THE PHYSIOLOGICAL AND BIOCHEMICAL CONSTRAINTS
ON ACTIVITY IN SPIDERS

BY
KENNETH NEAL PRESTWiCH

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL

OF THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1982

Dedication: To Jumper, Mix, Cuca, and Frisca, all of whom
like to smash spiders.

ACKNOWLEDGEMENTS

Portions of this work were carried out with the support of a grant
from Sigma Xi and generous grants from the B. K. McNab and K. N.
Prestwich Research Foundations. Special thanks to Dr. F. C. Davis,
S. G. Zam, and G. C. Karp of the Department of Cell Science and
Microbiology, Dr. J. L. Nation of the Department of Entomology and
Nematology and Drs. J. F. Anderson, B. K. McNab, and F. G. Nordlie of
the Department of Zoology of the University of Florida for their timely
loans of equipment and materials and for their helpful advice. Further
thanks is given to Dr. Wendell Stainsby of the Department of Physiology,
University of Florida, and to Dr. P. W. Hochachka of the Department of
Zoology, University of British Columbia, for their interesting and
helpful suggestions. Finally, 1 wish to thank Drs. Anderson, Nordlie,
and Nation once again for their suggestions on improvement of this
manuscript, Mrs. Donna Epting for typing the manuscript, and Ms. Nancy
Ing for suggesting the lay-out of Figure I 1-1. Finally, thanks to
Dr. J. F. Anderson for his patience, understanding and always available
ass i stance .

I I i

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES v

LIST OF FIGURES vii

ABSTRACT x

CHAPTER

I INTRODUCTION 1

The Problem ]

Approach k

II ANAEROBIC PRODUCTS IN SPIDERS 10

Summary 10

Introduction 10

Materials and Methods 1^

Results 16

Discussion 22

III THE RATES OF ANAEROBIC AND AEROBIC METABOLISM

DURING ACTIVITY 30

Summary 30

Introduction 31

Methods 32

Results 38

Discussion A8

IV ACTIVITY AND RECOVERY IN SPIDERS 58

Summary 58

Introduction 59

Methods 59

Results ^2

Discussion 8?

IV

1 02
V THE HYDROSTATIC FATIGUE HYPOTHESIS

102

Summary 102

Introduction 1Q3

Methods 106

Results 116

Discussion

VI THE METABOLISM OF PHOSPHAGENS , ADENOS I NE^PHOSPHATES ,

AN

D SOME GLYCOLYTIC INTERMEDIATES AND SUBSTRATES .... 121

121

Summary 122

Introduction ^23

Materials and Methods ^^^

Results l^r

Discussion

VII ACTIVITY IN SPIDERS: A REVIEW ^53

153

Summary -^r-i

Introduction ,r^

Methods 1 c^

Discussion

191

APPENDICES

I THE ESTIMATION OF CARDIAC OUTPUT AND STROKE VOLUME ^

IN SPIDERS

192

Summary 1Q2

Introduction ; • • - — , iq-j

Calculation of Resting Q and SV. in tarantulas 1^^

Calculation of Maximal 0 and SV Under Steady-

State Conditions. ._^

Estimation of Maximum SV Based on Non-Steady

Conditions

II THE REGULATION OF GLYCOLYSIS IN SPIDERS 197

197

Summary ^ny

Introduction ^ on

Methods and Results 202

Discussion

. . 206
LITERATURE CITED

212
BIOGRAPHICAL SKETCH

LIST OF TABLES

Table

1-1 Species used and a brief summary of the relevant features

of their life histories and respiration 5

1-2 A description of the tissue contents of the legs,

prosoma, and opisthosoma of spiders 7

1-1 Changes in concentration of D- (-)- lactate as a
consequence of activity in F. hibemalis and
L. lenta 17

1-2 The concentrations of three possible anaerobic

products as a function of activity 18

1-3 The concentration of lactate (non-enzymatic

determinations) as a function of activity in spiders 20

1-1 Anaerobic metabolism in spiders that struggled for

two minutes in respirometer flasks 46

1-2 Oxygen and lactate metabolism during exercise and

recovery In spiders ^7

1-3 Estimated and actual recovery oxygen volumes (\/02)

derived from Table I I 1-2 52

\-k Estimated anaerobic dependences during a two

minute struggle in a respirometer flask 5^

1-5 Estimated anaerobic dependence of Neoscona domiciliovi,wn

during a two minute struggle and 1 hour of web-building. ... 56

V-l Running speeds and recovery in spiders and a scorpion 68

\/-2 Heart rates in two species of spiders as a function

of activity and temperature 73

\/-3 Lactate concentrations and accumulations as a function

of activity, recovery and T^ for prosomas plus legs o'*

M-h Lactate concentrations as a function of activity

in the opisthosomas of spiders °5

VI

Table

IV-5 Lactate concentrations during activity and recovery

in whole spiders and a scorpion 86

\\l-6 Total distance traveled in two minutes of maximal

activity as a function of temperature 90

IV-y The effect of temperature on locomotion in

F. hibemalis and L. lenta 91

Vl-l Methods of analysis (Analyses #6 and 7 were only made
on a few samples due to insufficient volume of
homogenates) 125

VI-2 The amount of carbohydrate present in spider prosomas
at the start of exercise compared to the amount needed
to produce all the intermediates and lactate found
after two minutes of activity 1^1

VI-3 Total adenosine phosphate concentration in prosomas

during rest, exercise, and recovery 1^2

Vl-'t Partition of phosphates during activity and

recovery 1^6

VI-5 The changes in high-energy phosphates, AMP, and Pj

during exercise in spiders and a fly 1^7

VI 1-1 Values for several physiological parameters in

resting (alert) and active spiders 157

VI 1-2 The effect of temperature on 25°C acclimated

Filistata and Lycosa 184

AII-1 Relative activities of glycolytic enzymes and

3 Krebs cycle enzymes 200

AII-2 Equilibrium constants and mass action ratios [P]

for four reactions 201

VI 1

LIST OF FIGURES

Figure

ll-l A simplified schematic of glycolysis including

three possible anaerobic schemes 13

11-2 The relationship between anaerobic capacity and

booi< lung surface area 27

I I 1-1 Oxygen consumption, before, during, and after a

two minute struggle in respirometer flasks at 15°C ^0

I I 1-2 Oxygen consumption before, during, and after a

two minute struggle in respirometer flasks at 25°C hi

I I 1-3 Oxygen consumption before, during, and after a

two minute struggle in respirometer flasks at 33°C hh

IV-l Running speed at 25°C during two minutes of forced
activity and after five and ten minutes of recovery
(+ 5 and + 10) 65

\\J-2 Running speed at different temperatures in 25°C

acclimated F. hibemalis 67

l\/-3 Heart rates at 25°C in active and recovering

F. hibemalis and L. lenta 71

lV-4 The effect of temperature on the heart rates of
active and recovering F. hibemalis (acclimated
to 25°C) • . . . 75

IV-5 The accumulation and removal of lactate during

exercise and recovery in three species of spiders

at 25°C 78

Wl-G The accumulation and removal of lactate in

F. hibemalis at three different temperatures 81

IV-7 The accumulation and removal of lactate in L. lenta

at three different temperatures 83

JV-S The accumulations of lactate at 25°C during the first

30 sec of activity in L. lenta 96

IV-9 Anaerobic capacities in 25°C acclimated F. hibemalis

and L. lenta 98

V i i i

Figure

V-l Pressure generation and muscle group movements In

F. hihemalis 108

V-2 Same as Figure V-l 110

\/-3 Prosomal carapace movements and leg hemolympin pressures
in Filistata during and after five and ten minutes of
recovery 113

\/-4 Leg hemolymph pressures i n a F. hihevnali-s with a

ligatured pedicel 115

Vl-l The metabolism of carbohydrates in active and recovering

spiders at 25°C 129

\/l-2 Metabolism of gl ucose-6-phosphate (G6P) and fructose-l,

6-diphosphate (FDP) during activity and recovery 131

VI-3 Metabolism of gl ucose-6-phosphate (G6P) and fructose-l,

6-diphosphate (FDP) during activity and recovery 131

VI-4 Malate metabolism 131

VI-5 D-lactate metabolism during exercise in FvZtstata

and Lyaosa 13^

VI-6 The metabolism of glycerol -s-phosphate (G3P) and

dihydroxyacetone phosphate (DAP) 13^+

VI-7 The metabolism of arglnine phosphate (AP) in

Filistata and Lyaosa 137

VI-8 ATP metabolism in Lyaosa and Filistata 137

VI-9 Changes in concentration of ADP and AMP in Lyaosa 137

VI-10 Changes in the concentration of inorganic phosphate

(P;) during activity and recovery 139

Vl-Il Energy charge during activity in Filistata and

Lyaosa 1 39

V I 1 - 1 Changes in running speed, lactate, ~P stores, prosomal
pressure and heart rate during a two minute maximal
struggle in F. hihemalis at 25°C 16^

VI 1-2 Changes in running speed, lactate, ~P stores, and
heart rate during a two minute maximal struggle in
L. lenta at 25°C 166

I X

Figure

VI 1-3 Total ""P use during two minutes of maximal exercise

in Filistata and Lyaosa 172

M\\-k The changes in utilization of ~P from stores and

aerobic and anaerobic metabolism during a two minute

maximal struggle 17^

VI 1-5 Recovery in F. h-ibevnal-is at 25°C after a two minute

bout of maximal activity 178

VI 1-6 Recovery in L. lenta at 25°C after a two minute bout

of maximal activity 1 80

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

THE PHYSIOLOGICAL AND BIOCHEMICAL CONSTRAINTS
ON ACTIVITY IN SPIDERS

By

Kenneth Meal Prestwich
May 1982

Chairman: John F. Anderson
Major Department: Zoology

Most spiders can be characterized as animals that fatigue rapidly,
especially when compared to insects. The maximum duration and rates of
a spider's burst activities [e.g., behaviors involved In attacks or
escape) are limited by biochemical factors. Rapid depletion of stores
of high-energy phosphate compounds (principally arginine phosphate and
adenosine triphosphate) early in maximal activities quickly results in a
marked slowing of the spider's movements. As the spider continues to
struggle, the build-up of anaerobic by-products (principally D-lactIc
acid) and associated effects result In complete fatigue within aa. two
minutes. Defects in the hydraulic leg extension mechanism of spiders
resulting in insufficient hemolymph to extend the legs (due to loss of
hemolymph from the prosoma and legs to the opisthosoma) do not appear
to cause fatigue directly. Instead, the high prosomal hemolymph
pressures needed for vigorous activity prevent the heart from pumping
freshly oxygenated hemolymph to the prosoma and Its active muscles. The

XI

result is that over a two minute bout of struggling probably less than
10^ of a spider's energy is derived from aerobic sources^ with the remainder
coming from anaerobic glycolysis and high-energy phosphate stores. This
contrasts with a aa. 3S% aerobic dependence in non-burst activity such
as web-building. The exact relative dependence of a given species on
each of these three energy sources during peak activity appears to be
correlated with respiratory surface area: the smaller this area, the
more dependency on anaerobic metabolism. Correlations probably also
exist with the circulatory system.

During recovery, lactate diffuses from the prosoma to the
opisthosoma where it is metabolized, probably to complex carbohydrates.
The reverse process occurs during activity. Recovery takes 30 minutes
or longer in completely exhausted spiders, the exact time being
correlated inversely with the respiratory surface area. Transport of
lactate from the prosoma is analogous to the process of blood transport
of lactate from muscles to the liver in vertebrates and is an adapta-
tion to allow quick recovery of a spider's running ability.

Temperature has less effect on spider locomotion than would be
expected based on the temperature dependence of a spider's aerobic
process. However, recovery is slowed in spiders that are at temperatures
away from that to which they are acclimated.

The metabolic abilities of spiders, both aerobic and anaerobic, are
low to moderate when compared to other groups of predatory animals
(vertebrates. Insects). However, a spi der ' s use of silk and poisons prob-
ably have been major factors in lowering the need for highly developed
metabolic capacities.

XI 1

CHAPTER I
INTRODUCTION

The Problem

Most spiders are incapable of prolonged periods of vigorous
activity. For example, two minutes of vigorous running or struggling
in many species results in complete exhaustion (Anderson and Prestwich
1982). This study examines both the proximal phys iolog i cal -biochemi ca 1
constraints and speculates on the ultimate ecological and evolutionary
reasons for this inability to sustain prolonged or high aerobic work
loads.

There are two suggestions as to the cause of the constraints on a
spider's activity. They are the fluid insufficiency hypothesis (Wilson
I97O; Wilson and Bullock 1973) and the anaerobic products limitation
hypothesis (LInzen and Gallowitz 1975).

The fluid insufficiency hypothesis argues that spiders are
constrained by problems related to their locomotory system. Spiders
lack direct extensor muscles in several of their leg joints (Petrunkev i tch
1909) and must rely upon hydrostatic pressure to extend legs (Ellis 19^^;
Parry and Brown 1959a, b) . High pressures (450-500 mm Hg) are generated
by the contraction of the prosomal (cephalothorac i c) muscles, principally
the musouli laterales (Wilson 1970; Anderson and Prestwich 1975). Con-
traction depresses the prosomal carapace towards the sternum resulting
in an i ncreased pressure of the fluid of the prosoma. The legs will extend

1

if flexor muscles are relaxed (Wilson 1970; Anderson and Prestv/ich 1975).
Unlike the prosoma, pressure in the op i s thosoma (abdomen) seldom exceeds
100 mm Hg. Since maximum heart pressure during systole is only slightly
greater than 100 mm Hg (Wilson 1962; Stewart and Martin 197^+; Anderson
and Prestwich 1975), Wilson (1970) argued that during maximum activity
there would be a loss of hemolymph from the prosoma due to the pressure
gradient. Wilson and Bullock (1973) presented indirect evidence for a
gain of volume by the opisthosoma with a concurrent loss by the prosoma
during maximal activity by the spider Amaurobius fevox. They argued that
this spider was largely limited in its maximal activity by the loss of
so much hemolymph to the opisthosoma that insufficient fluid for leg
extension remained.

Linzen and Gallowitz (1975) emphasized a different cause for fatigue.
They observed a small number of poorly developed mitochondria in the leg
muscles of a wolf spider, Cupiennius salei. They also found a well
developed glycolytic pathway along with a high activity of lactate
dehydrogenase (LDH) , a result confirmed by Prestwich and Ing (in press)
in eleven species of spiders representing diverse taxa.

High activities of LDH are associated with the ability to oxidize
cytosol -produced NADH under anaerobic conditions with the concomittant
reduction of pyruvate to lactate. Linzen and Gallowitz (1975) hypothesized
that since spiders possess relatively low aerobic abilities (also see
Anderson 1970; Anderson and Prestwich 1982) and apparently large anaerobic
capacities, they are limited in activity by anaerobic accumulations of
lactate and associated effects. This idea is consistent with observations

of Clouds ley-Thompson (1957). He found that spiders exposed to pure
oxygen could struggle longer and recover more quickly.

Wilson (1970) and Wilson and Bullock's (1973) fluid insufficiency
hypothesis does not exclude the possibility of constraints related to
anaerobic metabolism. They point out that one of the problems inherent
in the design of a spider's circulatory system is the inability of the
heart to pump freshly oxygenated hemolymph into the prosoma of a
vigorously active spider. Thus, they implied that two processes might
be involved in fatigue: f 1 u id (hydrostat i c) i nsuf f i ciency occurs quickly
(after less than 10 sec of struggle), slows the spider, and the second
constraint, a biochemical fatigue related to anaerobiosis is involved
1 ater in act i v i ty .

I suggest a third possible constraint, namely, depletion of stores
of high-energy phosphate compounds and substrates. Phosphagens (arginine
phosphate in spiders, Di Jeso et at. 1967) are depleted during the
initial phases of activity in both vertebrates and insects (Flock et at.
1939; Sacktor and Hurlbut I966). Also, carbohydrates are the only suitable
substrate for anaerobic glycolysis, but are not found in high concentrations
in spiders (Collatz and Speck 1970; Stewart and Martin 1970; Collatz and
Mommsen 1975; Rakotovao 1975). Shifts to usage of alternative sub-
stances such as fats and amino acids involve mainly aerobic processes.
It is unlikely that high rates of ATP production could be maintained
solely by aerobic processes, given the small number of mitochondria in
spider muscles (Linzen and Gallowitz 1975). It is possible that
phosphagen and/or carbohydrate depletion could result
in fatigue of maximally active spiders.

Approach

It is the purpose of this study to partition out the degree each of
these factors operates to constrain activity. To accomplish this and to
mai<e my results and conclusions as general as possible, I have used
seven species of spiders representing diverse taxa and very different
habits. A listing of spiders used and a brief description of their size,
respiratory exchange system, resting rate of oxygen consumption (VO^) and
prey capture mode are given in Table 1-1. These features are included
due to their association with the aerobic and anaerobic capabilities
and activity patterns of the spider. For comparison sake, some data
are included from a non-spider arachnid, the scorpion Centruroides
hentzi.

The most detailed analyses in the subsequent chapters will revolve
around three species: Fil-istata hibemalis , Lyaosa lenta, and Phidippus
audax. These species were chosen because of their differing habits,
respiratory anatomy, availability, and because they have been extensively
studied (Anderson 1970, 197^; Anderson and Prestwich 1975, 1980, 1982;
Harper 1970).

I have chosen to do the biochemical analyses on either the entire
animal or the functional subunits of prosoma, legs, and opisthosoma.
There is a disadvantage to this procedure in that each of these compart-
ments contains varying proportions of muscle and other tissues (Table 1-2).
Nevertheless, the method has the advantages of being (1) more rapid than
isolation of separate muscles for analyses and (2) it fits in line with
the general thrust of other studies on spider locomotion, i.e., other
work has focused on the relative roles of the legs, prosoma, and opisthosoma

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in terms of their separate contributions to movement in spiders instead
of the exact roles of different muscles (Parry and Brown 1959 a, b; Wilson
1962, 1965, I97O; Wilson and Bullock 1973; Stewart and Martin 197^4;
Anderson and Prestwich 1975; Prestvvich and ing in press).

The organization of this dissertation is in seven chapters: Chapter
II deals with the identification of anaerobic pathways in spiders and with
t he va 1 idat ion of a non-enzymat ic technique for lactate determination;
Chapter ill presents measurements of oxygen consumption in struggling
spiders and estimates the relative importance of aerobic and anaerobic
metabolism in two minute struggles and in web building; Chapter IV details
the actual pattern of fatigue as it occurs in spiders and a scorpion
and presents measurements of heart rates and lactate production during
exercise and recovery; Chapter V tests the fluid insufficiency hypothesis;
Chapter Vl presents measurements of exercise and recovery levels of
phosphagens, glycolytic substrates, and intermediates during exercise
and recovery; and Chapter Vll seeks to integrate this work with previous
studies and presents a model of the phys iologi ca 1 -biochemica 1 events that
occur in spiders when they are maximally active and during recovery.
Additionaly, I propose some correlations between behavior, physiology,
and the ecology of spiders. Finally there are two Appendices: Appendix
I deals with estimation of cardiac output in tarantulas and Appendix II
with the regulation of glycolytic metabolism in araneomorph spiders.

CHAPTER I 1
ANAEROBIC PRODUCTS IN SPIDERS

Summary

1. The major anaerobic by-product of spiders is D-lactic acid. Minor
accumulations of L-gl ycerol -3-phosphate may account for ca. five
percent of the anaerobic ATP production. The possibility remains
for the presence of other pathways, particularly one that results
in the accumulation of alanine and succinate.

2. Lactate accumulations after maximal activity are greatest in the
legs and prosoma and can be as large as 15 ymols/g; accumulations
of lactate in the opisthosomas seldom exceed k ymols/g and average
near 2.5 ymols/g.

3. At a common intensity of exercise, an inverse relationship exists
between lactate accumulation and book lung surface area (Fig. I 1-2)
This relationship is accounted for by a co-adapted respiratory-
circulatory system that is adapted to supply some oxygenated
hemolymph to the prosoma.

I ntroduct ion

The evidence for anaerobic metabolism in spiders is based on
enzymology. Long and Kaplan (1968) identified the presence of a D-optica'
isomer-specif ic lactate dehydrogenase (D-LDH) in spiders and scorpions.
This observation was confirmed for spiders by Gleason et at. (1970 and

10

11

Prestwich and Ing (in press). They showed, along with Linzen and
Gallowitz (1975), that LDH was present in high activity and could
therefore be regarded as important in anaerobic metabolism (Fig. Il-l).

Two other anaerobic pathways have also been proposed to operate
in spiders. Based on the activities of the cytosol form of malate
dehydrogenase (MDH) and the transaminases g 1 utamate-pyruvate transaminase
and g 1 utamate-oxa loacetate transaminase (GPT and GOT), Linzen and
Gallowitz (1975) proposed a pathway based on the work of Sacktor (1965).
This scheme results in the anaerobic accumulation of alanine and malate
at the expense of aspartate and pyruvate (Fig. Il-l). After examining
11 species of spiders, Prestwich and Ing (in press) found similar
activities for these enzymes and agreed that spiders in general might
use this mechanism to help maintain redox under anaerobic conditions.
Both studies suggested that the MDH-t ransami nase pathway was about 10
to 10% as active as the LDH pathway.

A third pathway was suggested by Prestwich and ing (in press).
They noted sufficiently high activities of cytosol ic glycerol-}"
phosphate dehydrogenase (GPDH) to suspect that it might play a role in
oxidation of NADH during anaerob ios i s . The products of this pathway
would be g 1 ycerol -3-phosphate and pyruvate (Fig. Il-l). Based on the
relative activities of GPDH and LDH, the GPDH scheme appeared to be only
about 10 to 20% as active as the LDH-cata lyzed scheme.

Enzymolog ical results such as these can only suggest the identity
of anaerobic products. Regulatory schemes can result in potential
anaerobic pathways being shut down under in vivo conditions. For example,
in tuna, both LDH and GPDH are found in high activities; yet, only lactate

Figure 11-1. A simplified schematic of glycolysis including three
possible anaerobic schemes. The first pathway (lower
left) is the reduction of pyruvate to lactate (solid
box) utilizing lactate dehydrogenase (LDH) . In the
second scheme (shown at the bottom of the figure),
NADH is oxidized in the production of malate from
oxaloacetate, mediated by malate dehydrogenase (MDH) .
Oxaloacetate is generated through two transaminations
mediated by the enzymes g 1 utamate-pyruvate transaminase
(GPT) and gl utamate-oxa loacetate transaminase (GOT).
Malate and alanine accumulate in equimolar amounts at
the expense of pyruvate produced during glycolysis and
asparate from reserves in the cell. This pathway and
the LDH mediated scheme result in a net gain of 3 ATP
per molecule of gl ucose-6-phosphate (G6P) entering
glycolysis. A third scheme involves the reduction of
d i hydroxyacetone phosphate (DHAP) to glycerol -3"phosphate
(G3P, dotted box, middle right of figure) with an equal
accumulation of pyruvate (lower left). This scheme
depends upon g 1 ycerol -3~phosphate dehydrogenase (GPDH)
and results in the net gain of 1 ATP per G6P metabolized.

A possible variation exists for the MDH- transami nase
pathway (not tested in the experiments described in
this chapter). In this scheme, malate is converted to
fumarate and then reduced to succinate. This scheme
would consume another pair of electrons and thus help
maintain redox. It could also explain the decrease of
malate under anaerobic conditions (Table M-2).

13

GLYCOGEN ^"-^GLUCOSE 6- PHOSPHATE

PYRUVATE

V' k2H

\ ^- GLUTAMATE-^^ /^^,A. ,^A,..T-r*T.-

TO \ ,;* , ^ ^-iKDXALOACETATE

MITOCHONORIA , f STI ^vloT

A
lALANINEl ^ <xKETOGLUTARATE " ''-ASPARTATE

/ \

14

accumulated under anaerobic conditions (Guppy and Hochachka 1978).
Therefore, the purpose of the experiments outlined in this chapter was
to determine the relative development of these three pathways by measur-
ing the quantities of product produced as a result of vigorous activity.
Additionally, I used the results to postulate a relationship between
the anaerobic capabilities of different species of spiders and their
respiratory systems and behaviors.

Materials and Methods

Animals

Spiders were collected in Alachua County, Florida, and were brought
into the lab at least two weel<5 prior to experimentation. They were
kept at aa. 25°C; this should be regarded as the acclimation temperature
(Anderson 1970). Spiders were maintained on a diet of crickets and were
last fed 7 to 10 days prior to their use to assure they were all in
comparable nutritional and metabolic states (Anderson 197'^). Three
species were selected for extensive biochemical analysis; Filistata
hibermalis, Lyaosa lenta, and Phidippus audax. They were selected due
to their different activity patterns and respiratory anatomies (Table
1-1).

