maintain high gut pH to extract
nitrogen formerly bound with tannins. Consuming leaves with the highest
nitrogen content or nutritional quality and efficiently utilizing the nitrogen
present in foliage or leaf litter are all adaptive strategies used by insects to
acquire dietary nitrogen. An additional feature, not examined in this study, was
the presence of microorganisms associated with detritivore digestive tracts
(Meitz 1975; Klug and Kotarski 1980). There appears to be on the potential for
gut symbionts to supply or recycle nitrogen for detritivore growth. Studies are
underway in these areas and may provide valuable insights to the nutritional

ecology of insects.

APPENDIX

THE UTILIZATION OF LATE SEASON FOLIAGE BY THE
ORANGE STRIPED OAKWORM, Anisota senatoria

(J. E. Smith) (Citheroniidae)

D. L. Lawsonl, R. W. Merrittl, M. J. Klugz, J. S. Martin3

1. Department of Entomology, Michigan State University, East Lansing, MI
48824 USA

2. Kellogg Biological Station, Michigan State University, Hickory Corners, Ml
49060 USA

3. Division of Biological Science, University of Michigan, Ann Arbor, MI
48109 USA

64

ABSTRACT

Larval Anisota senatoria (J. E. Smith) (Citheroniidae) were reared on 6 oak
leaf species which differed in concentrations of leaf water, nitrogen and tannin.
Larval growth did not differ significantly among most leaf species while relative
consumption rate and efficiency of conversion of ingested food were
significantly correlated with leaf nitrogen concentration. Alterations in relative
consumption rate in relation to the nitrogen concentration of the diet maintained
uniform relative nitrogen consumption rates, nitrogen utilization efficiencies and
relative nitrogen accumulations rates on nearly all leaf species consumed. Leaf
water and tannin concentrations among the leaf species examined did not affect

larval A. senatoria consumption, utilization or growth.

INTRODUCTION

Herbivory has been discussed in relation to the nutrient content of the leaf
(Mattson I980): principally leaf moisture (Scriber 1977) and leaf nitrogen (Feeny
1970); Slansky 6c Feeny 1977; Fox 6c Macauley 1977). These factors vary
seasonally (Feeny 1970; Mattson 1980) with high concentrations of water and
nitrogen in spring foliage sustaining greater herbivore diversity than low
nutrient, late season foliage (Feeny 1970). Secondary plant compounds synthe-
sized during leaf development (i.e., tannins) are postulated to further inhibit late
season herbivory through the formation of indigestible complexes with protein
(Feeny 1969; Bate-Smith 19710. Although many insects are known to utilize late
season leaves (Baker 1972), the mechanisms involved in their ability to assimilate
the diet are not fully understood. The objective of this study was to determine
the consumption, utilization and growth of a late season herbivore, Anisota
senatoria, fed on a variety of oak species which contained differing concentra-

tions of leaf water, nitrogen and tannin.

METHODS AND MATERIALS

Collection and Rearing. Eggs of the orange striped oakworm, A. senatoria
normally hatch in late July and complete larval development by early September.
Second instars were collected from the field on 30 August 1979 from Quercus
Mg L. and reared at 24° under a 12/ 12 hr photophase/scotophase. Larvae were
fed red oak (9. M) foliage from the second to the onset of the fourth instar;
whereupon, they were randomly divided into 6 groups and fed one of the
following 6 leaf species; white oak (Q. 3133 L.), swamp-white oak (Q. biocolor

Willd.), burr oak (Q. macrocatfi Michx.), pin oak (Q. pallustris Huenchh.), red

66

67

oak (Q. Luge), black oak (Q. veluntina Lami). Twenty freshly molted fifth
instars reared on each leaf species were transferred individually to 0.47 l waxed
paper cups with a hole punched in the bottom. The petiole of a freshly harvested
leaf was placed through the hole and the cup was placed within another cup filled
with 20 ml of distilled water to provide a continuous water supply to the leaf.
Larval and leaf fresh weight were determined and then converted to dry weights
(Walbauer 1968). Fresh leaves were added and feces removed at 24 hr intervals.

Chemical AssaJS. After excision of the midrib, leaves were lyophilized and
R

 

ground to 250 microns in a Wiley mill before assaying.

Approximately 6 mg of leaf was extracted in 2.0 ml of 50% methanol at
80° for 10 min. Following centrifugation (12,000 x g, 4°, 20 min), the
SUpernatant was collected. The pellet was resuspended in 10 ml of 50%
methanol, centrifuged as above, the supernatant collected and combined with the
original extract. Aliquots of these extracts were used in the phenol and
proanthocyanidin assay.