Enzymatic Determination of Anaerobic Products

Spiders were placed in large beakers. After they remained motion-
less for at least one half hour (T = 1S°0 , they were instantly frozen

d

using liquid nitrogen. Other spiders were stimulated to maximal activity
by prodding with a metal rod and air puffs for tv^o minutes and then

15

frozen. This procedure generally produces maximal activity (Anderson
and Prestwich 1982). The frozen spiders were broken into sections,
weighed rapidly to reduce condensation, and homogenized in ^°C 0.6M
HCIO^ (1 ik or 1:8 w/v) .

Cold homogenates were neutralized using h°C 6m KOH , the solution
was thoroughly mixed, allowed to stand several hours at k°C and then
filtered using a 93^+ AH glass f i ber Whatman Fi 1 ter. The filtrate was
analyzed for the following substances using the method cited after each
substance: L-alanine, Williamson (197^); L-glyceroI 3-phosphate (Michal
and Lang 197^); D-lactate (Gawehn and Bergmeyer 197'*); L-malate (Gutmann
and Wahlefeld 197't); and pyruvate (Czok and Lamprecht 197'*). Readings
were at 3^+0 nm using a Beckman DU spectrophotometer.

Colorimetric Analysis of Anaerobic Products

Spiders were treated as described for enzymatic analysis except
some individuals were run for periods that did not result in exhaustion.
After freezing, spiders were homogenized in ]0% TCA and filtered as
described above. Analysis was according to the technique of Harrower

nd Brown (1972) with the exception that each sample was determined in
duplicate along with an individual blank that did not receive the
p-phenyl phenol indicator. Samples were read on a Model 6/20 Coleman
Jr. II Spectrophotometer at 585 nm.

The technique was validated by comparison of the results obtained
against those obtained enzymat i cal 1 y . I was unable to compare homogenates
directly by analyzing them both enzymat i cal ly and color imetr i ca 1 1 y because

f residual CIO, which strongly interferes with the colorimetric test.
Even after precipitation of this ion with KOH, some CIO. remains in

a

o

16

solution (K for KCIO, at pH 7-0 at 0°C = 0.0029) and 1 found that even
minute quantities of CIO, affected the amount of color developed from
lactate standards. Conversely, samples in TCA could not be used for
enzymatic analysis since TCA inhibits LDH. Comparison of samples in
both acids, when taken from spiders under identical conditions, revealed
an overestimate of ca. 20%. Corrections were made for substances known
to affect the intensity of color (Barker and Summerson 19^0 if they were
known to either accumulate anaerob ical ly (Tables M-2, Ch. Vl) or be
present in spiders (Collatz and Speck 1970; Stewart and Martin 1970;
Collatz and Mommsen 1975)- The resultant values were consistently 10^
greater than those obtained enzymat i ca I 1 y . Nevertheless, given the
advantages of low cost and repeatability, I felt justified in using
the colorimetric method.

Results

Since lactate (presumably the L isomer since a Boehringer test kit
had been used) was reported for an agelenid spider (Collatz and Mommsen
1975), first analyses were for L(+) lactate. I found none in any of my
samples. This is in agreement with Long and Kaplan (1968), Gleason
et al. (1971) and Prestwich and Ing (in press), all of whom found only
D-LDH in spiders.

Subsequent enzymatic analysis for D-lactate confirmed its presence.
Table ll-l shows the results for F. hibermalis and L. lenta. Values for
P. audax are not included since I was only able to analyze the prosomas
plus legs of five animals. Of these, resting (N = 2) levels of lactate

Table I 1-1. Changes in concentration of D- (-)- lactate as a
consequence of activity in F. hibernalis and
L. lenta (P = prosoma, L = legs, t = time of
activity in seconds).

17

Species (N)
Mass at t = 0; t = 120 sec

D- (-) -Lactate Concentration umols/g
(+ SE)

0 sec

120 sec

I ncrease
Ar I tnmet i c
(Factor i al )

F. hi-bernalis (6)

0.270 g; 0.251 g (P + L)

L. lenta (6)

0.523 g; 0.^4^5 g (P + L)

0.293 g; 0.2A6 g (P)

0.230 g; 0.200 g (L)

1

.7

(+

0

.A)

0,

.9

(+

0,

.0)

0,

.7

(+

0,

.0)

(+ 0.1

16.2
(+1.5)

10.9
(+1.16)

9-3
(+0.7)

M.h
(+ 1.3)

1A.5
( 3.hX)

10.0
(12. 2X)

8.6
(13.1)

11.3
(11 .2X)

Op i sthosoma 1 D-Lactate Concentrations

F. hibernalis (6)
0.318 g; 0.186 g

L. lenta (6)

0.302 g; 0.186 g

0,

.7

(+

0.

.3)

0.

,6

(+

0.

■ h)

2.7
(+ 1.1)

3.5
(+ 0.7)

2.0

3.9X)

2.9
5.8X)

F. hibernalis (6)
0.588 g; O.A37 g

L. lenta (6)

0.825 g; 0.631 g

Whole Spider D-Lactate Concentrations

8.9
( 7. IX)

(+

1

0

.5
.4)

(+

0,
0,

.7
.0)

10

.h

(±

1

.3)

9

.6

( +

1

.0)

8.9
13. 6X)

Arithmetic scope = anaerobic capacity (Bennett 1978)
factorial scope = (t = 120 sec)/(t = 0 sec)

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19

were similar to those of the two other species while active (N = 3)
levels were 1Q% less than those of L. lenta.

I also analyzed the same homogenates for the products of the other
proposed anaerobic pathways (Fig. il-I). The results are reported in
Table 11-2. Malate showed a significant decrease during activity in
both species, dropping 0.25 ymols/g in Filistata and 0.2 in Lycosa.
Small, non-significant increases in alanine occurred in both species.
The largest changes were in G3P where significant increases of 0.55 and
Q.k ymols/g occurred in Filistata and Lycosa, respectively. Pyruvate
concentrations were below the limit of detection (0.05 ymols/g). There
were no s i gn i f leant changes in any of these substances in the opisthosoma.

Color imetrica 1 ly Determined Lactate

Table 11-3 shows the color imetr i cal ly determined lactate concen-
trations of seven species at rest and after exercise. Lactate concen-
trations in resting spiders ranged as follows: 0.3 to 1.5 ymols/g (whole
spider); 1.0 to 2.2 (prosoma plus legs); and 0.5 to 2.6 (op i sthosomas) .
The resting concentrations were usually higher (within any species) in
the prosoma plus legs. The range of lactate concentrations at the
completion of exercise was 1.6 to ]0.h ymols/g (whole spiders); 5-2 to
16.2 (prosoma plus legs); and 1.2 to 5.2 (op is thosomas) . The increases
in lactate concentration after activity were significant in all species
for both whole animals and for the prosomas plus legs. Also, concentra-
tions after exercise were always significantly greater in the prosoma
plus legs than in the opisthosoma.

In the opisthosoma, lactate increased but this was usually not
statistically significant. Lactate concentrations in this compartment

20

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22

were negatively correlated with its relative size (relative size range
was 29 to 75'o of total mass). These differences in relative opisthosomal
size are a function of interspecific differences (orb weavers tend to
possess larger op i sthosomas) and differences in nutritional history
(Anderson 197^) •

increases in lactate concentration varied as a function of exercise
level. The greatest increases resulted from activity culminating in
exhaustion. These increases are referred to as anaerobic capacities
(Bennett 1978) and range from 3-0 to 8.9 ymols/g for whole spiders.
Accumulations resulting from sub-maximal exercise are given for A. avantia
and /V. clavipes at 120 seconds and P. audax at 200 seconds.

Pi scuss ion

Development of Anaerobic Pathways in Spiders

Barring the presence of other anaerobic pathways not investigated
in this study, D-lactate appears to be the main by-product of anaerobic
metabolism in spiders. Resting and exhaustion levels of this substance
are within the range reported for L-lactate in reptiles and amphibians
(Bennett 1978) .

Lactate accumulations in each body compartment (Table 11-3) are
correlated with LDH activity (see Prestwichand Ing in press). Pierson
product-moment correlation coefficients were 0.68 (leg LDH) and 0.71
(cephalothorax LDH) with the leg plus prosomal lactate accumulations
and 0.89 between opisthosomal LDH and lactate accumulation. Only the
opisthosomal correlation is significant (P < 0.05); however, the other
results are suggestive.

23

Two other anaerobic pathways have been proposed to operate in
spiders (Linzen and Gallowitz 1975; Prestwich and Ing in press). One of
these schemes, catalyzed by MDH and the transaminases GOT and GPT should
result in the equal production of alanine and malate (Fig. I 1-1). The
data on Table 11-2 for prosomas show that malate concentrations decrease
during activity while alanine remains constant. These results argue
against the use of this pathway in spiders. They are suggestive of the
possibility that malate itself becomes an electron acceptor and is
thereby converted to succinate. Glycerol phosphate dehydrogenase (GPDH)
appears to help maintain cytosol redox. Use of this enzyme to oxidize
NADH results in equal production of G3P and pyruvate. Since pyruvate is
a substrate for LDH, its accumulation is not an accurate measure of the
activity of this pathway. However, the only possible sinks for G3P during
anaerobiosis are fat or glycerol synthesis (Fig. Il-l). Since neither
seems lil<ely, the G3P accumulation should be a good measure of the develop-
ment of this pathway. Prosomal accumulations of G3P are 0.55 (F.
hibernalis) and 0.^ (L. lenta) pmols/g compared to simultaneous lactate
accumulations of 12.8 and 9-1 ymols/g respectively (Table Il-l). Thus,
G3P accumulations are. only about S% of those of lactate in both species.
The ratios of the activities of GPDH to LDH are, on the other hand,
slightly larger (Prestwichand Ing in press) beingabout 10^ in Filistata
and \G% in Lyaosa. This discrepancy could be due either to experimental
erroror inhibition of GPDH during anaerobiosis in order to prevent
competition between the two enzymes for NADH (Guppy and Hochachka 1978).

A weighted calculation should be used to evaluate the relative
contributions of the LDH- and GPDH-catalyzed pathways to the total

Zk

anaerobic production of high-energy phosphate bonds {-?) ^'assuming, of
course, that no unknown pathways are involved). This is necessary since
1.5 "P sre gained per lactate that accumulates and 1 "P per G3P (both
calculations assume glycogen as the starting point for glycolysis: Stewart
and Martin 1970; Collatz and Speck 1970). The proportion of the total
anaerobic production of ~P due to the GPOH pathway is then only three
percent of the total in both species and can be ignored.

The question arises as to why the lactate scheme is favored. Its
superiority over the G3P"Pyruvate scheme is obvious: formation of lactate
yields three times as much ATP as the alternative scheme (Fig. Il-I) and
it produces smaller osmotic and pH changes in the cell on a per ATP
basis. The differences do not exist between the LDH and the MDH-
transaminase schemes as ATP gains and osmotic effects are comparable.
The lack of an important MOH-transami nase pathway may relate to both its
complexity and its requirement for a substrate (asparate) that is not
part of glycolysis (Fig. Il-l).

Determinants of Lactate Accumulations

The increases in lactate in the prosoma and legs are associated
with muscular activity. The smaller increases in opisthosoma! lactate
concentrations could be due to two factors: anaerobic activity of
opisthosomal muscles indirectly associated with locomotion (Wilson 1970;
Anderson and Prestwich 1975) or the transport of lactate through the
hemolymph to the heart and digestive diverticulum for reoxidation (Long
and Kaplan 1963) and perhaps gl uconeogenes i s . The inverse relationship
between lactate concentration and relative opisthosomal mass is consis-
tent with both possibilities.

25

Lactate accumulation is related to both the intensity of activity
and the ability of a spider to deliver oxygen to its muscles, i.e. its
aerobic capacity. The interplay between activity level and the aerobic
and anaerobic capacities of animals is complex (Taigen et at. in press)
and are. the subject of the remainder of this chapter.

Aerobic capacity in spiders is proximal ly determined by the same
factors as in vertebrates; respiratory surface area, ventilation, and
circulatory capacities. Respiratory surface areas aro. known to vary
i nterspec i f ica 1 ly in spiders (Anderson 1970; Anderson and Prestwich 1982;
also see Table 1-1). At a common intensity of activity, anaerobic
accumulation should be negatively correlated with respiratory surface
ar&a. I tested this hypothesis by constructing Figure 11-2 for the
five species (Table 1-1) where these data are available.

Figure 11-2 is consistent with the expected relationship for both
whole animals and prosomas plus legs. The only exception is that the
whole animal anaerobic capacity of P. audax is higher than predicted from
the other species, probably due to a proportionally smaller abdomen
(Table 11-3). Figure 11-2 indicates a distinct difference between orb
weavers versus non-orb weavers. Phidippus audax, a non-orb weaver, has
a book lung surface area similar to N. clavipes , an orb weaver; yet it
accumulates more than twice the lactate in one third the time (Table
11-2). The orb weavers and non-orb weavers represent two distinct,
homogeneous behavioral groups. While the relative speeds (prosoma
lengths/sec) within either group were the same, the non-orb weavers were
excellent runners. The orb weavers were simply not morphologically
adapted to move as rapidly. As they ran or climbed they spent a great

Figure 11-2. The relationship between anaerobic capacity and book

lung surface area. Data are plotted on a mass-specific
basis due to differences in size (Table 11-3) and on
both on a whole animal (solid circles) and a prosoma
plus legs (open circles) basis. The latter approach
is favored due to a "dilution effect" from the largely
inactive abdomens of widely variable size (Table 11-3)-
The figure indicates two important trends: first,
larger mass-specific book lung surface areas are
associated with smaller lactate accumulations since
proportionately more 0~ should be delivered to the
tissues in these species; and secondly, no obvious
influence of tracheal development is apparent since
P. audax has a much larger tracheal system than any of
the other spiders but it falls on a line predicted by
other non-orb weavers possessing greatly reduced tracheal
systems. Finally, the lower lactate accumulations in
orb weavers are interpreted as due to the slower move-
ments of these spiders.

27

15 —

10

:5
< -o

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<

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I

—

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—

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-

1 t 1 1

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-

500

BOOKLUNG SURFACE AREA

( mm^/g)

28

deal of time flailing about with their legs. This behavior is probably
not very costly since the spider does not need to pull its entire mass.
Its function is probably related to searching out paths through the
foliage into which these spiders may retreat when continually threatened.

One surprise from Figure 11-3 was that P, audax prosoma plus leg
lactate accumulations fell on a line predicted by the accumulations and
bool< lung surface areas of the other two non-orb weavers. Since ?. audax
alone possesses an extensive cepha lothoraci c tracheal development (Table
11-2), I expected 0- delivery in this species to be more constant than in
species without significant trachea (Anderson 1970). Given greater 0^
availability and a common level of activity, I expected much smaller
lactate accumulations, but they were predictable based on bool< lung
surface area. The role of trachea in spiders needs further study. One
possibility relates to aerobic scope. Since the aerobic scope of jump-
ing spiders is higher than that of F. hibevnal'is and L. lenta (see Ch.
Ill), the trachea may allow a higher threshold for the transition between
aerobic and anaerobic conditions. Also, they could help decrease the
time needed to repay an oxygen debt.

The presence of a relationship between book lung surface area and
lactate accumulation implies a co-adapted respiratory-circulatory system.
Increased respiratory surface area will not by itself provide more oxygen
to the tissues. More circulatory transport of 0^ is required. This can
be achieved via enhanc ing both the cardiac output and hemolymph 0- capacity.

To minimize anaerobic accumulations, the proposed co-adapted
respiratory-circulatory system must be capable of delivering well-
oxygenated hemolymph at least intermittently to the active prosoma. In

29

spiders, this implies a correlation between the book lung surface area,
the strength of the myocardium, and the opisthosomal sub-cut icu 1 ar muscle
sheet. The muscle sheet and heart are vital in this scheme because they
operate together to produce the hemolymph pressures great enough to pump
blood to the prosoma of active individuals (Stewart and Martin 197^).

There are no comparative data on heart strength in spiders. How-
ever, Wilson (1970) showed marl<ed differences in the development of the
opisthosomal sub-cut i cul ar muscle sheet that correlated directly with the
activeness of a given species of spider. He interpreted this as an
adaptat ion to reduce fluid loss from the prosoma to the opisthosoma during
maximal exercise (see Chs. I and V) . I propose that an equally (if not
more likely) function is to develop high opisthosomal pressures to assure
hemolymph flow for a greater proportion of activity and thereby reduce
the need for anaerobic metabolism. Studies on the relative development
of opisthosomal and heart musculature, prosomal pressures, the book lung
surface area, and anaerobic accumulations are needed to test this
hypothes is .

CHAPTER I I I

THE RATES OF ANAEROBIC AND AEROBIC METABOLISM

DURING ACTIVITY

Summary

1. The relative Importance of aerobic and anaerobic metabolism In
active spiders was investigated In four species, Fitistata hibemalis,
Lyoosa tenta, Phidippus audax and Neoscona domicitiorium.

2. Peak \/02 varied from 2.3 to 5-8 times the resting V0~ . Within any
one species this ratio decreased with T . Aerobic capacities were
directly related to book lung surface area.

3. Estimates of the relative Importance of anaerobic metabolism to total
power generation during short, maximal struggles (less than two
minutes) varied from between 55 to Sh% of the total power generation.
Anaerobic dependence was inversely associated with respiratory surface
area.

A. The anaerobic contribution to construction of orb webs in Neoscona
domiailiorium was estimated to be aa. \Z of the total cost of the
web.

5. Calculations based on lactate accumulations, recovery oxygen, and
the known sto ich iometry of gl uconeogenes I s and complete oxidation
of lactate suggest most of the lactate accumulated during struggle
is reconverted to hexose during the recovery period.

30

31

I ntroduct ion

Many spiders rely on a mix of aerobic and anaerobic metabolism to
fuel their activities. The relative importance of these two sources of
high energy phosphates (~P) have not been studied in spiders.

Spiders are characterized by relatively limited aerobic capabilities.
Their resting rates of oxygen consumption (V0„) are low, ranging from 2k
to 122°^ of the predicted VO- for ectotherms of their size (Dresco-Derouet
I96O; Anderson 1970; Greenstone and Bennett I98O; Anderson and Prestwich
1982). Their maximum \102 values are likewise low: factorial aerobic
scopes (the ratio of maximal to resting VO-,) are below 10 and most are
below 6 (Seymour and Vinegar 1972; Peakall and Witt 1976; Ford 1977a,
b; Prestwich 1977). These compare with factorial aerobic scopes of 20X
in mammals and several hundred in insects (McArdle I98I; Weis-Fogh 1964).

To compensate for their low aerobic abilities, spiders have
reasonably wel 1 -developed anaerobic capacities (Linzen and Gallowitz
1975; Prestwich and ing in press; Ch. ll). Anaerobic accumulations in
spiders may be negatively correlated with both respiratory surface area
and rest-ing VO. (Fig. 11-2; and see Anderson and Prestwich 1982), given
a common level of activity. Thus, spiders with the poorest 0^ exchange
ability, being less able to generate ~P compounds aerobically, rely to
a greater degree on anaerobic metabolism.

This study seeks to partition the contributions of aerobic and
anaerobic metabolism to total ~P production during both maximal and
sub-maximal activities. Three species {FiZistata h-ibemal'ts, Lyaosa
lenta, and Phidippus audax) that possess different respiratory surface

32

areas and anatomies were used to study near-maximal activity. Evaluation
of relative anaerobic dependency in these species allowed for testing of
the hypothesis that anaerobic dependence is inversely correlated with
respiratory surface area. Because the relative anaerobic dependence of
poi ki lotherms is frequently temperature dependent (Bennett 1978), these
measurements were made at three different temperatures. Estimation of
the anaerobic contributions to long-term activities in spiders were
obtained by measuring the anaerobic accumulations during orb-weaving In
Neoscona domiciliorin/n and comparing these with the estimated aerobic
costs. Finally, the data on oxygen consumption and lactate removal
during recovery allowed speculation as to the metabolic fate of lactate
in sp iders .

Methods

An i ma 1 s

individuals of the species Filistata hibemalis, Lyoosa lenta^ and
Phidippus audax were used for the experiments involving maximal struggles.
These spiders were brought into the lab and treated as described in
Chapter II. Lactate accumulations Incurred during orb weaving were
made by freezing individuals of the araneld species Neoscona domic-iZioviim
as they finished construction of their webs in the field. A summary of
the characteristics of these four species is given in Table 1-1.

Oxygen Consumption (VO2)

Oxygen consumption was measured manometr i cal ly using a G i 1 son
Differential Resp i rometer . Spiders were placed in either 15, 50, or

33

120 ml respirometer flasks depending on their size. Tin i s permitted
roughly equal areas for spiders to move about during their struggles.
Each respirometer flask contained a measured amount of soda lime as a
CO- absorbant and also a number of glass beads or ball bearings.

After several hours of temperature equilibration, resting VO^
was measured using the shortest feasible time intervals (five or ten
minutes). Exercise was initiated by manually shaking each flask for
two minutes. This caused the balls to bounce about the flask and induced
the spiders to struggle.

The shaking was done with the flasks out of the water bath in order
to observe the spider's activities. At 15 and 33°C it was necessary to
periodically reimerse the flasks for ca. 10 sec (with swirling) in order
to prevent manometer fluid overflow. Blanks were treated identically
to the animal flasks.

Most spiders struggled for the majority of the two minutes. How-
ever, some spiders wedged themselves into the neck of the flask to avoid
the balls. If a spider avoided moving for more than 20 sec, it was
dropped from the experiment.

At the end of the two minute activity period, recovery VO^ was
recorded for the next 30 to 55 min. Measurements of VO^ were made every
five or ten minutes depending on my ability to discern a measurable
change in the manometric level. All data were converted Into y 1 0„/(g-h)
at STPD and mean VO2 and standard errors (SE) for each interval were
calculated. The results were plotted as a function of rest, exercise,
and recovery.

An interesting phenomenon occurred during many measurements. Near
the completion of exercise or more often, early in the recovery period.

34

many individuals of all species expelled large (> 100 \j 1 ) volumes of
gas. The gas release caused a manometer deflection opposite to that
produced by consumption of 0^. Some of the deflections persisted for
five to ten minutes and thereby obfuscated any changes in V0„ . If the
deflections lasted more than five minutes, the experiment was terminated
and the data discarded. These deflections were never observed in the
blanks .

To determine if these gas pulses were due to a burst release of
CO- [perhaps related to 1 actate-caused changes in hemolymph pH
(Angersbach 1978)], I loaded the flasks identically to the oxygen
consumption experiments, except that in place of the spider there were
two small vials containing solutions of NaHCO, and HCl. The quantity of
NaHCO, was adjusted to result in the evolution of aa. 150 yl of CO2
when the solutions were mixed. I then recorded the time course for
reabsorption of the gas. The removal of C0~ generally took less than
3 minutes and was faster than for a similar volume of gas produced by
the spiders. There are two likely explanations for this: (a) the
spiders released CO^ over a long time span and/or (b) other gases were
also released. Option (a) requires that volumes of CO- in excess of
100 yl be frequently released by the exhausted spiders. A calculation
based on the estimated blood volume (Stewart and Martin 1970), CO2
carrying capacities (Loeweand deEggert 1979), and estimated tissue VCO2
(based on VO- measurements) shows this to be impossible. There is not
enough CO-, stored in the hemolymph or being produced via respiration
to account for such a large release of C02- Thus, the most plausible
explanation is that spiders release a mixture of gases during these
pulses. However, their exact compositions remain unknown.

35

The practical implication of these observations was that it
introduced both a degree of uncertainty and also made the measurement
of VO- more difficult. Given the different time courses for the removal
of these evolved gases, it is likely that the exact gas composition was
variable. if CO^ composed a relatively small proportion of a gas pulse,
the result would be an artificially low VO- (because this gas would not
be absorbed). Thus, I decided to discard the data from any run where
the positive pressure deflection persisted for over five minutes.

Due to the common occurrence of pulsed gas release, I was usually
unable to obtain a measurement of VO-, immediately at the end of the
activity period. Therefore, I used a five or ten minute interval for
measurement of the VO- for activity: five minute intervals were
possible in Lycosa and Phidippus at all temperatures because they had
high VO-, but were feasible for F. hibemalis only at 33°C. Thus, the
exercise interval also includes the first three to eight minutes of
recovery. However, this was not a problem in Lycosa and FiZistata. In
cases where no gas release was apparent, noticeable increases in \/0„
in both Lycosa and Filistata did not occur until one or two minutes after
the completion of exercise (see Results and Discussion). However,
increases in VO- in P. audax did occur during activity periods and their
full magnitude may have been partially obscured by gas releases.

Recovery oxygen (recovery VO-) was difficult to measure precisely
in Phidippus and Filistata due to the common occurrence of activity
during recovery (see Results). I estimated recovery V0-, as the total
oxygen used above resting levels between the end of the exercise-early
recovery VOj measurement (at +3 minutes of recovery) until either

36

resting VO- was reached or until a time when activity caused VO, to
increase (see Results and Discussion). Other experiments will show
that in Filistata and Lycosa lactate concentrations remain constant
during the first 3 to 5 minutes of recovery (Ch. IV). The principal
uses of the first few minutes' recovery 0-^ may be to fully saturate
the spiders hemocyanin (Angersbach 1978) and resynthesize depleted stores
of -P compounds. Thus, the recovery VO- I report are probably good
measures of the Oj needed to remove the lactate.

Anaerobic Accumulations

Spiders were placed in respirometer flasks and treated identically
as in the VO-^ measurements. After either two minutes of stimulated
activity or after two minutes of activity followed by 15 minutes of
recovery, the respirometer flasks were opened and the spiders instantly
frozen in liquid N„. This operation required less than 15 sec and the
spiders did not move significantly during the time required to open the
flask and freeze them. The frozen spiders were homogenized and
analyzed using the colorimetric method of Narrower and Brown (1972) as
modified and described in Chapter II. Resting levels of lactate were
not measured given the constancy of these values for individuals within
any species. To obtain anaerobic accumulations, resting lactate concen-
trations given in Table il-3 were subtracted from the values I obtained
for this chapter. Factorial increases in lactate were calculated by
dividing active or recovery lactate concentrations by resting concen-
t rat i ons.

37

Heart Rates

Heart rates of resting and recovering F. hibemalis and L. lenta
were measured at 25°C. After measuring resting heart rates, these
spiders v^/ere exercised identically to those in the V0„ experiments. At
the end of the activity period, they were quickly placed in glass cages
and their heart rates monitored by the use of a laser as described in
Anderson and Prestwich (1932). These rates were compared with those
given for free-running or restrained spiders in Chapter IV.

Calculations of Energy Equivalents of Aerobic and Anaerobic Metabolism
and the Energetics of Lactate Oxidation and Gl uconeogenes i s

Assuming that the substrate for energy metabolism during exercise
is glycogen (Stewart and Martin 1970; Collatz and Speck 1970), the total
number of high energy phosphate bonds ("P) synthesized per hexose residue
cleaved from a glycogen polymer can be estimated as follows:
Aerobic metabolism: results in (maximally) a synthesis of

38 ~P per hexose residue. This requires six 0_; therefore,
0.282 ymols ~P are produced per ul 0„ used.
Anaerobic metabolism: results in a net synthesis of 3 ~P per
hexose residue removed from glycogen. Thus, 1.5 Umols ~P
are formed per pmol of lactate produced.
To calculate the amount of 0„ needed to drive gl uconeogenes i s from lactate
to g 1 ucose-6-phosphate (this ignores the final costs involved with
glycogen synthesis) or the amount of 0- needed to fully oxidize lactate,
the following relationships can be used:

G 1 uconeogenes i s : Three -P per lactate are needed to make

gl ucose-6-phosphate. These could be formed aerobically

38

by the use of 1/2 of a mol of 02- Therefore, gluconeo-
genesis requires 11.2 yl 0_ per mol of lactate.
Ox idat ion : This process requires 3-0 mols of 0~ per mol of
lactate or 67-2 yl 0- per ymol lactate.

Anaerobic Contribution to Orb-Web Building

Lactate contribution to orb-web construction was measured by
collecting adult Neoscona dom'icil-Lorn.um in the field as they finished
building their webs. They were dropped into a large volume of swirling
liquid N- and kept frozen until returned to the lab. There, they were
thawed while being homogenized in cold TCA. Lactate analysis was by the
colorimetric method (Ch. II).

Resul ts

Oxygen Consumption

The spiders struggled vigorously during most of the two minute
stimulation period but they reduced their movements in the last minute.
Plots of VO- as a function of exercise and recovery are shown in Figs.
Ill-I, 2, and 3 for temperatures of 15, 25, and 33°C respectively.
Generally, resting and active VO^ were highest in P. audax and lowest in
F. hihemalis.

Increases in VO- during the two minutes of activity were evident
mainly in P. audax. In the other two species increases in metabolic
rate were usually not seen until one to five minutes after the end of
activity. Delayed increases in VO- were especially obvious in

o

CSI

O

a.

o

0)

O

0)

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F. hibemalis. This was most evident at 33°C (Fig. I I 1-3) where it was
possible to use five minute measurement intervals. Peak \/0„ was not
reached until after three minutes of recovery.

The length of the recovery period appeared to be shortest in
P. audax, intermediate in Lyaosa, and longest in Filistata especially
where P. auda:c recovery data are not complicated by activity (Fig. Ill-l).
Recovering individuals of Filistata and Phidippus tended to become active
before their VOj had returned to pre-struggle levels. In P. audax this
occurred at 25° and 33°C as individuals continued activity after the
end of stimulation and never calmed down. This accounts for the continued
high V0„ shown for this species in Figures I II -2 and 3. In Filistata
exploratory and grooming behavior commonly appeared after hO minutes of
recovery. This prevented me from fully measuring the recovery 0^ in
this species. Therefore, the data for recovery 0„ in Filistata
represent a minimum estimate. Recovery and activity VO. for all three
species are given in Table 111-2. Finally, the resting V0-, in Neosoona
domiailiorium measured at 25°C was 2^45 + 22 yl 02(g'h) at STPD (N = 20,
mass = 0.^*92 g + 0.003 g) . These rates were taken from spiders hanging
in "webs" (silk attached to wooden rods in the respirometer flasks).

Anaerobic Metabolism

Lactate concentrations Immediately at the end of exercise and 15
minutes later are shown for F. hibemalis, L. lenta, and P. audax In
Table Ill-l. In Neosoona, lactate concentrations at the completion of
web-building (T. = 23°C, field collected samples) were A. 7 + 0.7,
prosoma: 1 .5 j^ 0. 1 op I sthosoma; and 2.1 j^O.25 ymol s/g whole an imal ,

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Table I I 1-2. Oxygen and lactate metabolism during exercise and

recovery in spiders. Exercise \/02 is calculated for
a five minute interval including the two minutes of
exercise and first three minutes of recovery (see
Methods). Data are derived from Figures IM-1, 2,
and 3 and represents the amount of oxygen used
above the resting \/02 {i.e. the net recovery O2) .
Lactate concentrations are all changes from the
previous reading. Positive readings are increases
in lactate.

A \/02 (vil 02/g) A Lactate (ymols/g)

2-15

■1.7

Ta

Act i ve

Recovery

Species

(°c)

(0-5 min)

(5'

-kO min)

0-2 mi n

F. hibermalis

15

1.7

26.0

2.2

25

2.5

^0.8

5.2

33

1.9

60.6

5.1

L. lenta

15

9.0

28.8

3.0

25

12.1

29.2

k.]

33

S.h

A3. 3

3.9

P. audax

15

15.6

21.2

—

25

lit. 8

59.2^

3. A

33

18.5

90.7^

--

-1.3

Estimated by straight line extrapolation. This was necessary due to
large amounts of activity starting 10 to 15 min after the end of
forced activity.

48

N = ^ for all measurements. Anaerobic accumulations and factorial
increases (values in parentheses) of lactate during web-building were
as follows: prosoma, 2.5 jimols/g (2. IX); opisthosoma 0.6 ( 1 . 7X) ; and
whole spiders 0.7 (1 .5X) .

Heart Rates

Heart rates of alert Filistata and Lycosa at 25''C were ^0 and ^48
beats per minute (bpm) respectively. At the completion of exercise the
respective rates were 1 1 't and 122 bpm. After 25 minutes of recovery
the rates had dropped to about kS bpm in both species.

Pi scussion

Aerobic Metabolism

The pre-exercise (or routine) V0„ shown in Figures I I 1-1, 2, and
3 agree with resting rates calculated for these species using the
appropriate V0-, and Q.^ values from Anderson (1970) and Anderson and
Prestwich (1982).

The factorial aerobic scopes vary between 2.3 to 5-8X resting and
are within the range reported for sp i ders of 2 to 6X resting VO2
(Seymour and Vinegar 1973; Peakall and Witt 1976; Ford 1977a, b;
Prestwich 1977). Aerobic scopes and capacities (maximum '^0^) are
consistently largest in P. audax and smallest in F. hibemalis.

in all three species aerobic capacity increases with T^. However,
aerobic scope declines with temperature because resting MO^ increases
at a faster rate than active MOy- This is consistent with the idea
that maximum 0, exchange is somewhat temperature dependent due to changes

^3

in ventilation and circulation, but is finally limited by the respiratory
exchange surface area (Ultsch 1973)-

It is important to know how close VO^ was to the maximum for each
species. It was partially for this reason that heart rates and lactate
accumulations were measured. Accumulation of lactate implies insufficient
delivery of 0« to the muscles and, therefore, near maximal VO-^ . Heart
rates in spiders are elevated after activity in order to (a) redistribute
hemolymph between prosoma and opisthosoma (Wilson and Bullock 1973), (b)
fully saturate the hemocyanin with 0_ (Angersbach 1978), (c) to provide
0„ necessary for reactions that replenish depleted pools of ~P, and (d)
to circulate lactate from the muscles and to provide 0^ to metabolize
this lactate (Ch. IV). Factors a through c are probably resolved during
the first few minutes of recovery (Wilson and Bullock 1973; Stewart and
Martin 197^4; Angersbach 1978; Chs. IV and VI) while the oxidation of
lactate may take longer than one half hour (Ch. IV).

Together, the heart rate and lactate data indicate that exercise
was near maximal. Heart rates at the completion of activity were aa.
35% those found in free-running spiders and lactate concentrations ranged
between 35 and 10% those found in exhausted spiders. Any large accumu-
lations of lactate such as these indicate a relative insufficiency of
Op delivery to active muscles. This implies 0- consumption is at peak
level and further increases in work could only be fueled by greater
production of lactate.

In spiders not possessing extensive trachea, high work loads are
not compatible with the highest rates of oxygen consumption. This is
because maximal activity is accompanied by high prosomal hemolymph

50

pressures that prevent delivery of oxygenated hemolymph to the active
prosomal muscles (Wilson 1970; Stewart and Martin 197^; Anderson and
Prestwich 1973; Ch. V) . Thus, the physiological state of a spider's
prosoma during vigorous activity resembles the situation in diving
animals: muscles reach peak activity while relying only on 0^ already
present in the animal. In spiders this 0_ is bound to the hemocyanin
already present in the prosoma. This is probably a small amount of 0_
because (a) in resting spiders the arterial hemocyanin may be only half
saturated (Angersbach 1978) and (b) the 0^ capacity of spider hemolymph
is low. Thus, peak VO^ for maximal activity will be measured late in
exercise as prosomal pressure drops (Stewart and Martin 197'*; Anderson
and Prestwich 1975; Ch. V) and normal gas exchange and circulation is
resumed (Angersbach 1978). This unusual arrangement in part explains
why peak VO^ is observed late in the exercise period or early in recovery
(Figs. I I 1-1, 2, and 3)- The highest rates of oxygen consumption in
spiders may occur when they are engaged in less than maximal activities
that require low prosomal hemolymph pressures and thus permit constant
circulation and exchange of 0^ (Anderson pers. comm. ; see Appendix l).

Estimated total mass-specific VO^ (ul 0„/g) for activity and
recovery are shown in Table I 11-2 (see Methods for a description of the
measurement interval). The differences in the exchange abilities between
the three species are evident with F. hibermalis using the least and
P. audax the most 0„ in what appeared to be comparable activity. The
opposite pattern occurred in recovery. Filistata hihemalis generally
used the greatest amount of 0^ , consistent with its usually largest
lactate accumulations (this ignores the 25° and 33°C data for P. audax

because continued activity of these spiders prevented measurement of
recovery VO^) . Tlie actual difference in recovery VO- between F.
hibemalis and the other two species is even greater than indicated
in Table I I 1-2. At the end of ^0 minutes Filistata still had a slightly
elevated VO- while Lyaosa and Fhidippus (15°C only) had returned to
rest! ng va lues .

Lactate Removal

In Lyaosa (and Phidippus at 15°C), it is probable that all lactate
was removed after 35 minutes of recovery because V0„ was back to resting
levels by this time and because this Is more than sufficient time to
remove greater amounts of lactate from free-ranging spiders (Ch. IV).
Total recovery V0„ is probably a reasonable estimate of the amount of
0- required to remove the accumulated lactate.

In Filistata, the V0„ was still elevated at the end of hS minutes
(Fig. I 11-1-3) when spontaneous activity increased the VO^ of most
Individuals. However, some of the lactate accumulation was probably
still present at this time given that lactate removal Is slower In
free-ranging individuals of this species than it Is In Lycosa and
Phidippus. Therefore, recovery VO^ on Table I I 1-2 is probably an
underestimate of the actual requirements for recovery In this species.

The pathway(s) used to remove lactate during the recovery period
cannot be actually demonstrated without the use of labelled compound.
However, a reasonable guess as to the fate of lactate can be made by
calculating the amount of 0- required to remove lactate through either
gl uconeogenes i s or oxidation and then comparing these numbers to the
measured recovery oxygen. This Is done in Table I I 1-3 for each lactate

52

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53

accumulation. The measured V0„ are close to those required to use most
of the lactate in gl uconeogenes i s and completely oxidize only a small
portion. If this is indeed the case, then spiders resemble vertebrates
where about 80?^ of the lactate is reconverted to hexose and 20% is
completely oxidized (Bennett 1978).

Relative Contributions of Aerobic and Anaerobic Metabolism During
Peak Activity

High-energy phosphate compounds for burst activity come from three
sources: aerobic metabolism, anaerobic "lactacid" metabolism, and
depletion of phosphagen stores (pr i nc ipal ly arg in ine phosphate and ATP).
Calculations of the relative importance of aerobic and lactacid power
generation for the cond i t ions used in this study are given on Table 111-^.
At 25°C estimates of total high-energy phosphate production are similar
in all three species. This is consistent with the observation that the
spiders all underwent similar intensities of struggles during the two
minute activity period.

Consistent differences in anaerobic dependencies exist between the
species: Filistata is definitely the least aerobic spider deriving
between 8? to Sk% of its power from lactate production. The other two
species have lower anaerobic dependencies, utilizing anaerobiosis for
ca. S5% {Phidippus) and 65% {Lycosa) of the total. These figures compare
to a range of 58 to 96% anaerobic dependence for burst activities in
lower vertebrates (Bennett 1978). The calculations indicate that the
relative importance of anaerobic power generation does not change as a
function of temperature.

For three reasons these estimates should be treated with caution:
(a) aerobic inputs during activity are to a degree uncertain (see

Table lll-A. Estimated anaerobic dependences during a two

minute struggle in a respirometer flasl<. High-
energy phosphate figures are derived from the
VOo and lactate accumulations on Table 11-2.

~P Bonds Formed

5k

Spec i es

F. hibevmlis

15

0.5

25

0.7

33

0.5

L. lenta

15

2.5

25

3.^

33

2.7

P. audax

15

k.2

naerob

Ic

Total

% Anaerobic

3.3

3.8

87^o

7.8

8.5

92^0

7.6

8.1

Sk%

^.5

7.0

6k%

6.2

9.6

esz

5.8

8.5

68%

5.1

9.3

55%

55

Methods and Discussion), (b) free-ranging spiders have lactate accumu-
lations that are about twice as great as those used in the calculations
in Table I I 1-4, and (c) inputs from phosphagens are ignored. Biases
due to factors a and b tend to cancel each other. Factor c could be
significant. Using values for whole spider concentrations of arginine
phosphate (AP) reported by Di Jeso et at. (1967) and assuming all AP
is hydrolyzed during a struggle, the proportional dependence on lactate
drops to between 43 and 10% with AP providing between 21 and "iSX of the
power. Thus, the contribution of phosphagens may be important.

Respiratory Surface Area and Anaerobic Dependence

All results are consistent with the hypothesis that an inverse
relationship exists between anaerobic dependence and respiratory surface
area (Fig. 11-2). Relative dependencies of Lycosa and FiZistata are
also consistent with the observations of Anderson and Prestwich (1982)
on the abilities of the two spider's respiratory-circulatory systems
to deliver 0„ to the tissues. They found that at 20°C it took resting
Filistata an average of 28 heart beats to deliver 1 ul of 0„ compared to
16 beats for Lycosa. These two species have similar maximum heart rates;
therefore, the rate of 0^ delivery to the tissues should be less in
Filistata in agreement with the much slower recovery and lower peak
VO2 in this species (Table 11-2; Figs. Ill-l, 2, and 3).

Anaerobic Dependence During Orb-Web Building

Long-term activities such as orb-web construction contrast sharply
with the burst activities discussed above. Table I! 1-5 shows the estimated
anaerobic dependence of orb-web building to be low in Neosaona

56

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djom-ic-hliovlvm. In this species, web-building is essentially entirely
aerobic if costs are estimated at the moment the web is completed.
In contrast, burst activity is approximately AO^ anaerobic (using data
for Neosoona from Table 11-3). By reliance on aerobic metabolism, an
orb-weaver uses a more energy- and substrate-efficient process and
maintains a reserve (anaerobic) power generating capacity for use if
it is threatened during web-building and needs to escape.

CHAPTER IV
ACTIVITY AND RECOVERY IN SPIDERS

Summary

1. The relationship between lactate accumulation, heart rate, and
fatigue in maximally active spiders was Investigated to learn the
cause of fatigue in spiders.

2. Spiders that were forced to run for two minutes show two stages of
fatigue. In the phase i, they lose nearly two thirds of their maximal
speed in 20 to 30 seconds {Lyoosa and Filistata, respectively). This
striking decrease in speed is followed by a slower phase of fatigue
that takes the rest of the activity period and Is characterized by a
much slower rate of decrease in speed. Scorpions have a similar
pattern of maximal activity (Fig. IV-1).

3. Maximum heart rates are not reached until late in the two minute
activity bout or, more commonly, early in recovery.

4. Lactate accumulations in the prosoma similar to levels known to
cause fatigue in other animals are reached after about one minute
of maximal activity (Figs. IV-5, 6, and 7). However, lactate
accumulations in Lyaosa during the first 20 sec of activity (Fig.
IV-8) are not high enough to explain the rapid fatigue that occurs
during this time (phase I; see above).

5. During recovery, lactate is apparently circulated to the opisthosoma
from the prosoma. In the opisthosoma It is probably either completely

58

59

oxidized or resynthes i zed into glucose. This arrangement allows
rapid removal of lactate from the muscles where it is probably
responsible for fatigue to an area where it can be metabolized
without affecting the ability of the spider to locomote. This
decreases the time necessary for recovery.

I ntroduct ion

In the preceding chapters I have demonstrated that the major
anaerobic by-product in spiders is D-lactate (Ch. II) and that anaerobic
metabolism is the dominant energy supply during maximal activity (Ch.
III). The purpose of the experiments described in this chapter are to
detail the changes in running speed, lactate concentration, and heart
rate that occur during two minute struggles and during the recovery
period following these struggles. Specifically, the data will be used
to test the hypothesis that lactate accumulations are the main limitation
on maximal exercise in spiders.

Methods

An i ma 1 s

Three species of spiders (F. hibemalis, L. lenta, and P. audax)
and one scorpion (C. hentzi) were used. All individuals were maintained
in plastic boxes or Petri dishes in the lab at a temperature of aa.
25°C. They were last fed 7 to 10 days before use in an experiment
(Anderson 197^*). Water was freely available to all individuals except
F. htbemal'ts (this species dies when exposed to moist conditions).

60

Running Speeds

Animals were exercised in an area measuring 1.0 x 0.3 x 0.2 meters
having an interior mari<ed in 0.1 m grids. Spiders were placed in the
runway and then prodded with a blunt rod to force them to run. Speeds
were determined by measuring the distance the spider traveled over a
five second period and were expressed as either cm/s or prosoma length/s.
In spiders the latter measure is preferable to body lengths/s since the
size of the opisthosoma varies greatly in different species and according
to the nutritional status of an individual (Anderson 197^+). Prosomal
lengths were determined on CO--anesthes ized spiders after the completion
of speed measurements by using a Gaertner measuring microscope. All
measurements were made to 0.01 mm.

Not all spiders ran continuously during the two minute exercise
period. Some individuals spent a great deal of time attacking the rod.
However, the attacks were usually brief. If any attack lasted more than 10
sec the data were discarded. At the end of the two minute exercise
period most spiders moved only very slowly and only when sharply prodded.
I considered this to be exhaustion and it occurred in most spiders after
between 90 to 150 sec of activity.

At the completion of 120 sec activity, spiders were placed in a
vial for either five or ten minutes to recover. They were then put back
in the runway and their speed was measured over the initial five second
period of activity to provide an index of recovery.

Heart Rates

Heart rates were measured on both free and restrained spiders using
the methods of Carrel 1 and Heathcote (1976) and Anderson and Prestwich

61

(1982). Restrained spiders were glued to glass rods as described in
Chapter V Methods except that no other attachments were made to the
animals. Heart rates in alert spiders were about 1.5 to 2 times those
obtained for resting spiders of the same species (Anderson and Prestwich
1982).

Changes in heart rate in active and recovering spiders were
determined by mechanically stimulating the restrained spiders for two
minutes and then allowing them to remain undisturbed for up to one hour
while they recovered. As a control, several L. lenta were exercised at
25°C in the runway described above, quickly placed in a glass box, and
their heart rate during recovery was measured. No differences were
found between these and the restrained spiders heart rates during
recovery.

Lactate Determinations

Lactate concentrations were measured on spiders at rest and after
various intervals of running and recovery. Samples were obtained by
freezing the spiders in liquid N- and then dividing them into prosomal
and opisthosomal sections. These pieces were quickly weighed to prevent
condensation, ground to a fine powder under liquid N„ , and then
homogenized in cold 10^ TCA. This solution was filtered and later
analyzed according to the procedures for the colorimetric determinations
outlined in the Chapter I! Methods.

Stat i St ics

Standard errors were calculated for all means. Tests of significance
were based on Student's T-test and the 1 evel of s i gn i f icance was def i ned at 0. 05.

62

Resul ts

Runn i ng

Prodding results in activity patterns that, while maximal, are
nevertheless somewhat abnormal, especially in P. audax. In the field,
Phidippus actively hunts in shrubbery using vision to locate prey and
avoid enemies. A dragline of silk is constantly laid down by the spider
as it runs and jumps about. When placed in a running arena and touched,
they often sidestepped and dodged the prod, frequently making 180° turns
instead of running. While turning, they often became entangled in their
own draglines. The result of these maneuvers was to slow them down. How-
ever, these jumping spiders were maximally engaged in constant activity
and most individuals refused to move more than minimally at the end of
the two minute stimulation period.

Lyaosa tenta apparently also has good vision but individuals of
this species attacked the prod instead of sidestepping it. These attacks
were frequent and violent: the rod was tightly seized and repeatedly
bitten, reminiscent of prey capture (Rovner I98O). When these spiders
were shaken loose, they would run rapidly. This pattern was similar to
what I observed in the field. When I disturbed a L. lenta, it fled to
its burrow or some nearby cover. If continuously pursued, Lyaosa attacked.
Thus, the forced activity in the arena approximated field behavior in this
spec i es .

Finally, F. hihermatis individuals ran continuously as long as
prodded. After repeated prods some assumed a tight, ba 1 1 - 1 i ke posture
and never attempted to attack. In field conditions, a threat is met by

63

retreat into the tubular sanctuary of the web. If a spider cannot locate
the retreat, it will run a considerable distance.

The results of forced running at 25°C are shown in Figure I V- I ,
Speeds are expressed as prosomal lengths per sec. Integration of each
curve gives the total relative distance (prosomal lengths) traveled in
two minutes. By making the total distance traveled by Lyoosa equal to 100^,
the relative distances covered by Filistata and Phidippus were 3(>% and 10A°'
respect ively with nostatisticallysignificantdifferences amongst these spec ies

The effect of T on running speed is illustrated for Filistata in
Figure IV-Z and summarized for all species on Table IV-1. For the table,
the running speeds have been fitted to an equation for exponential decay:

Ioq^qS = i + d(t) (1)

where S is the absolute speed in mm/sec, i is the log,-, of the initial
(maximum) speed, d is the rate of change in speed and t is the total time
since the start of running in seconds. Coefficients of determination (r )
range from 0.75 to 0.96 with most above 0.86 indicating that the data fit
the model .

The values of i can be used to compare the initial running speeds.
In each species, the initial speed increas'es with T and L. lenta always

d

has the fas test i n i t ia 1 speed. The rate of change of speed (d) is generally
similar in all species at any common temperature. In both Lycosa and
Filistata, the smallest decreases in speed with respect to running time
occur at 25°C and the greatest rates of decrease are at 33°C.