Total phenols were measured using the Folin-Denis procedure (Swain 6c
Hillis 1959; Ribereau-Gayon 1972). Tannic acid (Sigma) was used as a reference
standard. Proanthocyanidins were assayed using the method of Hillis 6c Swain
(1959) with an 80% butanol-hydrochloric acid reagent containing 15.4% (w/v)
ferrous sulfate (Govandarajan 6c Mathew 1965).

Initially, total nitrogen was determined on subsamples of each leaf species
and larvae from each experimental treatment. At the conclusion of the
experiment, nitrogen concentration was determined on each remaining larva.
Larvae and leaves were prepared prior to assay as decribed above, weighed and

analyzed for nitrogen on a model 1102 Carlo-Erba' elemental analyzer.

68

Growth Indices. The following indices of consumption, utilization and

 

growth were calculated utilizing the procedures detailed by Waldbauer (1968),
and are reported as dry weight. Mean larval biomass is defined as the average of
the sum of larval initial and final dry weights.
Relative Consumption Rate: RCR (mg/mg/day) 2 food ingested per unit mean
larval biomass per day
Relative Growth Rate: RGR (mg/mg/day) = biomass gained per unit mean larval
biomass per day
Approximate Digestibility: AD (96) = 100 (food ingested -feces)/food ingested
Efficiency of Conversion of Digested Food: ECD (96) = 100 (biomass gained)/food
ingested - feces)
Efficiency of conversion of Ingested Food: ECI (96) = 100 (biomass gained)/food
ingested
Relative Nitrogen Consumption Rate: RNCR (ug/mg/day) = biomass nitrogen
ingested per unit mean larval biomass per day
Relative Nitrogen Accumulation Rate: RNAR (ug/mg/day) = biomass nitrogen
gained per unit mean larval biomass per day
Nitrogen Utilization Efficiency: NUE (96) = 100 (biomass nitrogen gained/nitro-
gen ingested

Statistics. To investigate the effects of leaf chemistry on the consump-
tion, utilization and growth of A. senatoria, the above indices were related to
concentrations of leaf nitrogen, water, proanthocyanidin and total phenol by a
Pearson Correlation procedure. Mean differences were derived from standard

ANOVA precedures

69

RESULTS

The RGR of larval A. senatoria did not differ significantly among most leaf
species (Table A.1), despite the observed differences in leaf water, nitrogen,
proanthocyanidin and total phenol concentrations among different leaf species
(Table A.2). RCR, ECI, and AD, however, varied significantly among the 6 leaf
species (Table A.1), in relation to the leaf nitrogen concentration of the diet.
RCR was negatively correlated with leaf nitrogen concentration (r = -0.92, p is
less than 0.050) (Fig. A.1d). Leaves with low nitrogen concentration were
consumed at a greater rate than leaves with high nitrogen concentration. Leaf
nitrogen was positively correlated with ECI (r = 0.92, p is less than 0.05) (Fig.
A.1b) and AD (r = 0.72, p is less than 0.052) (Fig. A.1a) indicating increased
larval assimilation of leaves with high nitrogen concentration. ECD varied
between leaf species (Table A.1) and was not correlated with any of the leaf
chemical parameters measured in this study (Table A.2).

In contrast to dry weight consumption and utilization, NUE demonstrated
little variation among leaf species (Table A.1). The uniform RNCR's and N UE's
were due to the differential RCR displayed on leaves differing in nitrogen
content (Fig. A.1), and resulted in similar nitrogen accumulation rates (RNAR)
on most leaf species (Table A.1). The low RGR of larvae fed white oak and black

oak is most likely a result of the low NUE and RNAR on these leaf species.

DISCUSSION

Foliar water concentration has been demonstrated to affect the digestive
efficiency of herbivores (Scriber 1977); yet, in this study, leaf water content

varied proportionally little between leaf species and was not shown to reduce

70

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Table A.2. Leaf chemical characteristics of six oak leaf species.
Values are X t SE.*

 

 

Water Nitrogen Total Phenols Proanthocyanidins
(96) (96) (ug TAE/mg (abs units AASSO/
dry wt) mg Dry Wt)
White oak 51.5b 2.26a 175.d 0.07oa
(0.8) (0.11) (2.) (0 . 001)
Swamp- 45.93 2.82b 125.b 0.1102f
white oak (0.9) (0.09) (2.) (0.0011)
Bur oak 43.43 2.92b 161.C 0.300e
(0.5) (0.12) (1.) (0.004)
Pin oak 54.6b 2.27a 162.C 0.167C
(0.6) (0.10) (1. ) (0.002)
Red oak 52.2b 2.22a 9235‘l 0.235d
(0.2) (0.09) (5.) (0.003)
Black oak 52.0b 2.11al 13.7.b 0.128b
(0.8) (0.09) (5.) (0.003)

 

*Values followed by different letters differ significantly (p < 0.05)

(Student-Newman-Keuls procedure).