Another measure of the decrease In speed as a function of running
time is the time required for running speed to decrease to a value that
is about a third of the initial speed (see Table IV-1 for a more rigorous
definition of this measure of exhaustion). By this measure, L. lenta

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Table IV-1. Running speeds and recovery in spiders and a scorpion.
The data are fitted to a mathematical model of the
form: logio S = i + d(t) where S is running speed in
cm/sec, i is the initial speed (average over 0 to 5
sec), d is the rate of change of speed, and t is the
running time in seconds. Coefficients of determination
(r ) are given for each regression. The last columns
are measures of fatigue and recovery and are explained
below and in the text.

Percent of

Species

N

Ta

Regression Equation
i d r2

Length
Phase
(seconc

of
is)

Max imum
+5

L \J 1

Speed^
+ 10

C. hentzi

10

25°C

2.1151

0.0077

0.93

28

F. hibemalis

10

15°C

2.1653

0.0051

0.94

37

54%

71%

12

25°C

2.2951

0.0048

0.87

30

49°^

76%

9

33°C

2.4222

0.0059

0.92

31

60%

74%

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12

15°C

2.2067

0.0051

0.89

38

61%

78%

12

25°C

2.4957

0.0044

0.97

20

73%

100%

12

33°C

2.5998

0.0056

0.86

18

63%

84%

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7

25°C

2.1889

0.0042

0.96

45

84%

—

^Phase I is defined as the time required for a spider to exhaust such that it
is only running at 133% of its final speed. Thus, it has decreased in speed
2/3s of the way to a minimum (phase II fatigue) speed.

Percent of maximum speed is the maximum running speed after five or ten
minutes of recovery divided by the initial (maximum speed, see Figs. IV-1
and 2) times 100.

69

slov^/s the most rapidly, especially at 25 and 33°C, having reached this
speed in 20 sec or less. This is largely a reflection of the high
initial running speed in this species. By contrast, it takes P. audax
hS sec to have slowed by an equal proportion (also see Fig. IV-1).

By the time the speed of Lyaosa and Filistata had slowed by two
thirds, aa. \0% of the individuals were plainly dragging their IVth
pair of legs. When this occurred, it had always happened before the
completion of 20 sec of running. There was no correlation between initial
running speed and the development of leg dragging behavior. In the
remaining 90°o of the individuals of both these species, this behavior
was not observed.

Comparison of running speeds after five or ten minutes of recovery
with the initial (0-5 sec) running speeds gives a measure of recovery
(Figs. IV-l, 2; Table IV-l). Although all spiders did comparable amounts
of exercise over two minutes (except at 33°C--see Table iV-6), P. audax
and L. lenta tend to recover the most rapidly. Recovery rates (speed
after resting/initial running speed) are similar at all three tempera-
tures in F. hibermalis; however, in Lycosa recovery is fastest at 25°C.

Finally, the running and recovery performances of "C. hentzi, a
scorpion, is similar to the three species of spiders in terms of both
relative speed (Fig. IV-l) and total distance covered (91% of Lycosa).
However, the rate of decrease in speed is greater in this species
(Table IV-l).

Heart Rates

Figure IV-3 shows the effect of exercise and recovery on heart rates
of restrained L. lenta and F. hibemalis at 25°C. Maximum rates are

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similar in both species and occur either near the end of exercise or
early in the recovery period. However, the magnitude and rate of the
increase in heart rate is greater and the recovery is slower in F.
hibemalis. During recovery, interspecific heart rates are significantly
higher in Filistata after five minutes of recovery and remain so until
+33 min into the recovery period. At this time, Lyaosa has a rate below
its routine heart rate while Filistata' s rate is still slightly (but not
significantly) elevated.

The effect of temperature on heart rate is shown for F. hibenmalis
in Fig. \y-h and the data for both species are summarized in Table IV-2.
Maximum rate and pattern of recovery is similar in both species {Lyaosa
and Filistata) at all three temperatures. Both species recover most
rapidly at their acclimation temperature (25°C) , although Lyaosa recovers
sooner than Filistata. Finally, the Q.^ for both alert and maximal heart
rates are between 1.3 and 1.8.

Lactate Production

Figure IV-5, bottom panel, shows change in lactate concentrations
for whole spiders at 25°C. At any given time there is usually no
significant difference in lactate concentrations between species. It is
not until 15 to 25 min into the recovery period that lactate concentrations
are significantly lower than those found at the end of exercise. Complete
removal of lactate (recovery) probably takes over 30 minutes in all
three species.

Because the relat ive opisthosomal sizes differ in these three species,
whole animal lactate concentrations are misleading as they can obscure

73

Table \\l-2. Heart rates in two species of spiders as a
function of activity and temperature. All
spiders were acclimated at 25°C.

Percent of

Heart Rates Maximum

(beats/min) Rate ,.„, ^^ jb
^ 66^ Decreased

Species N T Alert Maximum +5 +10 Time (min)

F. hibemalis

10

15°C

27

75

99%

87%

> 30

\h

25°C

ko

135

in

69%

19

8

33°C

Ih

180

88%

7^4%

30

L. lenta

10

15°C

--

76

87%

71%

--

17

25°C

56

138

62%

49%

7

10

33°C

70

170

85%

86%

> 30

Heart rate after 5 or 10 minutes of recovery divided by the
maximum observed rate times 100.

The time in minutes required for the heart rate to decrease from
maximum to a value that is 133% greater than the alert heart
rate.

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76

interspecific differences in lactate in the working muscles of the prosma
and legs (Table 11-3)- Partition into the prosoma (and legs) and
opisthosoma of the same individuals as shown in Figure IV-SC points out
interspecific differences.

In the prosoma and legs (Fig. IV-S, top panel) the resting and
exhaustion (120 sec) lactate concentrations and also the maximum rate
of lactate production (anaerobic scope, Bennett 1978) are different in
all three species, being distinctly higher in F. hibemalis . By contrast,
the highly active jumping spider P. audax was the lowest in all of these
categories. A detailed look at the changes in lactate concentration that
occur during the first 30 sec of activity in L, lenta is shown in Figure
IV-8. In all species, recovery is characterized by a steady decrease in
lactate concentration over 15 to 25 minutes (Fig. l\/-5; the initial rise
in concentration in L. lenta during the recovery period is not statisti-
cally s i gn i f icant . )

The handling of lactate by the opisthosoma (Fig. l\/-5) is
different than in the prosoma. Lactate accumulations during activity are
much smaller than in the prosoma and are negatively correlated with
opisthosomal size. Lactate concentrations during the recovery period
were generally significantly higher than the pre-exercise concentrations.
However, there are no statistically significant differences between
opisthosomal lactate concentrations after 15 to 25 min of recovery
compared to the concentration at the end of exercise. While prosomal
lactate concentrations dropped in all species during this time,
opisthosomal concentrations remained unchanged and at an elevated
level .

Figure l\/-5. The accumulation and removal of lactate during

exercise and recovery in three species of spiders
at 25°C. Note that during recovery, prosomal
lactate concentrations drop while those of the
opisthosoma remain elevated or increase. Standard
errors are shown as bars. (N values are for each point.)

Key: ■ F. hibermalis N = 9

O L. lenta N = 12

A P. audax N = 6

All spiders were acclimated at 25°C.

73

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79

The effect of temperature on lactate accumulation is shown in
Figures IV-6 and 7 for F. hibernalis and L. lanta, respectively. A
summary of the most pertinent data on lactate concentrations in activity
and recovery is shown given in Tables IV-3, ^, and 5. The resting
lactate concentrations increase with T in all tagamata and in both
species. However, maximum lactate concentrations are the greatest at the
acclimation temperature. Temperature affects the rate of accumulation of
lactate differently in different species. In Filistata, the maximum rate
of lactate accumulation (anaerobic scope) is highest at the acclimation
temperature of 25°C and is lower at both 15 and 33°C, while in Lyaosa
the anaerobic scope increases with T. between 15 and 33°C. Recovery in
both species is most rapid at the acclimation temperature. At 15 and
33°C the whole spider lactate concentration increases or remains constant
for the first ten minutes of the recovery period. However, the increases
are not significant statistically. (During this time, prosomal lactate
concentrations decrease.)

A different picture is presented in the opisthosoma (Table IV-A)
where there is an increase in lactate concentration over the first five
minutes of recovery. At temperatures other than 25°C, opisthosomal
lactate concentrations continue to increase for at least 15 min (Figs.
IV-6 and 7, middle panels). Thus, the total body lactate concentration
remains constant since as the prosomal lactate concentration decreases,
the opisthosomal concentration increases.

Table I\/-5 contains data for the scorpion C. hentzi. Although
lactate concentrations seem low compared to the spiders, this is
partially due to the large mass of non-motion generating tissues.

Figure IV-6. The accumulation and removal of lactate in

F. hibernalis at three different temperatures.
The spiders were all acclimated at 25°C. Note
the very slow recoveries at non-acclimation
temperatures .

Key: 33°C

25°C

15°C

Standard errors are not shown but were consistently
less than 12^ of any mean value except for the
opisthosomal data at 15 and 33°C. Here SE approached
20^.

o

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TIME (M"N)

Figure IV-7- The accumulation and removal of lactate in
L. lenta at three different temperatures.
The spiders were all acclimated at 25°C.
Note the very slow recoveries at non-acclimation
temperatures .

Key: 33°C

25°C

15°C

Standard errors are not shown but were consistently
less than ]h% of each mean value with most less than
10^ of the mean.

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87

The non-prosomal mass (abdomen and telson) of the scorpion averages 63%
of the total body size.

Pi scuss ion

Running Speeds

One problem with the previous discussions of exhaustion in spiders is
the lack of a quantitative description of fatigue in running spiders (Millot
19^9; Wilson and Bullocl< 1973; Linzen and Gallowitz 1975). The data pre-
sented in Figures IV-l and 2 partially remedy this situation. However,
they are somewhat misleading in terms of their description of the earliest
moments of activity. During the initial 15 to 20 sec of struggle, spiders
jump about alot, especially L. lenta and P. audax. Thus, the data reported
in these figures and table represent minimal estimates of the work the
spiders are doing. Given this underestimate, the figures do show that
spiders generally slow to aa. one-third of their initial speeds before 30
sec of activity are complete. Thus it is reasonable to divide activity into
two phases: an early, rapid-fatigue period (phase l) and the period that
follows, which is characterized by a slower decrease in speed (phase II).
Mathematically, I have demarked these periods as being before and after
the moment that a spider has slowed to a speed that is 133% of its final
speed. This mathematical definition corresponds well with the cessation
of any jumping behavior in Filistata and Lycosa. Thus, phase I corresponds
to the first 15 [Lycosa) to ^0 sec [Phidippus) of the two minute activity
per iod .

The fact that about 10% of L. lenta and F. hihermalis drag their IVth
pair of legs during late phase I is significant. This is consistent with

88

the hydrostatic insufficiency hypothesis of Wilson and Bullock (1973).
Forward motion is produced in the first through third pair of legs
through either flexion (pair I) or rotation (pairs II and III) while in
pair iV it is due to extension (Parry and Brown 1959; Wilson 1970).
Since much or all of extension of pairs l-lll is done while the legs
are off the substratum, the work required is relatively small. Extension
of these legs could be accomplished by a low hemolymph pressure (Manton
1958). However the IVth pair of legs require hemolymph
under high pressure in order to extend rapidly while in contact with the
substratum. Furthermore, these rear legs would be expected to be crucial
in providing much of the forward thrust in spider locomotion (Parry and
Brown 1959b). The spiders that were dragging their legs during late
phase I could be suffering from inadequate pressures and/or amounts of
prosomal hemolymph. This possibility and its relationship to fatigue will
be specifically discussed in Chapter V.

Phase II is characterized by relatively slow movements of all
limbs and a gradual decrease in speed (compared to phase I). After one
minute of activity, many spiders refuse to move unless constantly prodded.
This corresponds to a time when the prosomas contain considerable amounts
of lactate (Figs. \\I-S through 7). The final running speeds shown in
Figures IV-l and 2 are biased towards high speeds since spiders that had
refused to move for much of the previous 30 seconds (by assuming a ball-
like posture) often burst into activity.

Suggestive similarities exist between fatigue in spiders and
scorpions. Scorpions do not use a hydrostatic skeleton for leg extension
(Manton 1958). However, their pattern of running when subject to

89

continuous stimulation resembles that of spiders (Fig. IV-1). This
argues against the notion that fatigue in spiders might be largely related
to hydrostatic insufficiency (Wilson and Bullock 1973).

Recovery of running ability in spiders is a slow process. I used
a measure of recovery that applied only to the first five seconds of
phase I activity. Spiders forced to run longer than 15 sec, even after
10 min of recovery, exhausted within kS sec. This is strong evidence
that complete (ultimate?) "locomotory collapse" (Wilson and Bullock
1973; Linzen andGallowitz 1975) has nothing to do with hydrostatic
insufficiency. Given the high heart rates of spiders during recovery
(Figs. IV-3, ^; Table l\/-2) hemolymph would seemingly be adequately
redistributed after 10 min of recovery. If hydrostatics are the main
limit to activity, running after recovery would resemble the running of
completely rested animals. Instead, this limitation on activity is
probably due to continued large concentrations of anaerobic byproducts
(Figs. IV-5, 6, 7; Tables IV-3, ^, and 5).

The effect of temperature on locomotion in L, lenta and F.
hibemaZis can be evaluated in two ways: comparison of maximum speeds
and total distance traveled. Maximum speeds are synonomous with initial
speeds while total distances traveled are obtained by integrating
each speed versus time curve. In terms of these two measures, temperature
effects are presented in Tables l\/-6 and 7. The Q,_ values (Table IV-7)
are nearly all below two indicating that the spiders' locomotory patterns
are somewhat independent of T . As such, an 18°C increase in T results
in a 210/3 increase in distance traveled over 2 min and a 315^ increase
in maximum speed in Lycosa and respective increases of 170 and 195^ in

90

Table IV-6. Total distance traveled in two
minutes of maximal activity as
a function of temperature.
Distances (total prosomal length)
for Lyoosa and Filistata were
converted to percentages by
defining the distance Lycosa
traveled at 25°C as 100^.

Species

Relative Distance Traveled
15°C 25°C 33°C

F. hibemalis

68°^

%%

\\5%

L. tenta

68%

100%

160%

91

Table IV-?. The effect of temperature on locomotion in
F. hike'pnalis and L. lenta. Spiders were
acclimated at 25°C; the temperature range of
15°C to 33°C is possible during a spring day
in Gainesville, Florida.

Temperature
Species Range (°C)

^10

Ini

tial

Total

Sp

eed

Di stance

F. hibemalis

15-25

1.49

1.23

25-33

1.40

1.52

15-33

1.45

1.35

L. lenta

15-25

1.75

1.38

25-33

2.1

1.67

15-33

1.9

1.50

92

Filistata. Increases of 350/? would be expected over this range if the
Q.Q were equal to 2. Thus, a spider threatened at temperatures below
those to which it is accustomed can move faster than would be expected
based on a Q ^ of between 2 and 2.5 calculated from its resting V0_
(Anderson 1970).

Heart Rates

The change of heart rate as a function of exercise and recovery
varies between species in spiders. Angersbach (1978) reported peak
heart rates in a tarantula, Dugesiella calif ormicum , within 2 min after
the completion of a struggle of one to two minutes. Wilson (1967)
reported a similar phenomenon in Heteropoda. These observations agree
with mine for L, tenia (Fig. IV-3). However, in F. hibematis the rate
rapidly approaches the maximum and remains at an elevated value longer
than in L. tenia at the same exercise level. This continued, elevated
rate could be associated with the equal or larger lactate accumulation
coupled with its smaller respiratory exchange capacity (Anderson 1970;
Anderson and Prestwich 1982; Fig. 11-2).

Temperature affects heart rate in the same manner as it affects
running speed and total distance, and lactate production and removal.
The Q. ^ for alert and maximal heart rates varies between 1.3 and 1.8
(Table \\J-2) indicating these processes are less temperature dependent
than are most chemical reactions. As a result, the spider gains a small
degree, of thermal independence.

Heart rate is not a totally adequate measurement of the ability
of the circulatory system to deliver 0~, remove lactate, or redistribute

93

hemolymph after exercise. Total cardiac output, the product of stroke
volume and heart rate, is the preferred measurement. However, I was not
able to quantitatively measure stroke volume in either Filistata or
Lyaosa. Visual observations were inconsistent although there did
appear to be a tendency for maximum stroke volumes to come after the
completion of activity (based on the intensity of the transmitted light
f 1 uctuat ions) .

Lacking direct data on stroke volume in this species, it is nonethe-
less possible to calculate the stroke volume for both alert and active
tarantulas from published data. These calculations are given in Appendix
1. Inasmuch as tarantulas can serve as a general model for spiders (see
problems with this approach discussed previously in this section) the
calculations indicate that while the stroke volume may increase slightly
after activity, the heart rate is the major determining factor in changes
in cardiac output. Estimated factorial increases in cardiac output
range from about A to 6 fold for the tarantula. This corresponds to up
to a ten fold increase in VO- (Anderson pers. comm.). The difference is
due to an increased loading and unloading of 0^ per volume hemolymph
(Angersbach 1978). The speculative nature of these calculations cannot
be over-emphasized.

Lactate Production

The color imetr i cal 1 y determined lactate concentrations reported in
this chapter are maximal concentrations for the conditions of time of
exercise and T» under which they were taken. Anaerobic scopes at 25°C
(maximum rate of lactate production) and anaerobic capacities (net lactate

9A

accumulations) are on the low end of the range of values reported for
terrestrial ectotherms (Bennett 1978). However, if only the prosoma is
considered, the anaerobic scopes and capacities are comparable to those
of vertebrates with highly developed anaerobic abilities. Thus, maximum
lactate concentrations in the motion-generating portion of the spider,
the prosoma, are consistent with levels that are associated with fatigue
in other animals. Ultimately, phase II fatigue is therefore related to
anaerobic accumulations. Phase I fatigue is probably not related to
lactate accumulation. In Lyaosa lenta the two-thirds running speed
reduction of phase I takes aa. 20 sec (Fig. IV-1; Table IV-l); however,
prosoma lactate accumulations are only aa. 20% of maximum during this
period (JFig. l\,'-8). Furthermore, during the first 10 sec of phase 1 when
most of the speed decrease occurs, only very slight lactate increases
occur. Thus, a non-lactate factor(s) is responsible for phase I fatigue.

In Chapter II, the hypothesis was advanced that the anaerobic
capacities of spiders are directly related to the intensity of activity
and inversely related to the book lung surface area (Fig. 11-2). This
hypothesis can be further examined using this chapter's data for the
effect of T on anaerobic accumulations and running speeds. Maximum
running speed and total distance traveled increase with T in both
Lyaosa and Filistata. These parameters increase faster in Lyaosa:
at 15°C both species' activity patterns are nearly identical while at
33°C Lyaosa is much more active (Tables IV-l, 6, and 7). However,
anaerobic capacities for both species follow a different pattern being
largest at the acclimation temperature of 25°C (Fig. WIS). Moreover,
unlike 25°C where Filistata has a larger anaerobic accumulation, the

Figure IV-S. Anaerobic capacities in 25°C acclimated F. hibernalis

( ■ ) and L. lenta ( — o — ). Anaerobic

capacity is largest at the acclimation temperature.

96

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99

anaerobic capacities of both species are nearly identical at 15 and 33°C.
While the 33°C data are not inconsistent with a relationship between
book lung surface area and anaerobic accumulation (since Lycosa runs much
further than Filistata at this temperature), the 15°C data do present
some difficulties for the proposed correlation. At this temperature,
both species' activity patterns are nearly identical and therefore,
lactate accumulations are expected to be much greater in FiZistata. This
inconsistency points out the need for further testing of the surface area--
anaerobic accumulation hypothesis. It does serve to remind one that other
factors may also be important and that their importance may vary at
different temperatures in different species,e.r.,different use of
stored forms of high energy phosphate such as arginine phosphate (Di Jeso
et at. 1967) .

Lactate Removal (Recovery)

For whole spiders, removal of lactate (recovery) occurs most
rapidly in P. audax and L. tenia at 25°C. Fifteen minutes after exercise,
lactate concentrations drop by over 50% while in FiZistata they hover
near 75% of the maximal value (Figs. IV-5-7; Table iV-3-5). At 15 and
33°C recovery is a very slow process, there being essentially no change
in whole spider lactate after 15 min of recovery.

Examination of the changes in prosomal and opisthosomal lactate
concentrations suggest how the lactate is subsequently handled. In the
prosoma (Table l\/-3; Figs. \\I-S through 7), lactate drops more rapidly
than in the whole animal. This removal is probably not entirely the
result of oxidation of lactate or gl uconeogenes I s . instead, the lactate

100

appears to be moving to the op i sthosoma as lactate concentrations in
this compartment either remain relatively constant or increase while the
spider is inactive and recovering (Table IV-'+i Figs. IV-S through 7).

The exact fate of the lactate in the opisthosma can only be guessed.
Evidence was presented earlier suggesting that most lactate is used as a
substrate for gl uconeogenes i s (Table I I 1-3). There are two likely
locations for lactate metabolism: the heart and digestive diverticulum.
The heart is probably always exposed to high POj (Angersbach 1978). If
spiders, like the horseshoe crab Limulus , possess an LDH that favors
oxidation of lactate (Long and Kaplan 1968), then the heart could be a
net oxidizer of muscle-produced lactate, much as in the chordate heart
(Hochachka and Somero 1973). However, ratios of recovery oxygen to
lactate removed suggest that most lactate is used for gl uconeogenes i s
(Table I I 1-3). The most likely site for this process is the digestive
diverticula and associated cells (Millot 19^9). Evidence for their
involvement will be presented later (see Ch. Vl).

The slow decrease in lactate at 15 and 33°C implies a slower recovery
at these temperatures. However, recovery of the ability to run is faster
than might be expected based on whole spider lactate concentrations.
This is because a considerable amount of lactate has been shifted from
the prosomal musculature to the opisthosoma. Additionally, the ability
to locomote at maximal speeds for brief periods of time returns nearly
as quickly at 15 and 33° as it does at 25°C (Table IV-1; Fig. IV-2).
Replenishment of phosphagen stores may be a major factor in the recovery
of the ability to run at maximum rates (see Ch. Vl). The ability to run
quickly, even if only for brief periods, doubtlessly has high survival

101

value for a spider facing danger. Transport of lactate from the prosoma
and legs to the opisthosoma helps in this process. Although it does not
result in an immediate decrease In whole animal lactate, transport
removes the substance from the muscles where it contributes to fatigue
and relocates the lactate in the tissues that may convert it to circu-
lating carbohydrate stores.

CHAPTER V
THE HYDROSTATIC FATIGUE HYPOTHESIS

Summary

1. Leg pressures were monitored in maximally struggling, restrained
Fili-stata hibemalis.

2. During the first few seconds of activity, pressures Increased rapidly
to 100-200 mm Hg. Peak pressures of ^50 mmHg are not reached until
after 20 sec of struggle (Fig. \l-3) •

3. In spiders with tight ligatures around their pedicels, pressures
reached high values sooner than in non-ligatured spiders.

k. The maintenance of high pressure throughout a period of time when

free-running Filistata rapidly slow down (Ch. IV) is contrary to the
Idea that defects in the hydrostatic leg extension mechanism of spiders
results in fatigue (Wilson and Bullock 1973).

5. The slower development of peak pressures in spiders without ligatured
pedicels supports the idea that prosomal pressures are partially
dependent upon the degree of filling of the op i stiioscma ! venous system
(Stewart and Martin 197^).

I nt roduct ion

Maximal activity in spiders can be divided into two phases (Ch. IV).
The second phase, which leads to nearly complete exhaustion, is probably
terminated by high lactate concentrations in the prosoma and legs. This

102

103

does not appear to be the case with phase I. Over the 15 to 30 sec
duration of phase I, lactate accumulations in Filistata and Lyaosa are
not large but these spiders lose about two thirds of their original
speed. Thus some other causative factor for the fatigue must be sought.
One possible explanation is "hydrostatic or fluid insufficiency."
Wilson (1970) and Wilson and Bullock (1973) showed there is a net loss of
hemolymph from the prosoma to the opisthosoma during the first eight to
ten seconds of vigorous struggles. They argued that insufficient fluid
would be available to force extension of the legs if too much fluid was
lost from the prosoma. Since spider legs are sealed, non-distensible
tubes, legs would not be expected to run out of fluid if the hydrostatic
insufficiency hypothesis is correct. Instead, insufficient prosomal
fluid would result in low pressures for leg extension, possibly as a
result of the prosomal muscles being forced to operate at lengths that
are increasingly shorter than optimal (< Lo) . Thus, rapidly decreasing
prosomal or leg pressure recordings (resulting from lowered contractile
force of the prosoma muscles) would support the hydrostatic insufficiency
hypothesis. To test this hypothesis, I simultaneously measured leg
hemolymph pressures and the movements of the major pressure-generating
muscle groups of the prosoma and opisthosoma.

Methods

Pressure and Muscle Movements

A saline filled catheter was connected by a length of polyethylene
tubing to a Sandborn Physiological Pressure Transducer, Model ISJB. The
saline was kkO mOSM consisting of 215 mM NaCl and 5 mM KCl (based on

104

Anderson pers. comm.). A series of valves allowed the entire system to
be flusiied with fresh saline. The pressure transducer was connected to
a Hewlett-Packard 31 lA Transducer-Ampl i f ier- i nd I cator which was in turn
connected to a Sanborn Model 320 strip recorder. A Narco pressure gauge
and mercury manometer were used for standardizing the pressure recordings
after each run. Recordings of muscle group movements were made using
myographs connected to a Narco Physiograph.

Preparat ion

Only large (mass >_ 0.45 g) Filistata hibemalis were used in these
experiments. The spiders were anaesthes ized with C0„ and then glued onto
glass rods using quick-setting epoxy cement. One day later the spiders
were again anaesthes i zed . A leg was severed near the middle of the femur
and a catheter was inserted into the center of the stub and glued into
place with epoxy. To avoid the introduction of air bubbles, the catheter
was introduced into the spider while it had a small drop of saline on its
tip. This drop merged with the hemolymph on the spider's leg and a good
liquid bridge was maintained. The spider was kept anaesthes i zed for the
half hour necessary to complete this procedure. A thread was attached
(using epoxy) to both the prosomal carapace and to the opisthosoma above
the anterior pericardium. These attachments permitted monitoring of
muscle group movements (Anderson and Prestwich 1975). No obvious ill
effects were produced by these procedures. Many of the spiders were
successfully removed from the apparatus at the conclusion of the experi-
ments and released several weeks later.

05

Record i ngs

Experiments were preceded by at least 20 min of baseline pressure
and myograph recordings. Occasionally it was necessary to break clots In
the catheter by gently squeezing the plastic tubing connecting the catheter
to the transducer. Activity was Initiated and maintained by lightly
scratching the spider's legs or chelicerae with a thin metal wire. This
produced vigorous struggles similar to those seen In the runway (see
Ch. IV). The main difference was that the spiders' legs were not fully
supported. Therefore, a smaller work load was involved in these struggles.

Lactate Concentrations

To evaluate the actual work done by the spider, I analyzed individuals
for lactate. For non-ligatured spiders this was done using different,
smaller individuals than were used in the pressure recording experiments,
(l felt that this was permissible since none of my previous experiments
had suggested any scaling relationship between lactate accumulation and
body size.) These spiders were mounted exactly as described for pressure
recordings and were stimulated in the same manner. At the end of two
minutes of struggle they were frozen by Immersion in liquid N„. The
ligatured spiders were killed in a similar manner except In this case the
same Individuals that were used for pressure recording experiments were
analyzed for lactate. In both cases, the frozen spiders were quickly
weighed and them homogenized in k°Z TCA and later analyzed for lactate
using the colorimetric method (Ch. II).

106

Results

Hemolymph pressures in the legs of resting FiZistata varied betv^/ee^
10 and 30 mm Hg and were independent of the leg measured. Also, since I
cannul ated the leg in a manner that reduced its ability to move, pressure
changes were not recorded in response to flexion of the leg itself (Stev/art
and Martin ISyt). Pressures during walking and the first few seconds of
vigorous activity seldom exceeded 70 mm Hg. Maximum peak pressures
occurred during violent struggles and were as high as 'tyS mm Hg and usually
were not achieved until 15 or 20 sec after the initiation of maximal
struggling activity.

Although both the prosoma and the opisthosoma appear to be involved
In the generation and maintenance of pressure (Figs. V-1 and 2) the
pressure pulses are most clearly associated with the prosomal carapace
depressions (Fig. V-1). However, not all equally forceful carapace
depressions result in equally large pressure changes. This is obvious
in Fig. V-l. The first two large carapace depressions are associated
with large but not maximal hemolymph pressure changes on the order of
100-200 mmHg (vs. 450 mmHg) . The third carapace depression of force
roughly equal to the first two, resulted in a much larger pressure pulse
of 450 mmHg. Op i sthosomal contract ions are increasing slightly in force
throughout this period of time.

The role of the opisthosomal musculature contractions are clearly
shown in Fig. V-2. A general correspondence between both opisthosomal
and prosomal contractions and leg pressure Is evident. The large shifts
in the baseline of this record occurring at the start of exercise and
about 40 sec later are due to slight jarring of the apparatus.

Figure V- 1 . Pressure generation and muscle group movements in
F. hibemalis. Note that maximum leg hemolymph
pressures are not reached until after nearly 30 sec
of activity. Also note the correspondence between
contractions of prosomal musculature and pressure
peaks.

108

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(MIN.)

Figure V-2. Same as Figure V-1. However, the figure more clearly
shows the role the op i s thosoma 1 musculature can have
in the generation of high prosomal pressures.

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Measurement of pressure and carapace depression under conditions of
maximal struggle over a tV'VO minute period and then after differing periods
of recovery are shown in Fig. V-3. The record is typical for eight
spiders. Peak pressures are not reached until about 30 sec into activity.
In the face of continued stimulation, maximum pressures dropped below
200 mm Hg over the next minute and a half and by the end of the activity
period were near 70 mm Hg.

After five minutes of rest, four of the spiders were again stimulated
for 30 seconds. Typical results are shown in Fig. V-3; peak pressures
were 100-200 mm Hg. In general, neither the pressure curve nor the force
of carapace contraction appear as high as in rested spiders. The final
trace represents leg pressures after ten minutes of rest (N = ^) . Pressures
now peak between 250 and 300 mm Hg and average pressures are higher than
those obtained after only five minutes of rest.

The final experiment involved tying a tight ligature around the
pedicel and thereby preventing any movement of hemolymph from the prosoma
to the opisthosoma. This was done on two spiders and the results for one
spider are shown in Fig. \l-k (results for the other individual were
essentially identical).

Upon tightening the ligature, leg pressures went to near 250 mm Hg.
[This is in contrast to spiders without the ligature where lower {ca.
100 mm Hg) pressures were found during the initial moments of activity.]
Peak pressures occurred between 20 and kO sec after tying the ligature and
were near ^00 mm Hg. During the first minute, pressures seldom dropped
below 100 mm Hg . After the first minute, movements by the spider became
very feeble in response to vigorous stimulation. At the end of the

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NOIRR3HH3a aOVdVMVO 3Un893U<i

Figure V-^. Leg hemolymph pressures in a F. hibemalis with

a ligatured pedicel. Note the faster initial rise
of pressure. Th i s i s consistent with the idea
proposed by Stewart and Martin (197^) that peak
pressures cannot be achieved until resistance to
flow into the op i sthosoma is maximal. Note the
lack of much pressure generation after one
minute. This is probably the result of severe
hypoxemia due to a complete block of circulation.
Prosomal lactate concentrations after two minutes
were high but not maximal (Chs. II, IV). This
might be due to the lack of a load on the leg
muscl es .

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116

two minute stimulation period the spiders legs were extended and pressures
of near 80 mm Hg were recorded. A gentle squeeze on the catheter showed
a normal pressure response indicating that the preparation was still
functioning normally. The catheter was then cut away by removing the leg
at the coxa and the recorded pressure dropped to zero (not depicted).
This showed that the high pressure recorded at the end of the two minute
runs was not an equipment artifact.

Lactate Concentrations

In non- 1 i gatured spiders, the mean whole body lactate concentrations
after 120 sec of activity was 3-63 + 0.22 ymols/g, N = 4, mass = 0.2^48 +
0.03. All of these spiders possessed large abdomens like those in the
Filistata used in Ch. II that certainly accounted for over half the total
body mass (Table II-3). The two ligatured spiders had an average total
body lactate of 3.2 ymols/g (mass = 0.567 g) with a mean prosomal concen-
tration of 6.0 ymols/g (mass = 0.256 g) and opisthosomal concentrations
of 0.9 ymols/g (mass = 0.311)-

Pi scussion

The hemolymph pressures agree with values obtained using different
techniques on Filistata (Anderson and Prestwich 1975) and other species
(Parry and Brown 1959a and b; Stewart and Martin 197^). Walking move-
ments occurred with pressures as low as 30 to 100 mm Hg; struggles were
accompanied by higher pressures of 80 to nearly 500 mm Hg. I could not
relate the intensity of the activity with the hemolymph pressure. In
fact, the highest pressures usually occurred well after the period of
most vigorous activity. In general, the myograph recordings {e.g..

117

Figs. V-1, 3) suggest that muscle tension was relatively constant through-
out this period of increasing hemolymph pressure.

The reason for obtaining the pressure data was to test the fluid
insufficiency hypothesis. Pressure is the most important index of power
for leg extension. The cross-sectional area of a spider leg is constant
(a slight increase in volume of the leg of an active spider occurs mainly
as the result of expansion of the arthritic membranes at each leg joint
(Parry and Brown 1959b). Also, the lever system determined by the/
sizes of the leg elements and the joints are fixed. Therefore, greater
pressures acting on the same area equate with greater force and result
in more forceful and/or rapid movements. The fluid insufficiency
hypothesis predicts that fluid loss from the prosoma to the opisthosoma
leads to insufficient hemolymph to extend the legs. For the reasons
mentioned above, any hydrostatic insufficiency must be reflected in low
prosoma-leg fluid pressures. If pressure remains high (even in the face
of a reduction of prosomal hemolymph volume), then there is no fluid
i nsuf f i ciency .

The data presented in the first three figures of this chapter show
that leg pressures remain high (above 100 mmHg) for the majority of the
two minute activity period. Furthermore, there was a marked tendency for
all individuals not to achieve highest hemolymph pressures until after
20 or 30 sec of vigorous struggles. During this time, pressures never
got low enough (below 100 mmHg) that recharge of prosomal fluid via the
heart would have been possible (Stewart and Martin 197'+). Thus, prosomal
hemolymph loss is not a constraint in Filistata during phase I of activity.

18

A possible criticism to this conclusion arises from the fact that
the spiders were mounted in such a way that they did not have to make
contact with the substratum. Therefore, the experimental situation was
not comparable to that of free running spiders. This was reflected in
the low lactate concentration found in both ligatured and non-ligatured
spiders (relative to free running spiders, Chs. II and I V) . The lower
load on the spiders legs actually allows for a more convincing demonstra-
tion that fluid dynamics are not limiting in spiders. Direct muscle
fatigue resulting from biochemical factors was lessened due to the
decreased load on the legs. The fact that the hemolymph pressures
remained high for long periods of time with accompaniment of low
lactate accumulations showed that hydrostatic collapse was not the cause
of fatigue since maximum volumes of prosomal hemolymph should have been
lost under these conditions.

The results of the ligature experiments are also not consistent
with the hydrostatic insufficiency hypothesis. If fluid loss is an
important constraint, then under the same experimental conditions the
hemolymph pressures found in the non- 1 igatured spiders would be lower
than in the ligatured spiders. There was no difference between the
pressures found in these two groups (Figs. V-l to V-'*) . Since the
intensity of struggling appeared to be the same in both groups, the
equal concentrations of lactate found in each group indicates there was
no flow of freshly oxygenated hemolymph from the opisthosoma to the
prosoma and therefore no recharging of the prosomal fluid levels.

Behavioral data is of significance in this context. The running
pattern of the scorpion, Centruroides hentzi, is identical to the three

119

species of spiders (Fig. I V- 1 ) . Scorpions do not, however, use fluid
pressure for leg extension (Manton 1958). Since rapid fatigue (phase I)
occurs in both hydraulic (spiders) and non- hy d ra u .1 i c (scorpions)
arachnids, it suggests that fluid constraints are not responsible for
fat i gue.

As mentioned in the Results of Chapter IV (see running speeds),
about ]0% of the Lyaosa and Filistata dragged their IVth pair of legs
after 20 sec of rapid running. This observation is the only support I
can offer for the hydraul ic insufficiency hypothesis. The fourth
pair of legs are the only legs in spiders that relay mainly upon
extension to propel the animal. The other three pairs mainly use
flexion (pair I), rotation-flexion (pair II), or rotation extension
(pair III) (Manton 1958; Parry and Brown 1959b). Insufficient fluid
volume and/or pressure is a likely explanation for the behavior of these
spiders .

Given the high pressure gradient that exists between the prosoma
and opisthosoma in active spiders, why is fluid loss not so large that it
becomes limiting? Stewart and Martin (197'*) suggested that the prosomal
hemolymph loss recorded by Wilson and Bullock (1973) was mainly due to
filling of venous sacs upstream from the heart (Comstock 19^8). Stewart
and Martin (197'») reasoned that the plateauing of the fluid exchange
between prosoma and opisthosoma that occurs in the first few seconds of
activity was a result of increased resistence to flow as the opisthosomal
venous sinuses filled to capacity. This explains several observations.
In both my records and those of Stewart and Martin (197'^), the highest
pressures occur well after the start of activity (Figs. V-l, 2, 3) even
though the spiders are no longer running rapidly (Figs. IV-l, 2). This

120

agrees with their suggestion that maximal pressure can not be generated
until resistance to fluid loss is maximal. Furthermore, very high
pressures may have a retardant effect on locomotion since the muscles
must work antagonistically to the large forces resulting from these
pressures (Stewart and Martin 197^). Finally, it is known that dehydrated
spiders quickly end up walking on their "knees" when forced to run
(Anderson and Prestwich 1975). Spiders with low hemolymph volumes would
have insufficient fluid to both fill the opisthosomal and prosomal sinuses
and also their legs. In a dehydrated spider, hydraulic insufficiency is
a very real constraint.

In most cases hydraulics Is not limiting to the activity of
healthy spiders. As Wilson (1970) pointed out, there is a positive
correlation between the degree of development of the prosomal pressure
generating musculature and the subcuticular opisthosomal muscle sheet
(which Is probably responsible for the development of much of the
opisthosomal pressure). This parameter also correlates with a spider's
habits: more active spiders have thicker musculature. Spiders are
designed to minimize any hydrostatic inhibition of their activity
patterns. Their unusual locomotory system does not directly constrain
spiders although it may exacerbate the hypoxemia found in the prosomal
musculature of active spiders (Wilson and Bullock 1973). Nevertheless,
the primary constraints of activity in spiders are biochemical, as in
many other species of animals (Ch. IV and Vl).

CHAPTER VI

THE METABOLISM OF PHOSPHAGENS, ADENOSINE PHOSPHATES,

AND SOME GLYCOLYTIC INTERMEDIATES AND SUBSTRATES

Summary

1. The changes in concentration during exercise of high-energy phosphate
compounds [arginine phosphate (AP) , ATP, ADP, and AMP], glycolytic
intermediates, and carbohydrate substrates were measured in the
prosomas of Filistata hihevnalis and Lycosa lenta.

2. The energy charge of resting spiders is high, being above 0.9.
However, during the first 20 sec of maximal activity there is a
near complete depletion of phosphagen stores (AP) and a lowering

of the energy charge to aa. 0.6 to 0.8. After this time, a steady-
state is reached between high-energy phosphate use and production.
This is reflected in a constant energy charge for the remainder of
the activity period.

3. Since the reactions involving the use of high-energy phosphate
compounds are potentially faster than those that supply such
compounds (such as glycolysis) the quick depletion of high-energy
phosphate stores seems to be the cause of the rapid phase I fatigue
of spiders (Ch. IV) .

k. The cause of phase II (Ch. IV) fatigue is probably related to the

effects of lactate accumulation.
5. Finally, the data suggest that carbohydrate availability is not

limiting during two minutes of activity. The results also suggest

121

122

that carbohydrates enter the prosoma from the opisthosoma during
moments of struggle when hemolymph can be pumped into the prosoma,

Introduct ion

Results presented earlier suggest that the ultimate cause of fatigue
in active spiders is related to accumulation of anaerobic metabolites.
However, the rapid fatigue that occurs early in exercise (phase I fatigue)
is one of the most characteristic features of vigorous activity in
spiders. In Chapter V, I showed that phase 1 fatigue is not normally
the result of hydrostatic insufficiency.

Four factors could contribute to phase 1 fatigue: (a) aerobic
limitations; (b) anaerobic accumulations; (c) phosphagen and adenosine
phosphate depletion; and (d) substrate depletion. The first two factors
are not important. Aerobic and anaerobic metabolism require time to
become fully activated as various metabolites are required for both
activation and de- i nhi b i t ion of their rate controlling enzymes. This
takes about five seconds in insects (Sacktor and Wormser-Shavi tt 1966;
Sacktor and Hurbut 1966). In L. Zenta, maximum anaerobic scopes are
not attained until after 10 sec of running (Fig. IV-S). While aerobic
and anaerobic metabolism are activated, the spider is slowing down
(Figs. IV-l and 2). Secondly, anaerobic accumulations are probably too
small during phase I to result in fatigue. In 15 sec of running, L.
Zenta and F. hibemaZis show respectively 64 and S^% reductions in speed
and yet their lactate accumulations are only 12 and 23% of their
respective anaerobic capacities. Thus, it is other factors that are
responsible for phase I exhaustion.

123

In mammals and in flying insects the main energy sources for the
first 10 sec of activity come from stores phosphagens, ATP, and ADP
(McArdle I98I; Sacktor and Hurlbut I966). A 100 meter human sprinter
relies almost exclusively on these substances for energy during a
race. Near the finish they are depleted and the sprinter may actually
be slowing down as he comes to rely on slower processes for ATP synthesis
(McArdle I98I). To test whether depletion of high energy phosphate
compounds could be related to phase I fatigue, I measured the levels of
arginine phosphate (AP; the phosphagen used in spiders, Di Jeso et at.
1967) and the levels of ATP, ADP, AMP, and inorganic phosphate (P.)
after different lengths of struggles.

Another potential cause of fatigue in spiders is carbohydrate de-
pletion. Spiders do not possess high concentrations of glucose or other
anthrone-react i ve substances (Collatz and Speck 1970; Stewart and Martin
I97O; Collatz and Mommsen 1975; Rakotovao 1975). I measured the changes
that occurred during periods of maximal activity in anthrone-react ive
substances, hexose-phosphates , and some glycolytic intermediates.

Materials and Methods

Preparation of Samples

Spiders were exercised in the runway described in Chapter IV. At
various intervals during activity and recovery, the spiders were quickly
frozen in liquid N_, the prosoma and legs separated from the opisthosoma,
and then the prosoma plus legs were stored frozen for several days at
-8C°C. Five spiders were used at each interval of activity except for

124

the plus ten minute recovery value in Filistata where N = 2. Homogenates
of frozen prosomas were prepared according to the methods of Lamprecht
and Trautschold (197^+) except that the ratio of t i ssue powder to frozen
HCIO, was maintained at 1 to 10 (Sacktor and Wormser-Shav i tt 1966) and
the samples were filtered to remove the protei naceous precipitant (see
Ch. II) prior to neutralization with ^M K-CO„. These extracts were
immediately refrozen at -80°C. Analyses were made on these samples within
seven days of refreezing (Walesby and Johnston I98O).

All biochemical analyses, except for anthrone-react i ve substances
and glucose, were carried out by coupling to indicator reactions based on
changes in concentration of reduced pyridine nucleotides. The absorbance
of these coenzymes was monitored at 3^0 nm using a Zeis m4QI I I spectro-
photometer with a Zeis PM I digital readout. The analyses were as listed
in Table Vl-l except for arginine phosphate (AP) , glucose, and glycogen.
Arginine phosphate (AP) was analyzed by placing between 30 and 200 yl of
sample in a cuvette containing the buffer, coenzymes, and enzymes needed
for the ATP and G6P analyses of Lamprecht and Trautschold (197^). ADP
was added to produce a concent rat ion of aa. 0.15 mM ADP. This mixture was
allowed to react at ^°C for ^+0 minutes to remove all G6P and ATP. This
step is important because the ADP suppl led by S i gma contained traces of
ATP. Attheendof the ^0 minutes, the samples were quickly brought to room
temperature and absorbance was read three times at two minute intervals
to obtain a correction factor for "creep" (Lamprecht and Trautschold
197^). Then, I added 20 \i\ of arginine kinase (AK) . The subsequent
react ion is slow and takes up to90minutes to compl ete (dependingon the
sample). Efforts to speed up this reaction through the addition of more

125

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AK resulted In the formation of an interfering precipitant of magnesium
ammonium phosphate. Future determinations of samples from individuals
suspected of having high AP concentrations should be done on much smaller
samples and/or following dialysis of commercial AK. The final volume of
all AP analysis reactions was 0.7 to 0.973 ml.

Glucose was analyzed using glucostat obtained from Worthington
Biochemical and total hexose was determined using anthrone reagent.

Biochemicals for all the above analyses were obtained from either
Sigma or Boehringer. These and all of the inorganic reagents were of the
highest purity available. All reactions were checked with appropriate
standards and blanks. Coefficients of variation were generally less than
20%; this is usually considered adequate for these types of analyses
(Sacktor and Wormser-Shavi tt 1966). The samples having higher variation
were usually those at the limit of detection (oxaloacetate , pyruvate, PEP,
GAP, glycerate-3-phosphate, and GIP). Other substances with larger
coefficients of variation were anthrone-react i ve substances (which would
be expected to show significant differences in concentration between
individuals) and FDP (reasons unknown).

Results are all expressed as mean +_ standard error. Tests of
significance were based on Student's t-test; the level of significance
was 0.05.

Results

Substrate and Hexose Phosphate Levels

Measurements of total anthrone reactive materials and glucose during
activity are presented in Figure VI-1 for L. lenta and F. hibemalis .

127

Glucose shows a slight but non-significant increase over the 120 sec
of activity in Lyaosa. Over the same period glucose concentration doubles
in the prosoma of FiZistata, a significant increase. During recovery in
Filistata, glucose levels are similar to those measured at 120 sec. A
non-significant decrease occurs between five and ten minutes into the
recovery period. The concentrations of glucose in L. lenta are about
half those of Filistata.

Total anthrone reactive substances are also higher in F. hibermalis
than in L. lenta (Fig. Vl-l). Two different patterns are seen. In
Filistata, an initial decline in prosomal anthrone reactive compounds is
followed by a more than doubling in these substances. Large variation
occurs in samples taken at 20, 30, and 60 seconds. However, by the 120 sec
measurements the increase in total hexose over resting levels is significant,
During recovery, a non-significant decrease occurs between minutes 0 and
10 of recovery.

In L. lenta total anthrone reactive substances decrease throughout
the 120 sec activity period although the change is not significant. At
120 sec nearly all the anthrone reactive substance is apparently glucose.

Figures \/l-2 and 3 show the changes in concentration of glucose-6-
phosphate (G6P) and fructose- 1 , 6-d i phosphate (FDP). During the 120 sec
activity period G6P increases over eight fold in both species, a highly
significant change. In Filistata, the G6P concentration continues to
increase during recovery. The concentration of FDP varied and the
apparently increased levels of this substance are not statistically
significant changes. Finally, the concentration of g 1 ucose- 1 -phosphate
(GIP) was below the level of resolution for the assay conditions I used
(0.01 ymols/g).

Figure Vl-l. The metabolism of carbohydrates in active and
recovering spiders at 25°C. Circles are for
F. la-tbevnalis and triangles are. for L. lenta.
In Filistata, significant increases in total
anthrone-react i ve substances occur by the end of
two minutes activity while in Lycosa these
substances decrease slightly in concentration.
The results suggest that in Fitistata carbohydrate
enters the prosoma from the opisthosoma during
exercise. Recovery values for glucose and
anthrone-react i ve substances show non-significant
decreases in concentration over the ten minutes
of recovery.

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ANTHRONE

GLUCOSE

129

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+5 +10

TIME

(MIN.)

Figure VI-2 and 3- Metabolism of gl ucose-6-phosphate (G6p) and
fructose-l, 6-d i phosphate (FDP) during activity
and recovery. Concentrations of G6P increase
significantly, there are no significant changes in
FDP concentration. Significantly higher concen-
trations of G6P in Filistata (circles) during
recovery (compared to the end of activity) may be
related to gl uconeogenes i s .

Figure VI-4. Malate metabolism. Significant decreases in malate
concentration occur in Filistata (open circles).
This is contrary to what would be expected under
hypoxemic conditions such as prevail in exercise
where an increase in malate is expected (see Ch.
II). The results may be explainable by the
conversion of malate to fumarate and/or succinate.