Figure A.l.

72

The relationship between leaf nitrogen concentration (96) and
approximate digestibility (a), efficiency of conversion of
ingested food (b), efficiency of conversion of digested food (c),

relative consumption rate (d), and relative growth rate (e) of Q.

senatoria (*) and _E. rapae ( ) (E. rapae data recalculated from

Slansky and Feeny 1977).

 

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711

digestion (i.e., AD, ECD, ECI). This does not imply that increased leaf water
concentration would not improve larval digestion efficiency; however, variations
in larval growth indices among the leaf species examined could not be attributed
to slight differences in leaf water content.

Leaf tannins have been implicated in reducing the digestibility of ingested
leaves (Feeny 1969, 1970; Rhoades 1979). In this study indices of leaf tannin
concentration (i.e., total phenols and proanthocyanidins) bear no relation to the
leaf's subsequent efficiency of utilization by A. senatoria. Martin dc Martin
(1982) have questioned the use of total phenol and proanthocyanidin assays as
indicators of potential digestibility reduction by ingested tannins and suggest
more direct measures of protein binding. Assays of protein binding capacity
(bovine serum albumin precipitated) on leaf samples from this study (Martin 6:
Martin 1982) again demonstrated no correlation with A. senatoria consumption,
utilization or growth. Burr oak leaves with high total phenol and proanthocy-
anidin concentrations (Table A.2) and high protein binding capacity (Martin 6:
Martin 1982) were more highly assimilated (i.e., AD, ECD, ECI) than leaves with
lower tannin concentrations (Tables A.l, A.2). The results are in agreement with
Fox dc Macauley (1977) and consistent with an emerging view of species which
are adapted to the presence of tannins when encountered as a normal dietary
constituent (Bernays 1981).

The importance of nitrogen to the consumption, utilization, and growth of
insect larvae is emphasized by the results of this study and those of Slansky 6c
Feeny (1977) and Fox 6t Macauley (1977). Larvae of A. senatoria and M
gag (Slansky 6c Feeny 1977) maintained a constant RGR on diets varying in

nitrogen concentration by adjusting their RCR. Associated with an increased

75

RCR on low nitrogen diets was a characteristic reduction in ECI. This could be
attributed to restricted digestion due to a lowered gut residence time (Slansky dc
Feeny 1977), and/or from lower concentrations of assimilable protein in leaves
low in nitrogen (Schroeder & Malmer 1980). Thus it follows that there exists a
lower limit to the nitrogen concentration of a plant which will maintain larval
growth. Fox 6: Macauley (1977) reported that Paropsis atomaria, when fed
leaves less than 0.996 nitrogen, lost weight and excreted more nitrogen than they
assimilated. However, 2. atomaria did not exhibit any changes in RCR observed
with A. senatoria and E. m. In contrast, larvae of E. atomaria maintained
uniform consumption rates on most leaves, regardless of nitrogen concentration,
but ECI and growth rate were positively associated with leaf nitrogen content.

Nitrogen concentration varies among leaves of a given species due to
natural variability and foliar age (Sampson dc Samisch 1935; Feeny 1970). As
leaves mature, non-nitrogenous cell wall materials disproportionately increase,
thus reducing the relative percentage of nitrogen (Tromp 1970). Gravid females
of A. senatoria oviposit on branch tips providing first instars with foliage that is
younger and higher in nitrogen content. As development proceeds, gregarious
larvae feed on more mature leaves as they move 93 m from the branch tip
inward, defoliating all leaves of various age classes. Alterations in consumption
rate aid larvae in maintaining growth rate on the nutritionally variable food
sources, insuring conspecific develOpmental synchrony.

Increasing concentrations of secondary compounds produced during decidu-
ous leaf development have also been implicated to complex with leaf protein and
herbivore digestive enzymes, thereby reducing digestive efficiency (Feeny 1969;

Goldstein dc Swain 1965). Although the majority of herbivorous species feed on

76

leaves low in tannins, many do feed on late season foliage (Feeny 1970; Baker
1972). High levels of proanthocyanidin and polyphenol concentrations and high
protein binding capacity (Martin 6: Martin 1982) had no effect on the assimilation
of NUE of A. senatoria fed Quercus in this study. The uninhibited assimilation of
nitrogen (NUE) in the presence of tannins is indicative of an adaptation by the
animal to metabolize high tannin leaves.