131

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TIME (MiN.)

132

Concentrations of Intermediates

The levels of glyceral dehyde-3"phosphate (GAP) varied between about
1 to 2k% those of d i hydroxyacetone phosphate (DAP, Fig. \J\-G) averaging
]2% of the concentration of DAP, or about 0.01 umol/g. Values for glycerate-
3-phosphate were near 0.7 ymol /g , p hos p hoe no 1 py r u va t e (PEP) was below
the limit of detection (0.005 ymol/g) and pyruvate concentrations were
at the level of detection of 0.05 ymols/g. However, for these three
compounds, only 6 total samples were analyzed; 2 at t = 0 sec and k at
t = 60 sec, all for Filistata. Finally, oxaloacetate levels averaged
near 0.08 ymol/g. For all of these substances the standard errors were
very large and approached 30% of the value of the mean in some cases.
Thus, they were not graphed. More reliable data will require the use of
more sensitive techniques involving enzyme cycling and/or flurorimetry
(Lowry and Passonneau 1972).

Other Intermediates

The concentration of L-malate (Fig. Vl-A) declined significantly
during the 120 sec activity period in both species. By the end of exercise
malate concentrations had dropped to 1/3 of their initial values. In
recovery in Filistata non-significant increases of malate occurred.

Enzymatical ly determined levels of D-lactate are shown in Fig. VI-5.
Throughout 120 sec of activity, Filistata maintains higher concentrations
of this substance than Lyaosa. The same is true of the other known
anaerobic by-product of spiders, gl ycerol -3-phosphate (G3P) • Initial
increases of G3P were not significant due to high variability but concen-
trations of this substance were significantly elevated after 120 sec of

Figure Vl-S. D-lactate metabolism during exercise in Filistata
(circles) and Lyaosa (triangles). The results are
consistent with those presented earlier (Chs. I! and
IV).

Figure Vl-o. The metabolism of gl ycerol -3"phospate (G3P) and
d i hydroxyacetone phosphate (DAP). increases in
G3P concentration are significant but are only
oa. 5Z of those of lactate.

15

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TIME (MiNj

135

activity (Fig. VI-6). The ratio of D-lactate to G3P was 19: 1 in
Filistata and 21:1 in Lyaosa. During recovery, there was a non-significant
decrease in G3P coupled with a non-significant increase in DAP in

Filistata.

High-Energy Phosphate Compounds

Changes in the concentrations of arginine phosphate (AP) and the
adenosine phosphates (ATP, ADP, and AMP) are shown in Figures VI-7, 8,
and 9 and fluctuations in inorganic phosphate (P.) levels are depicted in
Figure VI-10. The initiation of activity results in dramatic alterations
in the concentrations of all of these substances with the changes being
most extreme in Filistata. These changes will be carefully evaluated
i n the Di scuss ion.

Di scuss ion

Carbohydrate Metabolism

The prosomal carbohydrate concentrations of Lycosa and Filistata
are similar to the range of concentrations reported for other spiders.
Rakotovao (1975) reported whole spider trehalose concentrations ranged
between 0.3 and 5-8 ymols/g with the range for glycogen being 12.2 to
62.7 vmols/g (glucose equivalents) in an orb weaver, Collatz and Speck
(1970) reported a whole body glucose concentration of 1.8 and a sucrose
concentration of 1.2 ymols/g; glycogen was 100 ymols/g In an Agelenid
spider. Looking only at hemolymph, Stewart and Martin (1970) found a
tarantula's glucose concentration to be 0.28 ymols/g with total anthrone

Figure VI-7- The metabolism of arginine phosphate (AP) in
Filistata (circles) and Lycosa (triangles).
Note the very rapid depletion over the first
15 sec or less of activity.

Figure Vi-8. ATP metabolism in Lycosa and Filistata,

Figure \!\-S. Changes in concentration of ADP (open symbols)

and AMP (dark symbols) in Lycosa (triangles) and
Filistata (circles). Especially important are the
rapid increases in AMP which may have an important
role in activating glycolysis (see Appendix ll).

137

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AP

z

o

2 o
LU i
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o
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5 -

1.0

Figure VI-10. Changes in the concentration of inorganic phosphate
(P|) during activity and recovery. Resting levels
of P| are higher than expected and suggestive of some
hydrolysis of arginine phosphate (AP) during handling
of the samples. Note the significant increases in
P; that occur simultaneously with decreases of AP
(Fig. VI-7).

Figure VI-11. Energy charge during activity in Filistata (circles)
and Lyoosa (triangles). Energy charge is defined
mathemat ical ly as :

(2ATP + ADP)
Energy charge = 2(ATP + ADP + A^p)

An energy charge of 1.0 indicates all adenosine phosphates
exist as ATP and a cell possesses a large amount of
""P potential energy. Conversely, an energy charge of 0
means all adenosine phosphate compounds are. present as
AMP and there are no ~P available in the adenosine pool.
Note the high resting energy charge in both species,
its rapid decrease and then attainment of steady-state
after oa. 30 sec of activity.

139

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1 40

reactive substances of 0.72 ymols/g. They suggested the O.kk (jmols/g of
non-glucose material was trehalose since this substance Is found in
scorpions (Br i cteux-Grego i re et at. 1963). My data are for whole prosomas
and although the glucose data agree well, the non-explained anthrone
reactive materials (glycogen and trehalose?) are somewhat lower than those
summarized above. This may indicate that the opisthosoma is richer in
these materials than the prosoma.

In both Filistata and Lycosa the prosomal glucose concentration
increases with the onset of activity (Fig. VI-1). The source of this
glucose is not evident. The initial concentration of anthrone- react i ve
substances are not sufficient to explain the accumulations of different
substances that occur over 120 sec of activity (Table VI-2) in both
species. This conclusion is reached by subtraction of the t = 120 sec
concentrations of glucose and total hexose phosphates from the initial
total anthrone reactive substance concentration. The result must be at
least twice the accumulation of lactate since 2 lactates are
produced per hexose that enters glycolysis. Table \l\-2 shows the initial
hexose in Filistata is not even close to the required amount and that of
Lyoosa is also considerably less than needed.

Thus, it is reasonable that the opisthosomas of these spiders provide
some form of hexose to the prosoma during exercise via the hemolymph. This
is especially evident in Figure V I - 1 for Filistata: the concentration of
anthrone-react i ve materials significantly increases during activity. The
increase in glucose concentration in Filistata is about 2.5 Umols/g and
is not sufficient to explain the 7 pmol/g increase in anthrone reactive
substances. Based on the evidence from other species cited at the start
of this section, I suggest that some opisthosomal tissues may be a

]k]

Table \l\-2. The amount of carbohydrate present in spider
prosomas at the start of exercise compared
to the amount needed to produce all the
Intermediates and lactate found after two
minutes of activity. The differences should
be zero, because they are not, carbohydrate
must be being added to the prosoma during
exerc i se.

T - 120

Glucose Needed

Initial Anthrone- Hexose-P For

Species Reactive Substances Total Lactate Difference

F. hibemalis 8.3 6.8 -5-8 -6.1

1.8

-8.6

L. lenta 6.k 2.8 -5-2 -2.2

0.7

-3.5

Need: based on assuming that all carbohydrate is burned only to lactate.
Therefore, the increase in lactate concentration ^ 2 equals the
required hexose. This figure tends to err in favor of less
hexose needed that is really the case since it does not take
into account the increases in concentrations of trioses and
triose phosphates which in the case of DAP and G3P are
significant {aa. 1 ymol/g total).

Difference: initial ( [anthrone]-f i na 1 ( gl ucose] + [hexose-P] + ( 1 actate/2) .

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carbohydrate storage center, much like a vertebrate liver. During
activity, g 1 ycogenolys i s occurs and carbohydrates, such as glucose and
trehalose (Rakotovao 1975) or sucrose (Collatz and Speck 1970) are
released i nto the hemol ymph. Given the low carbohydrate concentrations of
tarantula hemolymph (Stewart and Martin 1970) compared to resting prosomal
concentrations (this study) these materials would then be transported into
the tissues. However, the problem remains as to how they are pumped to
the prosoma when a large pressure gradient exists (Ch. V) .

The data presented in Figure Vl-l suggest that Lyaosa is more
dependent on hexose already present in the prosoma than is Filistata.
This Is evident by the fact that the prosomal carbohydrate concentration
decreases to nearly zero by the end of activity. Thus, it Is possible
that Lucosa may be ultimately constrained by loss of hexose substrate
for glycolysis.

Finally the prosomal carbohydrate concentration remains high during
recovery In Filistata. This may indicate a prosomal role In gluconeo-
genesis or merely Indicate slow metabolism of accumulated G6P.

I ntermed iates

The resting levels of glycolytic and Krebs cycle intermediates are
consistent with those reported for the blowfly (Sacktor and Wormser-
Shavitt 1966). The on 1 y except ions to this were G3P which is less than 2/3
as concentrated In the spiders. The low concentration of G3P may be
re 1 ated to the lower aerobi c capacities of spiders (Anderson and Prestwich
1982). In Insect flight muscles, G3P is the major vehicle for transfer
of cytosol -produce/electrons to the mitochondrial electron transport
system. Spiders possess a less active cytosol GPDH (Prestwich and Ing

\kk

in press) and many fewer mitochondria (Linzen and Gallowitz 1975) and
therefore would not require or produce as large amounts of this substance
as would insects. Similarly, the higher malate concentrations in
insects may reflect the greater proportion of mitochondria and therefore
higher cellular concentrations of Krebs intermediates. Finally, I was
only able to set upper limits on the concentrations of several substances
(GIP, F6P, gap, 3PGA, PEP, pyruvate, and oxa loacetate) . All of these
substances except 3PGA and pyruvate are normally found in only trace
amounts in flight muscle (Sacktor and Wormser-Shav i tt 1966) and mammalian
tissue such as brain (Lowry et at. 1964). The maximum possible concen-
trations I have found are consistent with other literature values.

Intermediates tend to increase in concentration early in activity.
Some of these substances (that could be measured accurately), such as
FDP and DAP, quickly level off or decrease by the end of activity.
Another trend is found In the Krebs cycle intermediate malate which
decreases significantly during activity. The cause of this reduction is
not known but one possible explanation is that it is anaerobica 1 1 y
metabolized to another substance(s) such as succinate and fumarate.

The intermediates that increased in concentration during activity
were G6P, G3P, and lactate. The increases of G3P and lactate are related
to the maintenace of cytosol redox (Ch. II). Lactate increases reported
in Fig. Vl-S closely resembles those reported in Chapters II and IV.
Filistata accumulates lactate faster than Lyaosa and the rates of
accumulation in both species are like those reported in Chapter IV.
The accumulation of G3P suggests two possibilities. Either the pathway
is inhibited after the initial phase of exercise or/and the mitochondria
are better able to oxidize G3P to DAP later in activity. Given no

^5

dramatic Increase in DAP (even though earlier glycolytic intermediates are
in high concentration) it is not likely that G3P oxidation is significantly
enhanced later in the exercise period.

The continued increase of lactate throughout the activity bout
coupled wi th constant [G3P] after the i n i t ial few seconds of activity suggest
that in the presence of continued glycolysis GPDH must be inhibited
(Guppy and Hochachka 1978). Generally, the rate of lactate production
appears to increase between t = 0 and 30 sec, reaching a maximum value
(anaerobic scope, Bennett 1978) between 10 and 30 sec and thereafter
decreasing or remaining constant (Table \l\-k). At the same time, G6P ,
the starting substrate continues to accumulate at a constant rate in
both species. This suggests that regulation of the process is still
limited by PFK since G6P (and therefore probably F6P, Newsholme and
Start 1973) increases, FDP remains essentially constant or increases
onl y s 1 i ght 1 y.

Arginine and Adenosine Phosphates and Pj

There is some evidence of partial hydrolysis of AP and possibly
ATP from the initial, resting state, samples. First, the P. values are
somewhat higher than normally reported in resting muscular tissues where
concentrations are on the order of 8 ymols/g or lower (Newsholme and
Start 1973; Lehninger 1975; and see Table Vl-S). Secondly, Di Jeso et al.
(1967^ report whole spider P. values near 7-^ ymols/g and a ratio of AP
to ATP of about 't : 1 . Since phosphagens are normally highest in muscle
and nervous tissues (Lehninger 1975) such as those found mainly in the
prosoma (Table 1-2), I expected a ratio of at least A:l in the prosoma.

1^46

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However, my data show AP to ATP ratios of 1.75 to 1 and 2.75 to 1 with
resting P. concentrations of 12.7 and 9.0 in F'ilistata and Luaosa
respectively. Given the lability of AP and the high energy charge of
resting spiders (Fig. V I - 1 I , suggesting little ATP hydrolysis), I assumed
that the AP concentrations reported for rest (t = 0) are minimal
estimates for resting conditions and that the elevated P. is due to
hydrolysis of this substance. There are two possible causes of low AP :
(a) minor activity (less than one sec) occurred when some individuals
were frozen and (b) the fact that the t = 0 samples had to be analyzed
twice over a 1.5 day period due to problems in the AP assay encountered
when high concentrations of AP were present (see Methods). (These
difficulties were not experienced with any of the other samples.)

There is only one other discrepancy in the high-energy phosphate
compound data. In Table \/l-3, the total adenosine phosphate concentrations
are summed for each interval during the activity period. These sums should
all be roughly the same or decrease slightly with time due dephosphory-
latlon of AMP to adenosine (Lowery et al . 196^) or deamination to inosine
monophosphate (Lowenstein 1972). In Lyaosa, the summed concentrations
are constant. However, in Filistata the t = 0 and 30 sec summations are
much greater than any of the others. Thus, a positive bias to the
concentrations of one or more of the adenosine phosphates is possible.
However, overall the agreement is good and the possibility of some of
the fluctuations being due to conversion of adenosine needs investigation.

The general consistency of all of the data relating to the metabolism
of phosphorylated compounds is shown in Table \l\-h. Here, the changes in
bonded phosphate concentrations (both in intermediates and in high-energy

149

compounds) are compared with the changes in P.. Excellent correspondence
is obtained in all intervals except early recovery in Filistata and the
0 to 15 sec interval in Lyaosa. However, even these deviations are small
in light of the large standard errors associated with some of the com-
pounds used in obtaining these results (Figs. \/l-3, 6, and 10).

One very important result shown in Table Vl-^ is that the rate of
phosphate cleavage reaches an equilibrium after 10 to 15 sec of activity.
This is reflected in the relatively small changes in total AP , ATP, and
ADP after this time (there may actually be a net ATP synthesis in
Filistata, see Fig. VI-8). Another indication of equilibirum is shown
on Fig. Vi-11 which graphs the prosomal energy charge with respect to
time. Energy charge is a measure of the ability to do chemical work. A
value of 1.0 equates to maximal energization in the cell due to the
presence of all adenosine compounds in the form of ATP. An energy charge
of 0 indicates all adenosine compounds exist as AMP. In spiders, the
energy charge drops prec ip i t ious 1 y from 0.95 (an extremely high value)
to a minimum of 0.56 or 0.8 within 20 sec. From then on, the level is
constant. An even more sensitive measure of the ability of the cell to
do work is the phosphorylation potential (F) . It also drops rapidly
at the start of exercise and thereafter changes very little. Both
measures indicate an equilibrium between the use and production of ~P.
Finally, the energy charge quickly returns to a higher value during
recovery (O.8O and 0.93 after five and ten minutes of recovery).

It is evident that spiders are different from insects. This point
was made most recently by Anderson and Prestwich (1982) in reference to
the aerobic capacities of spiders. Another reflection of this is in

150

Table Vl-S where the metabolism of high energy phosphate compounds in
running spiders is compared to a fly in flight.

Examination of the resting (t = 0) values in the table points up
several interesting differences. First, ADP concentrations in spiders
are quite low compared to Phomria (a fly) and also to other animal tissues
where they normally are between 1 to 2 ymols/g (Newsholme and Start
'973; Lehninger 1975; Sacktor and Hurlbut 1966). The spider values are
only 1/10 as much. This low concentration is correlated with the high
energy charge {'^ 0.95) found in spiders compared to the ratio of 0.85
to 0.9 normally seen in other organisms (Lehninger 1975). Secondly, the
ratio of AP to ATP in spiders is less like that of the fly, and more like
the CP (creatine phosphate) to ATP ratio of mammals. In mammals the
CP:ATP ratio of muscle is typically about 5 to 1 (Lehninger 1975) and in
spiders it is at least 2 or A to 1 . By contrast, Phormia has an AP
to ATP ratio of I to 2.25. This indicates spiders and vertebrates have
a much higher reliance on phosphagen stores during muscular activity than
do insects.

The differences between insects and spiders are even more impressive
when the changes that occur in the concentrations of metabolic and energy
intermediates at the onset of flight and running are examined (Table iV-
5). in spiders, within 15 sec of the onset of vigorous activity AP is
completely depleted and ATP has dropped by 33 to 50%. By contrast, in
Phormia AP concentrations drop 33? ( a small absolute change) and ATP
decreases \1%. Clearly, spiders are more dependent upon stored phosphate
during the early phases of movement. This is analogous to the process
as seen in a human sprinter where tremendous CP and ATP depletions occur
during short runs (McArdle I98I).

la
o

151

The Relative Contributions of Stored High-Energy Phosphates and Glycolysis
to Phase I of Activity

Glycolysis is completely over-shadowed as an energy source by the
useof AP and ATP stores during the first 10 to 15 sec of activity. During
this time, phosphate is cleaved from stores at a rate of 65 ymols/g min
in Filistata and 37 ymols/g min in Lyaosa (10 and 15 sec periods,
respectively). These rates might be higher since it is possible the AP
and ATP is used in a shorter time than the measurement intervals. During
these same time periods, ~P is produced from anaerobic glycolysis at the
rate of about 13 ymols/g min in Filistata and 10 ymols/g min in Lyaosa
ssuming 1.5 ~P per lactate and 1.0 per G3P; Ch. II). Thus the cleavage

f stores occurs 4 to 5 times faster than its production via glycolysis.
Finally, aerobic processes can probably be dismissed as a significant
source of ~P due to the low number of spider muscle mitochondria (Linzen
and Gallowitz 1975), low PO2 (Angersbach 1 978) , and the time necessary to
achieve full activation of the Krebs Cycle (See Appendix 11, and Sacl<tor
and Wormser-Shavi tt I966).

The relative roles of anaerobic glycolysis and ~P stores depletion

re crucial to the understanding of the rapid slowing in running spiders.
The summary presented above clearly shows that ~P stores are the major
energy source during phase I. The depletion of these stores is mainly
responsible for the rapid fatigue and change In gait (from jumping and
running to walking) that occurs near the end of phase I (Ch. IV). In
summary, I conclude that phase I slowing is the direct result of the
spider being forced to rely upon processes (anaerobic and aerobic
metabolism) that cannot deliver ~P to drive muscle contractions at as high

a

152

a rate as is possible by catabolzing AP and ATP stores (see previous
paragrapin) . The reason for the slowness of delivery undoubtedly relates
to: (a) these pathways large number of steps, (b) the low activities
of their rate- 1 imi t i ng enzymes compared to that of arginine kinase
(Prestwich preliminary data), and (c) poor availability of O2 and
poorly developed aerobic pathways (Angersbach 1978; Linzen and Gallowitz
1975; Prestwich and Ing in press).

CHAPTER VI I
ACTIVITY IN SPIDERS: A REVIEW

Summary

1. The ranges of a number of important physiological parameters that
change during exercise (hemolymph pressures, heart rate, VO- , lactate,
phosphagen metabolism, pH , P0„ , and % saturation) are summarized
(Table VI 1-1).

2. A model of exercise energetics in spiders suggests maximal struggles
are limited first by phosphagen depletion and later by anaerobic
accumulations. Overall, anaerobic metabolism produces most of the
~P used during activity; aerobic contributions are responsible for
less than ]Q% of the total ~P used (Figs. VI 1-3 and k) .

3. The interrelationships between prey capture technique, resting V0-,
and anaerobic metabol i sm are d i scussed . Spiders are characterized

as animals of moderate power production capability; aerobic abilities
are low while anaerobic capacities and phosphate stores are not
unusually high. Use of silk and poison helps make it unnecessary
for spiders to be able to generate ~P at high rates.

I ntroduct ion

This chapter will review the physiology of movement and recovery in
spiders. As part of the review, a model of the energetics of spider
locomotion in the prosoma is presented. The overall approach

153

155

comparable to the other results); (c) lactate, Chapter IV; (d) ~'P from
the sums of the concentrations of AP + ADP + 2x(ATP), Chapter Vl; (e)
pressure, the mean pressure from an integration over 15 sec intervals
of the results of the experiments in Chapter V; (f) heart rates,
Chapter IV. In Figures VI 1-5 and 6 the running speed percentages are
based on the first ten seconds of running after five and ten minutes of
recovery (see Methods, Ch. IV) and the hemolymph pressures during
recovery are based on 30 sec activity periods like those shown in Figure
V-3 (measurements integrated over 15 sec periods to give mean pressures).

Estimation of ~P Turnover

In Chapter III the relative importance of phosphagens, aerobic,
and anaerobic metabolism to the energetics of struggling in spiders in
respirometer flasks was estimated. However, there were several problems
with these estimates. The most serious were that (a) the animals were
performing at high but not maximal work loads and (b) assumptions were
made as to the degree of phosphagen metabolism and aerobic metabolism
during act i vi ty.

The data presented in Chapter VI allow an independent assessment
of the relative importance of these three sources of ~P to maximal
activity. However, Chapter VI data apply only to the prosoma. This is
not a serious defect since the prosoma generates most of the power used
during activity (Chapters II and IV). The only drawback to using the
Chapter VI data deals with the necessity of estimating aerobic metabolism.
To calculate aerobic power, 1 assumed that aerobic ~P synthesis could be
estimated from knowledge of the spiders heart rate and resting VO2 .

15^

of this chapter will be comparative and deals with how the circulatory,
respiratory, and locomotory systems have evolved in different species of
spiders faced with different environmental pressures.

Methods

The ranges of values for several physiological functions that are
important in activity are presented in Table Vll-l. These values were
obtained from the earlier chapters of this dissertation and from the
literature. Also, the data presented in earlier chapters have been
summarized in Figs. Vll-l through k and Tables VI 1-2 and 3- Figures
Vll-l, 2, 5, and 6 show changes in various physiological parameters
during exercise in terms of percentages. The percentage scale fs based
on the maximum and minimum values observed for the variable in question.
Percentages are expressed either as percent of maximum value (pressure,
heart rate, lactate concentration, VO^) or minimum value (running speed,
~P concentration). Thus, all values are calculated according to an
equation of the form:

I = Ll (LSl^^x 100

(AM) (1)

where V is the value of the parameter in question at some time t, M. or

M- is either the maximum or minimum value of that parameter and AM is the

difference between the maximum and minimum value of the variable.

The calculation of all of the percentages in Figures Vll-l, 2, 5,

and 6 is from the following sources and uses 25°C data only: (a) running

speed (fatigue). Chapter IV--relative speeds; (b) VO- , Chapter III (note:

these involved submaximal activity and therefore are not strictly

156

Factoria] increases in HR above resting were taken as factorial
Indicators of VO^ above resting (Anderson and Prestwich I982). The
estimated activity VO- for some time period was converted to 'f^mols ~P
produced by assuming gl ucose-6-phosphate was the initial substrate and
that 38 jimols of ~P were synthesized per mol of G6P used. Because 6 mols
of 0- are required to oxidize 1 mol of G6P and 38 mols of -P are yielded
per G6P oxidized, then 6.3 mols of "P would be synthesized per mol of 0^
consumed. Thus, 1 yl 0^ (STPD) = 0.28125 ymols ~'P synthesized. As an
example of a calculation, if the resting VO- = 22. A yl 0-/g min at a HR
of 20, then at a HR of 40 the estimated VO- = ^^.8 yl 0„/g min and estimated
"P production = 12.6 ymols/g min.

Calculation of ~P contributions from stores and anaerobic metabolism
are straightforward: Stores: were calculated from the changes in the sum
of AP + (ATP X 2) + ADP (Ch. Vl); Anaerobia Contributions: are based on
the differences in the concentration of lactate and G3P accumulations
from one time to the next (Fig. VI-2) by assuming a net gain of 1.5 ""P
per lactate and 1.0 -P per G3P (Ch. 11).

Discuss ion

Values for some physiological parameters under varying exercise
conditions are given in Table VI 1-1. These values will be the basis for
much of the following discussion.

Rest and Moderate Activity

It Is well-documented that spiders have low resting VO2 (Dresco-
Derouet 1969; Anderson 1970, 197^, 1978; Anderson and Prestwich 1982;

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Greenstone and Bennett I98O). Rates of oxygen consumption in spiders
correlate strongly with book lung surface areas (Anderson 1970; Anderson
and Prestwich 1982). This implies that resting VO^ must also correlate
with resting cardiac output (Q) since large lungs by themselves cannot
deliver 0- to the tissues. Thus, species with relatively high VO2 must
have large book lungs and Q (assuming that hemolymph 0^ capacity is
roughly the same in all species).

Cardiac output is determined by both stroke volume and heart rate
(HR) . Greenstone and Bennett (I98O) and Anderson and Prestwich (1982)
have shown that no good interspecific relationship exists between resting
HR and VO-. Therefore, different species must rely on different stroke
volumes. However, within a species, HR probably is a good indicator of
VO- since available data and calculations suggest that stroke volume
changes little at different HR and, thus the main determinant of changes
in Q is the HR (Appendix I; Ch. IV; Stewart and Martin 197'*; Anderson
and Prestwich 1982) .

Besides the cardiac output and respiratory surface area, several
other factors affect the amount of 0„ available to spider tissues. One
very important factor appears to be ventilation of the lungs. Assuming
tarantulas are a good model for all spiders, then it appears that in resting
spiders there is little ventilation of the lungs. As a result, the P
(measured in the heart) is only about 30 torr and the hemolymph is only
half saturated, containing about 1 vol % 0„ (Angersbach 1978). There is
a time lag between the initiation of activity and an increase in ventil-
ation. After ventilation begins, the PaOo ^^y soar to 8O mm Hg and
the hemolymph will completely saturate and contain about 2 vol % 0-

a02

160

(Angersbach 1978). Additionally, other factors may operate to increase
the availability of 0_ to active tissues. Spider hemolymph shows a Bohr
effect and the A-V PO- difference may increase (Angersbach 1978). Also,
spiders that possess extensive trachea in addition to their lungs may be
able to use these structures to boost 0- availability to their tissues
(Anderson 1970).

Taking HR and ventilation as the main factors that can influence
increases in VO- during activity, then maximum rates of VO- may be cal-
culated. In mygalomorph spiders such as tarantulas, the maximum increase
should be about 8X resting VO2 since these spiders can increase their
heart rates a maximum of four times (Stewart and Martin 197^; Angersbach
1978; Anderson and Prestwich 1982) and the saturation of their hemocyanin
can double (Angersbach 1978). This estimate agrees with observed \/02 for
active tarantulas by Anderson (pers. comm.). For araneomorphs , the
maximum increase in VO- should be about l8X resting since HR can Increase
about 9X resting (Anderson and Prestwich I982) and assuming that saturation
of hemocyanin can double.

While the maximum V0_ of spiders may be nearly 18 times resting,
the data that have been obtained on spiders performing routine activities
suggest that normally VO- during these activities seldom exceeds 3 or
k times resting (Seymour and Vinegar 1973; Peakall and Witt 1976; Ford
1977a, b; Prestwich 1977). Working at levels far below their aerobic
maxima helps spiders avoid the necessity of reliance on anaerobic
metabolism. They preserve the ability to increase their aerobic metabolism
and to begin to rely on anaerobic metabolism.

The metabolism of stored ~P In moderately active spiders is not
known. However, it is not likely that these compounds are greatly

161

depleted during such activities: if a moderately active spider is
threatened or presented with prey it can still move rapidly. Data
presented in Chapter Vl suggests that if phosphagens were depleted, this
rapid movement would be impossible. It can be concluded that abundant
~P compounds are present in sub-maximally active spiders. However, this
observation needs biochemical confirmation.

Thus, spiders wori<ing at moderate rates do not utilize anaerobic
metabolism and phosphate stores to the large degree that these are
relied upon during maximal activity (see Chs. IV and Vl and also see
below). This is not to say that some utilization of ~P from these
sources does not occur. At the start of any activity, stores are almost
surely used until aerobic processes can be fully activated. Further-
more, there are good reasons to believe that regions of reliance on
anaerobic metabolism v;i 1 I exist even in moderately active spiders. First,
even at its maximum 0- carrying capacity, hemocyanin carries relatively
little 0- compared to other circulating pigments (Angersbach 1978).
This, coupled with an open circulatory system, implies that some hypoxemic
areas should develop where tissues are most active metabol ical ly .
Secondly, even when adequate 0- is present, "^P demands may exceed the
ability of the small number of muscle mitochondria to produce these
compounds. Therefore, more alternative pathways must be used to
supplement the production of ~P even in the presence of 0- . An example
of this may be orb-web construction where slightly elevated levels of
lactate are found in the prosoma after an hour of steady activity by
the spider (Ch. III).

in summary, the picture of a spider engaged in moderate behaviors
such as grooming, searching, and web-building is one of aerobic

162

steady-state activity. Reliance on aerobic metabolism allows for both
maximum energy extraction and the use of the full range of energy
substrates (fats, protein, and carbohydrates). It also allows the spider
to retain the capacity for bursting into higher levels of activity (via
anaerobiosis and the use of phosphagen stores) when circumstances require.

Previous discussion indicated that one of the two determinants of
cardiac output (and therefore 0~ availability) was stroke volume but that
normally stroke volumes are relatively constant. However,
there is an important exception to this. To a large degree, stroke
volume is determined by the sum of the pressures generated by the
opisthosoma and heart compared to that of the prosoma. in resting and
moderately active spiders, the slightly higher pressures found in the
prosoma are easily overcome during systole and hemolymph is pumped to the
prosoma (Table Vll-l). However, during vigorous exercise this is no longer
the case and the cardiac output into the anterior aorta suddenly drops
to zero. Prior to this point, increased levels of exercise could still
be fueled through aerobic processes as a result of increased HR and
ventilation. However, once prosomal pressures force the stroke volume
to zero, then the only possible sources of -P in the prosoma become
stored phosphagens and anaerobic metabolism. This event physiologically
demarks the difference between moderate and maximal activity in spiders.

Maxima 1 Act i vi ty

The physiological changes that occur In maximal struggles of ca.
two minutes or less are. shown in Table Vll-l and specifically for F.
hibematis and L. lenta respectively in Figures Vll-l and 2. The top

Figure Vll-l. Changes in running speed, lactate, ~P stores,
prosomal pressure and heart rate during a two
minute maximal struggle in F. hihemalis at
25°C. All changes are expressed as percentages
(see Methods and equation Vll-l). A full
explanation of this figure is given in the text,

Key for lactate panel:

B prosoma

O opisthosoma

O whole spider.

164

100-

o

Ui

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O
QC
LU

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100-

1001-

TIME (M'N.)

Figure \J\\-2. Changes in running speed, lactate, ~P stores,
and heart rate during a two minute maximal
struggle in L. tenia at 25°C. All changes
are expressed as percentages (see Methods and
equation V I I - 1 ) . A full explanation of this
figure Is given in the text.

Key for lactate panel:
A prosoma
O opisthosoma
H whole spider

16S

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167

panel of each of these figures shows that during the first 15 to 30 sec
of forced runs (referred to as phase I in Ch. iV), measured speed
decreases by 50 to 85Z- Also, the spiders' gait changes from a jumping,
boundino run to a slow run or walk. Simultaneously, heart rate (HR)
doubles, but this rate is still only 20 to 30% of the HR found near the
end of activity or during early recovery. It has not been possible to
measure VO-, during phase I. The inability to measure VO- is not surpris-
ing. The oxygen that the spider will use during phase I must already be
in the prosoma since little if any hemol ymph flows to the prosoma during
the first minute of activity (Stewart and Martin IS^t; Anderson and
Prestwich 1975; Ch. V this study). The low numbers of mitochondria and
low activities of Krebs cycle enzymes in spiders (s.g. NAD- i soci trate
dehydrogenase, malate dehydrogenase, and citrate synthatase; Linzen and
Gallowitz 1975; Prestwich and Ing in press) may be the result of
selection both for a low resting aerobic metabolism and in response to
the frequent denial of 0^ to the prosoma when the spider is active.

The pressure generated by the prosoma obviously helps determine
both the force and speed of a spider's movements. it does this by
allowing for more rapid and forceful extension at higher pressures.
High pressures also present the leg muscles with a load they must over-
come and could potentially slow the spider's movements (Parry aid Brown
1959a, b; Stewart and Martin 197^; Anderson and Prestwich 1975).

However, there are three lines of evidence that suggest that
direct hydrostatic effects (such as turgidity and fluid loss) do not
normally limit the degree activity in spiders. First, the only legs
that mainly rely on hydrostatics to produce forward motion are the

168

fourth pair (Parry and Brown 1959b). The other legs produce much of
their effect via flexion (pair l) or various mixes of rotation, flexion,
and extension (pairs II and III, Manton 1958). Since leg pairs l-lll
are not always in contact with the subst ratun when they extend, then
lower pressures are necessary for their rapid extension than in pair IV.
Much of the spider's propulsion can be supplied by legs l-lll with little
necessity for high pressure. Second, maximum prosomal pressures are not
reached until 30 to k5 sec into an activity bout (if no activity has
recently occurred) (Fig. VII-1). By this time, running speed has
decreased by 70 to 80 percent. Finally, as was shown in Fig. I V- 1 ,
scorpions (which lack a hydrostatic skeleton) show nearly identical
patterns of fatigue development to spiders.

The relatively low pressures of phase I (100 to 350 mm Hg vs.
the i»50+ mm Hg of early phase II) may logically be ascribed to a high
rate of prosomal hemolymph loss (Wilson and Bullock 1973) leading to the
filling of the anterior opisthosomal venous sinuses. As these are filled
to capacity for the high pressure regime found during activity, higher
pressures are measured in the prosoma (Stewart and Martin 197^) because
of the distended sinuses' higher resistance to additional hemolymph flow
from the prosoma.

The most important constraints engendered by a spider's hydrostatic
skeleton are the limitations placed on the delivery of O2 to the prosoma
and legs (Wilson and Bullock 1973). Given limited O2 availability to the
prosoma and legs during exercise, it is not surprising that spiders have
wel 1 -developed anaerobic capabilities. The generation of ATP via
anaerobic glycolysis is comparable to that of reptiles and amphibians

a

169

(Bennett 1978), the vertebrates that seem most analogous ecologically to
spiders. The accumulation of lactate is shown in the second panel of
both Figures Vll-l and 2. Maximum rates of lactate production occur
during the first 30 sec to minute. However, glycolysis takes time to be
fully activated. Figure IV-S showed that the rate of glycolysis in Lycosa
does not reach maximum until after the first 10 to 15 sec of activity.

Neura 1 -hormona 1 mechanisms for activation of glycolysis in spiders
are not known. However, one of the most important activators of the
glycolytic pathway is AMP (Appendix il). Increases in its concentration

re probably slowed by the action of arginine phosphate (AP) in decreas-
ing the rate at which ATP and ADP depletion occurs. Arginine phosphate
slows the depletion of these compounds through the transfer of ~P to ADP
formed from the hydrolysis of ATP by the myos i n-ATPase. This transfer of
~P from AP to ADP becomes thermodynami cal 1 y favorable as soon as ATP stores

re slightly depleted. Figures Vll-l and 2 show the rapid depletion of
phosphate stores, principally AP , during the first 20 sec of running
coincident with the full activation of glycolysis (Table \l\-?.. Figure IV-S)
and phase I fatigue (Chs. IV, VI; Appendix II). Thus, AP performs a
function analogous to creatine phosphate in the white muscles of
vertebrates by maintaining the levels of ATP and fueling the first
few seconds of muscular contractions. Furthermore, the rate at which
glycolysis is activated by AMP may be slower than would be the
case if little AP were present.

a

Energy Usage

A model of the use of ~P stores, anaerobic, and aerobic metabolisms
in the prosoma and legs of Lycosa and Filistata for a two minute struggle

170

is given in Figures Vll-S and k. These estimates indicate that anaerobic
glycolysis accounts for over half of the total ~P used during a
struggle with only a slight dependence on aerobic metabolism. The
results agree with those in Chapter III that were for whole spiders and
were obtained from an entirely independent data base.

Earlier results (Ch. Vl) indicated that the relative importance of
the different sources of "P change with time. Figure \/ll-4 models these
changes in the prosomas of Filistata and Lycosa (see Methods). During
the first 10 to 15 sec of activity ~P stores accounted for 11% of the
total ~P used In Lycosa and 55? in Filistata. Over the same time period,
anaerobic glycolysis was of greater importance in Filistata, accounting
for k],% of the total ~'P versus 2]% in Lycosa.

Total energy use/production declines during exercise for two
reasons: rate limits on ~P availability and the results of the accumu-
lation of anaerobic by-products. Based simply on the activities of
rate- 1 imi t i ng enzymes, the availability of ~P from AP is much greater
than from anaerobic glycolysis which is in turn a potentially much faster
producer of '"'P than is possible from the mitochondria of spiders (Prestwich
unpublished; Prestwich and Ing in press; Linzen and Gallowtiz 1975).
Therefore, during the first 10 to 15 sec of activity (when AP is avail-
able), ~P cleavage rates of 30 to kO ymols/g min are found. Thereafter,
energy use trails off rapidly and maximum rates of ""P cleavage drop to
6-10 ymols/g min in Filistata and 6.5 in Lycosa. During this time
period (phase II), anaerobic metabolism becomes the dominant souce of
~P, being responsible for 2 to 6 times more ~P production than aerobic
metabolism. Stores of ~P are not depleted any more during the approxi-
mately 90 sec of phase II, in fact, they are actually replenished

Figure VI 1-3. Total ~P use during two minutes of maximal exercise
in Filistata and Lycosa. These totals represent
the sums of ~P obtained from stores of -P and
aerobic and anaerobic metabolism (see Methods).
The lower panel shows each species' relative
distance traveled (prosomas/sec) as integrated
for various time periods. Note the general
correspondence between the shape of each species
~P use graph and distance traveled. The main
disagreement is for the first 15 sec of activity,
-P use is higher per distance traveled than later.
This is an artifact of measurement: the spiders
jump a great deal during this time and this was not
quantified (Ch. IV).

172

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CO

<

o

LU
O

Z

O LU

> oc

I- K

<
-I
LU

20 - FILISTATA

101-

TIME

(MIN.)

Figure Vll-A. The changes in utilization of ~P from stores and
aerobic and anaerobic metabolism during a two
minute maximal struggle. Note that stores are
quici<ly depleted as a ~P source and rapidly
replaced by anaerobic metabolism. According to
the model used (see Methods), aerobic metabolism
becomes more important during late exercise because
prosomal pressures are lower (Ch. V) and bool< lung
ventilation has begun (Angersbach 1978). The
proportional amounts of ~P used during exercise from
each of the three energy sources are shown in
Table VI 1-2.

17A

■Vf-

FILISTATA

LYCOSA

LU
O

CO ^

in w

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TIME

(MIN.)

175

slightly. Thus, during late phase I and early phase II, steady-state
production/use of -P is achieved. By the time this steady-state is
reached, maximum activation of the glycolytic pathway has occurred and
doubtlessly, the potential for maximum aerobic metabolism has been
reached biochemically. Final decreases in the rate of "-P production
from these sources comes when anaerobic accumulations inhibit the
ability of muscles to contract and therefore ~P demand drops.

Support for the model in Figures \/ll-3 and k comes from agreement
between the use of ''P and the distances traveled during the exercise.
Distances traveled over 30 sec intervals are shown in Figure VI 1-3.
However, inspection of parts A and B of Figures VI 1-3 and k show that
changes in ~P usage and distance traveled do not exactly parallel each
other. The ratio of distance traveled (arbitrary units) to ~P (ymols/g)
can be calculated for two intervals: 0 to 15 and 15 to 120 sec. The
resultant ratios are measures of efficiency (power output in terms of
linear distance divided by power input in terms of ~P). For Lyoosa, the
ratio increases from 0.33 to 0.86; and in Filistata 0.^6 to 0.36. Thus,
the ratio is higher, that Is, the efficiency Is greater In phase II.
This result is at least partially artifact: jumping behavior, that I was
unable to quantify, occurs during phase I of activity (see Results,
Ch. IV). However, this result may also reflect a real difference In
efficiency of movement at different speeds.

In summary, the information presented in Figures VI 1-1 through k
support the hypotheses that (a) spiders are ultimately limited by fatigue
that is biochemical In nature due Initially to phosphagen depletion and
later to lactate accumulation and (b) that spiders are mainly dependent

176

on anaerobic metabolism and phosphagen stores as energy sources during
maximal activity. Any locomotory constraints that might be expected to
arise as a result of a spider's hydrostatic leg extension system do not
occur because of the design of the respiratory-circulatory systems (where
the book lungs provide resistence against prosomal hemolymph loss) and
the thickness of the opisthosomal musculature (which has coevolved with
the prosomal musculatures such that it is thicker in more active spiders)
(Wilson 1970; Wilson and Bullock 1973; Stewart and Martin 197^4; Ch. V) .

Recovery

Fatigue in spiders is related to depletion of ~P stores and to
lactate accumulation. Thus, the ability to run at maximum speeds is
dependent on both the resynthesis of phosphagens and removal of lactate
from the prosoma and legs. Figure \I\\-S shows partial recovery of phase
I speed in Filistata during a time when prosomal lactate is reduced to
about 60^ of the peak values and about half of the ~P stores have been
recovered. Average prosomal pressures also correlate approximately with
recovery of the above mentioned parameters.

In a spider with large anaerobic accumulations, removal of lactate
and resynthesis of ~P compounds is dependent upon aerobic processes. For
example, lactate may be removed from the prosoma by either physical
processes (that are ultimately aerobic process-dependent such as the
circulation) and/or biochemical processes (such as gl uconeogenes i s and
oxidation to CO- and water).

The results presented earlier in Chapters ill and IV suggested
that the rate of recovery is related to the respiratory surface area
(which is a direct reflection of the spiders aerobic abilities:

Figure VI 1-5. Recovery in F. hihernalis at 25°C after a two
minute bout of maximal activity. All physio-
logical values are presented as percentages
(see equation Vll-1, Methods). Pressures are
given with solid lines (bottom panel).

Key to lactate percentages:

B prosoma

O opisthosoma

O whole spider

178

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>

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m

CO
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c
a
m

■10

♦20

TIME

(MIN.)

Figure \/ll-6. Recovery In L. lento, at 25°C after a two minute
bout of maximal activity. All physiological
values are presented as percentages (see
equation Vll-l, Methods).

Key to lactate percentages:
A prosoma

0 opisthosoma

1 whole spider

130

100

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181

Anderson 1970; Anderson and Prestwich 1982). Recovery occurs more
quickly in P. audax (which possess a large book lung surface area and
tracheal system, Ch. l) and slowest in F. ht'bevndl-is (with small lungs
and vestigal trachea). Comparison of Ftlistata (Fig. VII-5) and Lycosa
(Fig. \/ll-6) again shows a rate of recovery is correlated with exchange
surface area. Every physiological parameter (VO^, HR, lactate concen-
tration, speed) returns to resting values at a rate that is 10 to 20%
greater in Lycosa than in Filistata.

Given a slower recovery in Filistata, nevertheless, similarities
exist in recovery between these two species and with other spiders. These
similarities deal with the overall pattern of recovery, especially as
pertains to the circulation. Peak heart rates are reached in both
species after the completion of exercise. This seems to be a character-
istic of spiders in general, having been reported for mygalomorphs
(tarantulas: Stewart and Martin 197'+; Angersbach 1978; Atypus Anderson
and Prestwich 1982) and araneomorphs (Heteropod i dae , Wilson 1967;
Filistata and Lycosa, Anderson and Prestwich 1982, and this study). The
highest rates appear to correspond to the peak VO^ (Ch. ill) and are
probably associated with the removal of anaerobic metabolites by both
physical (circulation) and biochemical (oxidation) means.

The exact mechanism of removal of lactate from the prosoma is
speculative. However, the data strongly suggest that it diffuses from
prosomal muscles to hemolymph and then is transported to the opisthosoma
where some isoxidized completely to CO^ and H„0 but nrrast is oxidized to
pyruvate and then converted into hexose (Ch. III). The evidence is
reviewed as follows: (A) Heart rates are high and prosomal pressure is

182

is low (Ch. V: Stewart and Martin ISy*; Angersbach 1978; Anderson and
Prestwich 1982). Therefore, maximum movement of hemolymph occurs.
(D) Prosomal lactate drops during a time when hemolymph pH falls suggest-
ing diffusion of lactate from the muscles (Ch. IV: pH data for a tarantula,
Angersbach 1978), while meanwhile opisthosomal lactate either remains
constant or rises, suggesting that at least some prosomal lactate
journeys to the opisthosoma (Figs. IV-S through 7). Removal of lactate
from the prosomal muscles by circulation is an important part of recovery
because it ameliorates the effects of high lactate more quickly than is
possible by direct biochemical oxidation of this by-product. Thus, the
muscles are able to worl< again even though the lactate has not been
removed from the spiders body. (C) High \/0„ is maintained while the
spider is inactive (Ch. Ill; Seymour and Vinegar 1973; Anderson
pers. comm. ) . The elevated VO^ is made possible by the high heart rates
and possibly increased stroke volume (Stewart and Martin 197'*; Appendix
l) coupled with maximal ventilation of the book lungs and a significant
Bohr effect (Angersbach 1978). (D) The amount of Oj used during recovery
corresponds to the amount used for gl uconeogenes i s of similar quantities
of lactate in vertebrates (Ch. Ill, Bennett 1978). (E) The primary
biosynthetic tissues associated with the opisthosomal digestive diverticu-
lum appear to be well-suited to perform this task (Millot 19^9; Prestwich
and Ing in press). (F) The opisthosoma may be a source of glucose
compounds that slip into the prosoma during activity (Fig. VJ-l). The
concept of spiders being organized into biosynthetic and locomotive
regions that is envisioned in the above scheme seems logical and is
found in other animals. However, it is well to remember that the above
scheme is only circumstantial and needs extensive validation.

183

Temperature

The effects of temperature (15° to 33°C) on activity in 25°C
acclimated Filistata and Lycosa are summarized in Table Vll-'*. These
spiders have a degree of thermal independence in terms of the rate at
which they can perform maximal activity. Most processes driven by
chemical reactions show a Q,„ of between 2 and 3; resting VO- in spiders
usually has a Q,, of between 2.0 and 2.5 (Anderson 1970). However, the
Q. for maximum running speed and total distance traveled in 2 min is
less than 2 in both species. Maximum rates of struggle (which are
important in prey capture and escape) are performed at a rate that
changes less with temperature than would be expected based on spiders'
Q,- value for aerobic metabolism.

Data on the effects of temperature on spider activity are
incomplete because there are no data for phosphagen metabolism at
temperatures other than 25°C. Earlier results have shown the critical
role played by these compounds during maximal struggles. Nevertheless,
the data for 15°C appear consistent and explainable. At this tempera-
ture, HR, rate of lactate production (L) , and \/0„ are all lower than
at 25°C. These observations all agree with the shorter distances
traveled and slower maximum speeds at this temperature. Likewise,
the slower whole animal removal of lactate can be ascribed to the
slow rate of oxidative processes at lower temperatures (Anderson
1970).

The 33°C results are not so consistent. The spiders ran faster and
further than at 25°C. However, L was greater than at 25° in Lycosa and
less than at 25° in Filistata. Heart rate and V0-, were both greater than

184

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185

at 25°C while recovery took longer. Perhaps the longer recovery is
caused by temperature effects on g t uconeogenes i s-spec i f i c enzymes. The
inconsistencies between lactate production and distance traveled may be
explained by two factors in Lyaosa. First, much of the extra distance
covered was in phase I. Since phase I is mainly fueled by -P stores,
it is possible that Lycosa relies more on ~P stores at this temperature.
Second, the very high L in Lycosa at this temperature could have led to
fatigue due to lactate accumulations within specific muscles before high
concentrations in the prosoma as a whole could occur. This could help
explain the rapid fatigue at 33°C and the refusal of most animals to
run after one and a half minutes. However, neither of these speculative
explanations can be invoked in the case of Filistata at 33°C. Clarifi-
cation of the effect of temperature on locomotion will probably have to
wait until data on the use of -? stores at non-acclimation temperatures
is in hand.

Interrelationships Between the Ecology, Behaviors, and Aerobic and
Anaerobic Abilities of Spiders

A vviovi , it seems that an interrelationship should exist between
a spider's behavior, its aerobic metabolism (VO-) , and anaerobic metabol-
ism. The bases for this supposition deal with the ways that resting VO-
on one hand, and anaerobic metabolic abilities coupled with the intensity
of activity, on the other, correlate with the book lung surface area. A
review of these interrelationships is necessary prior to discussion of
the factors that determine the mix of aerobic and anaerobic capabilities
possessed by a given species of spider.