Alkaline gut pH has been suggested as an adaptation of herbivores to obtain
protein from diets high in tannins (Feeny 1970; Fox 61 Macauley 1977; Berenbaum
1980). Protein-tannin complexes are stable at neutral or acidic pH's, but tend to
dissociate under alkaline conditions (Goldstein dc Swain 1965; Van Sumere gt a1.
1975). Many herbivores (Grayson 1951; Berenbaum 19800 and detritivores (Dadd
1975; Martin e_t a1. 1980) possess highly alkaline midguts. It has been reported
(Grayson 1951) and confirmed in this study that A. senatoria larvae also have
alkaline midguts with pH values between 9.0 and 10.5 over most of their lengths,
as well as a highly active, alkaline stable proteinase which may enable efficient
digestion of protein (i.e., nitrogen) at the pH's present in the insect's gut (M.
Martin dc J. Martin unpublished).

These findings are further emphasized when contrasted with those of
Slansky dc Feeny (1977) when leaves of a similar range in nitrogen content are
compared. Quercus leaves are as efficiently digested (AD) by A. senatoria as are
leaves of Cruciferae by _E. 13%; (Fig. A.1a). The generally lower ECI of A.
senatoria (Fig. A.1b) can be attributed to reduced ECD (Fig. A.1c), particularly
of leaves with less than 2.8% N. Reduction in ECD has been linked to metabolic
costs associated with low leaf water content. Scriber (1977) demonstrated a

reduced ECD in Hyalophora cecropia fed leaves with less than 75% water.

77

Lower concentrations of leaf water in late season oak leaves (46-55%), as
opposed to leaves of Cruciferae (81—8996), may account for a portion of
decreased ECD characteristic of A. senatoria.

Digestive efficiency alone is an incomplete indicator of the feeding success
of an insect on a food plant since consumption and growth rates are equally
significant measures of feeding proficiency. Overall differences between B.
M and A. senatoria RCR's and RGR's are apparent (Fig. A.1d,e). RCR of _E.
gag averages almost twice that of A. senatoria, and combined with a slightly
higher ECI, results in a RGR over twice that of A. senatoria. The high ECI
maintained by _13. ra ae, in spite of the high RCR, is significant in view of the
potential limitations imposed upon digestive efficiency by short gut retention
time (Slansky 6c Feeny 1977). High RCR and ECI result in the greater RGR
characteristic of early season non-tannin feeders (Scriber 6c Feeny 1979; Mattson
1980). These comparisons suggest that late season herbivores with high tannin
concentrations in their diet grow slower than their early season analogs as a
result of reduced consumption and metabolic costs incurred from low leaf water
concentration.

Therefore, we conclude that A. senatoria is most influenced by the
nitrogen concentration of the diet. Larval utilization (i.e. AD, ECI, ECD, N UE)
was not influenced by leaf tannin concentrations, suggesting that larvae have
adapted to the potential protein-binding of the ingested leaves. A. senatoria
grows slower than early season feeders due to reduced consumption rate rather
than assimilatory inhibition. Additional studies of other insects will be required
to establish whether there any general relationships between life history

parameters and assimilation efficiencies and rates of consumption and growth.

78

ACKNOWLEDGEMENTS

We would like to thank E. J. Grafius, M. M. Martin, and J. R. Miller for

reviewing the manuscript, and W. H. Taft, Jr., for technical assistance.

L I ST 0F REFERENCES

79

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aquatic detritivores. Oecologia 24: 94-104.

Baker, W. L. 1972. Eastern Forest Insects. U. S. Department of Agriculture,
Forest Service. Misc. Publ. No. 1175. 642 p.

Barlocher, F. and B. Kendrick. 1973a. Fungi and food preferences of Gammarus
psuedolimnaeus. Arch. Hydrobiol. 72:501-16.

 

Barlocher, F. and B. Kendrick. 1973b. Fungi in the diet of Gammarus
pseudolimnaeus (Amphipoda). Oikos 2#:295-300.

 

Barlocher, F. and B. Kendrick. 1975. Assimilation efficiency of Gammarus

pseudolimnaeus feeding on fungal mycelium or autumn-shed leaves. Oikos
26:55-59.

 

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