186

Convincing evidence has been presented that indicates that resting
VOj is largely determined by ecological factors; principly the quality,
abundance, and pattern of food availability (Anderson 1970, 197^, 1978;
Anderson and Prestwich 1982; McNab I98O). Thus, sp i ders that are long- I i ved
and/or may commonly experience extended periods of low prey availability
tend to have low resting VO- and the ability to survive long periods of
starvation. Examples of such spiders are Lycosa and Filistata (Anderson
197^). Other spiders, e.g. the orb weavers, have life cycles that are
tightly interfaced with the availability of prey. These spiders possess
relatively high VOo (Anderson and Prestwich 1982).

Anderson (1970) and Anderson and Prestwich (1982) have shown that a
strong direct correlation exists between book lung surface area and rest-
ing VO-. In a general sense, it is also reasonable that maximal VO-
should correlate directly with respiratory surface area assuming
ventilatory and circulatory adjustments are similar in all species.
Data suggesting that this is in fact the case in spiders is presented
in Chapter III; peak VO- was highest in P. audax, intermediate in
L. lenta, and lowest in F. hibemalis. The same ordering is found with
both resting VO- and book lung surface area (Table 1-1).

In the earlier chapters evidence was presented that the magnitude
of anaerobic metabolism for any activity correlates directly with the
intensity of activity and inversely with the book lung surface area
(Fig. 11-2; Chs. II, III, IV). Thus, at some common intensity of
activity a spider with small respiratory surface area shows a larger
anaerobic accumulation and a smaller aerobic dependence than a spider
with large lungs. The remainder of this section deals with the actual

187

mix of behavior and aerobic and anaerobic metabolisms found in spiders
and speculates on the forces that shape these interrelationships.

Web-Bu i 1 ders

Orb-weavers possess high resting VO- and large book lung surface
areas (Anderson and Prestwich 1982). Their aerobic capacities are large
compared to other spiders since even a small factorial increase in VO-
over resting levels will result in a large total VOj • Their major daily
activity is web-building which may take one hour to complete. It is
performed without rest periods and requires an approximate doubling in
\10~ over resting levels (Peakall and Witt 1976). Anaerobic contributions
amount to less than one percent of the total cost when measured at the
moment the web is complete (see Ch. 111).

The completed orb web simultaneously increases the area the spider
is able to sample for prey, minimizes the necessity for the spider to
move about to locate and capture prey (since it both ensnarls the victim
and informs the spider of its exact location), and enables the spider to
capture two or more prey, at the same time. The necessity for active
searching and intense activity Is further reduced by the use of silken
swathing bands which the spider throws on its victim from a distance.
The enshrouded prey may then be easily killed with a poisonous bite.
Furthermore, escape from predators by orb-weavers does not require
extended vigorous activity since these spiders either go to an off-the-
web retreat, or shake the web, or drop to the ground, form a ball, and
hide in the vegetation. Thus, orb-weavers can avoid the requirement for
anaerobic activity by possession of relatively large aerobic capacities.

188

an efficient trapping web, a separation of activity in to preparation
for prey capture (web-construction) and capture itself, and by using
relatively passive predator escape techniques.

F-ilistata hibematis offers an interesting contrast to the orb
weavers. Like the orb-weavers, it makes a trapping web. However, this
web Is not suspended but instead forms a sheet over the substratum. Un-
like the orb-weavers, Filistata lives for many years and may frequently
face times of low ."""^V availability (such as winter). These spiders
have very low VO- and small book lungs (Anderson and Prestwich 1982) and
high anaerobic capacities (Chs. II and IV). The large anaerobic capacity
is probably not related to web-making since it involves minimal activity
in this species. {Filistata stands in one place and combs out silk to form
its web. To enlarge its snare it occasionally moves to another place and
combs silk.)

Filistata probably requires its wel 1 -developed anaerobic abilities
for struggles with its prey. Unlike orb-weavers, it does not throw
swathing bands and instead timidly battles with its trapped victim.
Also, its poison does not seem especially potent (based on the time it
takes prey to succumb after being bitten). The total time required by
Filistata to completely subdue its prey may be several minutes. Lacking
the ability to perform these attacks aerobically, it must rely on
anaerobic capab i 1 i t i f s .

Finally, other spiders with low rates of \/02 that make webs such
as Agelenid spiders (Prestwich unpublished) require large anaerobic
capacities since their webs are often non-viscid and these spiders in
effect must hunt on their webs much like spiders that lack webs (Prestwich
1977; Prestwich and Ing in press).

189

Hunting Spiders

Jumping spiders such as P. audax do not make webs. Like orb-
weavers they possess high aerobic capacities but they achieve these
capacities differently. Jumping spiders have a moderately large book
lung surface area and moderate resting VO2 (Anderson 1970). They are
able to increase their rate of oxygen consumption many times, possibly
through the use of trachea (Anderson 1970; Ch. III). Their large aerobic
capacity is vital to their characteristically active prey pursuit. They
commonly either patrol vegetation or wait and then pursue any potential
victims (Hill 1979). Unlike most spiders they are frequently in motion
for extended periods and this activity is undoubtedly aerobic. Escape
is also highly aerobic. These spiders spot predators and actively
flee them. They also possess excellent anaerobic capacity. This is
vital in prey capture (especially of larger items or in cases where the
spider had been chasing its victim for some time) because they lack a
sticky web to hold their prey and therefore cannot attack incapacitated
prey at their leisure.

The more common pattern in hunting spiders is seen in Lycosa Zenta.
This is a "sit and wait" predator that spends most of its time motionless-
short activities of more than minimal intensity result in lactate accumu-
lations (Ch. IV). Given their low aerobic abilities and the intense
activity that accompanies capture of large prey (Rovner I98O) it is not
surprising that they have such high anaerobic capacities.

In summary, spiders like other animals, periodically rely on stores
and anaerobic metabolism to fuel their activities. This permits them to
accomplish feats that would be impossible were they forced to rely on

190

their feeble aerobic abilities. Hov^;eve^, even with their low aerobic
capability, they do not possess unusually wel 1 -deve loped anaerobic
capacities (Bennett 1978) or phosphagen stores (Lehninger 1975). These
non-exceptional metabolic abilities are probably related to the
extensive use of silk and poison by spiders. Used both offensively
and defensively, these materials lessen the demand for high peak rates
of ~P generation. In sum, these adaptations show spiders can be
characterized as having reached an "apex" in low energy life style
(Anderson 1970, 197^, 1978; Anderson and Prestwich 1982).

APPENDICES

APPENDIX I
THE ESTIMATION OF CARDIAC OUTPUT AND
STROKE VOLUME IN SPIDERS

Summary

A calculation shows that in active tarantulas over 90?: of the
increase in cardiac output over resting conditions is due to increased
heart rate. Stroke volume is roughly constant or may drop.

I ntroduct ion

The most complete data on spiders internal and external gas
exchange and hemolymph distribution exists for tarantulas. Using data
on oxygen consumption (V0«) and A-V P0-, differences, it is possible to
calculate cardiac output by the Pick Equation:

whe

re Q is the cardiac output, VO- the rate of oxygen consumption and

C n and CwDo 3re the arterial and venous 0^ contents respectively.
a02 ^2 2

Potential problems with the use of this equation to find Q arise from
two sources: (a) correct determination of the A-V difference and (b)
the assumption of steady state conditions. Correct A-V differences
depend upon having representative, fully mixed arterial and venous Cq-
values. In the spider, CaOo ^an be determined from the pericardial sac
or heart itself since the hemolymph has just passed through the book
lungs. The C^q^ is more difficult to find since most opisthosomal

192

193

hemolymph does not combine with hemolymph from the prosoma until after
passing through the lungs. However, If one assumes roughly constant A-V
differences throughout the resting spider and insignificant opisthosomal
VO^ during maximal activity, then estimates of C^^ can be made based on
the PO2 of the ventral sinus of the prosoma. Prosomal venous hemolymph
flov'js through this structure just before returning to the book lungs.
Steady-state assumptions can be met based on the pattern of the animal's
activity. Animals at rest or in long-term activity are assumed to be in
steady state.

Once Q has been calculated, it is a simple matter to calculate an
average stroke volume (SV) because:

Q = SV" (HR)
where HR is the heart rate.

Calculation of Resting Q and SV in Tarantulas

Assuming a mass of 30 g, then resting VO- = 840 yl 0-/h at 23°C
(estimated from Anderson 1970). Data from Angersbach (1978) and Stewart
and Martin (197^) indicate a resting heart rate of 30/min. According to
Angersbach (1978) the AC02 = 8.5^2 ul O^/ml hemolymph (if PgOo = 27.8,
PyOo ~ 5 torr, then 7-9^ yl O^/ml hemolymph are released from hemocyanin
and 0.6017 yl from physical solution).

Therefore:

Q= (8A0 yl 02/h) T (8.542 yl 02/ml)
= 98.3 ml/h
or Q = 1 . 64 ml/min

]3h

The mean stroke volume can now be calculated,
$7= (1.6A ml/min) t (30 b/min)
= 55 ul/b
If the total hemolymph volume in a 30 g tarantula is aa. 6 ml (Stewart and
Martin IS?'*), then ca 0.9^ of the total hemolymph volume is ejected per
beat.

Calculation of Maximal Q and SV Under
Steady-State Conditions

Using a flow through system, Anderson (pers. comm.) has data
indicating the peak sustainable V0„ in tarantulas exercised continuously
for ten minutes is aa. 4600 y'l 0„/h, a value that is between 5 and 9X
resting VO- (resting VO- varies greatly in tarantulas). Anderson's data
suggests the ventilation of the book lungs reaches a maximum after several
minutes of activity. This is consistent with Angersbach's (1978) P0„
measurements that showed maximal A-V differences were not reached until
after at least a minute of activity. Angersbach (1978) also reported
lowest hemolymph pH values (and largest Bohr effects) several minutes
after the start of activity, consistent with observations of lactic acid
accumulation presented in Chapter IV. Because Anderson's tarantulas
exhibited an 0» debt after the completion of 10 min exercise that was
far greater than the total 0- capacity of the spider's hemolymph, therefore,
it is reasonable to assume that anaerobic pathways were utilized. Thus,
exercise was maximal. Under these conditions, AC02 's aa. 15-6 yl 02/111
hemolymph (Angersbach 1978). Heart rate maxima for tarantulas are given
by Angersbach (1978) and Stewart and Martin (1975) to be 85 b/min; for

195

argument's sake I will calculate SV based on a rate of 120 b/min, a
figure consistent with measurements on other spiders (Ch. IV; and
Anderson and Prestwich 1982).
Thus ,

Q = (^600 yl O2) ^ (15.6 yl O^/min)
= 294.9 ml/h or aa. ^+.9 ml/min.
This represents a 3X increase in Q
--If the heart rate is 85 b/min;

S7 = 58 yl/b
--If the heart rate is 120 b/min;

SV = 40 yl/b.
Thus, average stroke volume remains constant or may actually decrease by
28% while the heart rate increases by 2.8X and 4X. Therefore, heart
rate probably accounts for nearly all of the Increase in cardiac output.

Estimation of Maximum SV Based on

Non-Steady State Conditions

During short bouts of activity, it appears that no increase In
ventilation and therefore no increase in CgQo occurs (the resting arterial
hemolymph is usually only about 50% saturated with 0„) . The CvOo does
decrease to nearly 0, however, this is only a slight change from resting
conditions since the ACq- is now 9-557 {vs. 8.542). Heart rates are
slightly elevated (Angersbach 1978). If anything, it is likely that SV
Is low during maximal running (up to 1 min) since the maximal systolic
pressure of the tarantula heart (with help from the opisthosomal muscles)
is aa. 100 mmHg while prosomal pressures during activity peaks may exceed

196

A50 mmHg and be above 100 mmHg for much of the activity period (Stewart
and Martin 197A; also see Ch. V) . Thus, during most of this time, pumping
of hemolymph through the anterior aorta is impossible. Hemolymph may
still exit through the posterior aorta (to the opi sthosoma) ; however,
this is a much smaller vessel and doubtlessly can transport less hemo-
lymph.

APPENDIX I I
THE REGULATION OF GLYCOLYSIS IN SPIDERS

Summary

1. Relative activities of enzymes associated with glycolysis in
Lycosid spiders were calculated from literature sources. The
results suggest that hexokinase (HK) , phosphof ructokinase (PFK),
aldolase, and gl ycerol -3"phosphate dehydrogenase may catalyze non-
equilibrium reactions (Table AII-1).

2. Calculations of mass-action ratios (F) for resting and active spiders
compared to K values suggest that control points for glycolysis

in spiders involve (a) PFK and (b) the feed-i n react ions respons i bl e
for providing gl ucose-6-phosphate (Table AII-2).

3. The role of AMP (Ch. Vl) in glycolytic regulation is discussed.

Introduct ion

The study of the regulation of biochemical pathways can be

approached many ways. Two relatively simple but valuable approaches

involve first, the characterization of the relative activities of

different enzymes used in a pathway and second, the calculation of the

mass-action ratios (F) of the individual steps of the pathway compared

to the equilibrium constants (K ) for those reactions.

eq

Comparison of relative activities of pathway enzymes is useful
because it points out the places in a pathway where rate- 1 imi t i ng

197

98

steps occui i.3. the reactions catalyzed by enzymes present only in

low activity (note: the activity of an enzyme takes into account many

factors including titer, thermodynamics of the reaction, "catalytic

efficiency" of an enzyme, and modulation of the enzyme). Activities of

an enzyme are measured -in vitro under conditions that are thermodynami ca 1 1 y

optimal and in the presence of optimal amounts of substrates and activators

(Lehninger 1975).

Measurement of mass-action ratios (F) is done under conditions of

both activity and inactivity of a given pathway. Thus, the resting and

active ? values for a given reaction can be compared. Regulatory reactions

are usually far from equilibrium (K ) at rest and move towards K when
' ^ eq eq

the pathway is activated (Newsholme and Start 1973).

Enough data are available for Lycosid spiders to permit a pre-
liminary study of the regulation of glycolysis in spiders. In most

animals the control points for glycolysis are at the feed-in points for

, ^ ^ / , phosphory lase „ , „ , , hexoki nase .^^x
substrates {e.g. glycogen-^- <■ >-GlP and glucose >-GdP)

, ^, ^. ^. r r ^ c. u i, ^ rriin phos phof r uc tok i na se (PFK)
and the activation of f ructose-b-phosphate [VbP — ^

FDP]). Other minor control points may exist at the cleavage of FDP to

triose phosphates by aldolase and the ~P transfer to form ATP as

phosphoenol pyruvate is converted to pyruvate in a reaction catalyzed

by pyruvate kinase (see Fig. 11-1 to locate all of these steps). It is

the purpose of this section to compare regulation of glycolysis in other

animals, especially insects, with the available data for spiders.

199

Methods and Results

Relative Enzyme Activities

The activities of nearly all of the glycolytic enzymes have been
measured in spiders. Linzen and Gallowitz (1975) measured activities
from prosomal, leg and heart muscle homogenates from the wolf spider
Cupiennius salei. Prestwich and Ing (in press) measured activities of
some of the same enzymes as did Linzen and Gallowitz; however, Prestwich
and Ing also measured the activities of several other enzymes. One of
the species studied by Prestwich and Ing was the wolf spider Lucosa lenta.
There are important methodological differences between these studies:
Linzen and Gallowitz (1975) studied isolated muscle not whole tagmata
and expressed their results as activity/g fresh weight of muscle (vs.
activity/g protein). However, the legs and prosomas of spiders are
mainly muscle (Table 1-2) and because it is unlikely that other tissues
make as large contributions to glycolysis as does muscle, then it is
reasonable to combine the data for Cupiennius and Lycosa to obtain a
rough overview of the relative activities of the glycolytic enzymes of
wolf spiders.

Combining the data from these two different species was accomplished
by compari sons of act ivi t i es of enzymes used in both studies [hexokinase,
(HK) --Prestwich and Ing, unpublished; gl ycerol -3-phosphate dehydrogenase
(GPDH) ; lactate dehydrogenase (LDH); malate dehydrogenase (MDil) ;
gl utamate-pyruvate transaminase (GPT) ; g 1 utamate-oxaloacetate trans-
aminase (got)]. The ratios of activities of these enzymes in Cupiennius
and Lycosa were similar. Therefore, a compilation ofactivities was made
using the activity of HK as the index to which the other enzymes were

Table AII-1. Relative activities of glycolytic enzymes and
3 Krebs cycle enzymes. All are based on
hexoklnase activity of 1.0.

200

Enzyme

Legs Prosoma

C. salei L. tenta C. satei L. lenta

Hexoklnase

Phosphogl ucose Isomerase

Phosphof ructok Inase

Aldolase

Triose phosphate isomerase

Glycerol -3~phosphate DH

G 1 ycera 1 dehyde DH

Phosphoglycerate kinase

Phosphoglycerate mutase

Enol ase

Pyruvate kinase

Lactate DH

Citrate synthetase

Isocitrate DH (NAD)

Malate Dh'^

1
90

34
1716

1
161
88
90
64
75
Sh

0.07

134

0.5

49'

34

0.4^
b

52

15
2000

2.4
107
38
48
48
49
37

0.05

95

1

0.5^

4. A'

34'

95

Data for C. salei are calculated from Linzen and Gallowitz (1975);
L. lenta from Prestwich and Ing (in press).

Range of activities from 11 species of spiders are PFK, 0.161 to 4;
G3PDH 1.5 to 10; LDH 16 to 77; ICDH-NAD 0.06-1.0; MDH 25-135
(Prestwich and Ing, in press)

'^Probably over 80% of all MDH activity is cytosolic in nature (Linzen
and Gal lowitz 1975) .

201

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202

compared. Because HK was assigned the activity of 1.0, then an enzyme
that had 10 times the activity of HK was given a relative activity of
10. The results of this compilation are given on Table A! 1-1. The
only enzymes closely associated with glycolysis that have not been
measured in wolf spiders and are therefore not included in the table
are phosphory ! ase (phosphate cleavage of glycogen) and phosphog 1 uco-
mutase (GIF to G6P) .

Mass Action Ratios

Using the data for Lyoosa presented in Chapter Vl, mass action
ratios for glycolytic reactions were calculated whenever possible. The
data in Chapter Vl are given as ymols/g fresh weight; for the purpose of
calculation these were converted to molar concentrations by assuming
a water content of 78%. For comparison, the blowfly data of Sacktor
and Hurlbut (1966) and Sacktor and Wormser-Shav i tt (1966) were converted
to molarity using the same assumptions as for the spider data. All
calculations were done using the equations presented in Newsholme and
Start (1973). The results of these calculations are given as Table
Al 1-2.

Di scuss ion

The activities of all of the enzymes associated with glycolysis
are quite high except for HK, FFK, and GFDH. The first two enzymes
have activities that are at best only 0.07 those of any other glycolytic
enzyme. Depending on the source of the sample, GPDH occupies a middle
ground between FFK and HK and the other enzymes.

203

Generally, enzymes that are integral parts of a pathway but that
occur with activities that are aa. 0.1 to 0.001 those of the other path-
way enzymes catalyze non-equilibrium reactions (Newsholme and Start
1973). Their low activities indicate that there are simply not enough
active sites available for the reaction to obtain equilibrium in the
face of the catalytic actions of the other enzymes that are in much
higher activities. Thus, by this criterion, HK, PFK, and GPDH are. all
good candidates for controllers for non-equilibrium reactions.

Non-equilibrium reactions are often the result of either low
enzyme titers {e.g. in reactions that are not important in certain cells)
or where control points exist. In the latter case, the activity of the
enzyme may be modulated In accordance with the physiological needs of
the cell. Identification of reactions that correspond
to control points is made on the basis of two criteria. First, the
reaction must at times be non-equi 1 ibr I urn. Secondly, when the pathway is
perturbed such as to increase the flux through it, the mass-action ratio
of the control reactions must increase. This second criterion eliminates
the possibility of simple thermodynamic control where with increased
flux through a pathway the mass action ratio will remain roughly constant
(Newsholme and Start 1973).

Calculations of V for resting and exercising spiders are presented

in Table AII-2. Large discrepancies (> 10 ) between reaction K and

^ ^ eq

resting T exist for HK, PFK, and aldolase controlled reactions. It was

not possible to calculate F for GPDH since the concentrations of NAD and

NADH were not known. The K and restinqF for the trio sei some rase (TIM) react ion

eq

differ only by a factor of 10. This difference could easily be due to

20i*

poor measurements of g I yceraldehyde-3-phosphate since its concentration
was at the limit of resolution (Ch. Vl). Overall, the pattern of resting
r values calculated for Lyaosa and Filistata agree well with those for
the blowfly.

During exercise VO^ increases 2 to perhaps 18 times above resting
(see Ch. VII). Based on the initial disappearance of anthrone-react i ve
substances and accumulation of lactate (Figs. Vl-'^ and II) it is obvious
that there is a large increase in glycolytic flux during exercise. High-
flux Fs were calculated for 15, 60 and 120 sec into activity; for com-
parison r was also calculated during recovery for Filistata. Table All-2
shows insignificant changes in F for the aldolase- and Tl M-cata 1 yzed
reactions. However, for PFK the F increased by a factor of IIX, this
is normally regarded as significant and indicative of a control point
reaction (Newsholme and Start 1973)-

The increases In the F for the PFK reaction parallels the increase
in AMP (Fig. Vl-S). During the first 15 sec of activity, AMP increases
by a factor of lOX. It is the factorial increase of this substance and
its absolute concentration that are important in understanding its role
as a regulator of various enzymes (Newsholme and Start 1973). Thus, the
situation in spiders appears to be like other animals; as AMP increases
the PFK reaction moves towards equilibrium, as it decreases (assuming
AMP changes in recovering Lyaosa are like Filistata), the PFK reaction
moves away from equilibrium. Other substances probably also help de-inhibit
the PFK. One of these is phosphate (P., see Fig. VI-9). After 15 sec
of activity, P. has more than doubled in Lyaosa and thus it may also be
important in the regulation of PFK (Lehninger 1975). There are several

205

other substances that are common)y thought of as activators of PFK;
especially important are increased Ca and 3'5' cyclic AMP (Newsholme
and Start 1973)- However, the regulatory roles of these substances in
spiders are not known.

Finally, the status of the HK-catalyzed reaction cannot be
interpreted unambiguously. A large shift of the F toward K may be
misleading since spiders can obtain G6P (used in calculation of V for
HK), from other reactions, especially the pathway leading from glycogen
to GIP to G6P (catalyzed by glycogen phosphory 1 ase and phosphog 1 uco-
mutase) . Another possible source of G6P is via the metabolism of
trehalose.

Given the decrease of anthrone-react i ve substances during exercise
(even after correction is made for glucose), it is likely that the con-
centration of G6P is determined by several reactions. Thus it is
impossible to comment on the importance of HK as a regulatory enzyme
with the present data. However, the increase of G6P does indicate
that the sum of the reactions leading to the production of G6P are
probably regulated and therefore important in the overall regulation of
glycolysis as in other animals. The presence, activity, and regulation
of glycogen phosphory lase in particular needs investigation due to the
major role this enzyme plays in other species and because of insights
it may give into the uses of Ca , cyclic AMP, and AMP in metabolic
regulation in spiders.

In summary, the results in this section should only be regarded
as preliminary. They suggest the major regulatory points of glycoylsis
in spiders are the same as in other animals. These results should be used
to guide future studies of the regulation of glycolysis in spiders.

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BIOGRAPHICAL SKETCH

Kenneth Nea 1 Prestwich was born on March 8, 19^9, in Inglewood,
California. He spent his youth moving about the country with his
parents and was especially intrigued as a child with the animals and
plants of north Florida. Upon graduating from Robert E. Peary High
School in Rockville, Maryland, in 1967, he attended Davidson College
In North Carolina where he received his B.S. in biology in 1971. In
I97A, he interrupted his studies to ride across the United States on a
bicycle with two friends. He received his M.S. in zoology from the
University of Florida in 1975. Since then he has continued his work
on spiders and crickets and has actively pursued hobbies of bicycling,
running, canoeing, and beekeeping.

212

I certify that I have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

^- 6?w^

'<U^Hry^

JO/KH F. And'ersoh', Chairman
Associate Professor of Zoology

I certify that I have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

4m

'm

Brian K. McNab
Professor of Zoology

1 certify that i have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Y)T7

James L. Nation

Professor of Entomology and Nematology

I certify that 1 have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

f^rank G. Nordl ie
Professor of Zoology

i certify that I have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

^ Re i ski nd

Tathan Reiskind
Associate Professor of Zoology

I certify that I have read this study and that in my opinion it

conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Wendell N. Stainsby ^
Professor of Physiology

This dissertation was submitted to the Graduate Faculty of the Department
of Zoology in the College of Liberal Arts and Sciences and to the Graduate
Council, and was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

May 1982

Dean for Graduate Studies and Research