Regeneration and autotomy in the black widow spider, Latrodectus variolus Walckenaer

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Title:
Regeneration and autotomy in the black widow spider, Latrodectus variolus Walckenaer
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Latrodectus variolus Walckenaer
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xiv, 86 leaves : ill. ; 28 cm.
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English
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Randall, John Brookes, 1949-
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Black widow spider -- Physiology   ( lcsh )
Regeneration (Biology)   ( lcsh )
Autotomy   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 77-84).
Statement of Responsibility:
by John Brookes Randall.
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Typescript.
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Vita.

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University of Florida
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REGENERATION AND AUTOTOMY IN THE
BLACK WIDOW SPIDER,
Latrodectus various Walckenaer.








By

JOHN BROOKS RANDALL


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


1979
















DEDICATION


This dissertation is dedicated to my mother, Norma B.

Randall, who taught me the values of study and striving to












REGENERATION AND AUTOTOMY IN THE
BLACK WIDOW SPIDER,
Latrodectus various Walckenaer.








By

JOHN BROOKS RANDALL


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














ACKNOWLEDGEMENTS


I would like to express my sincere appreciation to

Dr. H. L. Cromroy for his continual advice, encouragement

and friendship throughout the course of this study and for

furnishing me with the space and materials required to

complete this work.

I would especially like to thank Dr. Herbert Oberlander

of the Insect Attractants, Behavior and Basic Biology Research

Laboratory, USDA, Gainesville, Florida for his continual

counsel, encouragement and friendship and for his valuable

assistance in the preparation of the dissertation.

I would also like to acknowledge Dr. J. Nation and

Dr. J. Reiskind as members of my supervisory committee for

their advice and encouragement. I would also like to thank

Dr. D. L. Silhacek, also of the USDA Gainesville lab, for

employing me in his laboratory and Dr. M. S. Mayer of the

same laboratory for his help in assessing some of the data

I collected.

My sincere thanks go to my parents, Mr. and Mrs. John

A. Randall of Millersville, Maryland,and to Col. and Mrs.

Charles Foreman of McLean, Virginia, for their continual

support and encouragement in seeing that my family was

never without necessities.


iii








Finally, I wish to thank my wife Carol who worked hard

at a not so desirable job so that I could complete my

graduate studies. Without her support, love and encourage-

ment this goal could never have been attained.
















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

KEY TO SYMBOLS AND ABBREVIATIONS .

ABSTRACT . .

INTRODUCTION . .

LITERATURE REVIEW .

Regeneration .

Crustacea .

Insecta .

Arachnida .

Autotomy . .

Crustacea .

Insecta .

Arachnida .

L. Variolus and the Morphology
Arachnid Palpal Organ and Legs

METHODS AND MATERIALS .

L. various . .

Histology . .


Page

iii

viii

ix

xix

xiii

1

3

3

4

4

10

11

12

13

13


. 14

. 20

20

. 21


Amputation and Ligature


21









Page

RESULTS . . .25

The Palpal Organ of L. various .. 25

Development of the Papal Organ 25

Amputation of the Pre-penultimate Palp 30

Amputation at the mid-tarsus .. 30

Amputation at the tibia-tarsus joint 30

Amputation at the patella-tibia
joint . .. .32

Amputation at the femur-patella
joint . . 35

Amputation at the mid-femur and at
the trochanter-femur joint ....... 35

Amputation at the coxa-trochanter
joint .. . 36

Amputation of the Penultimate Palp .. 39

Amputation at the mid-tarsus 39

Amputation at the tibia-tarsus joint 39

Damage to the Palpal tarsus .. 39

Amputation at the patella-tibia joint... 41

Amputation at the coxa-trochanter
joint. . .. .. 41

Ligature of Pre-penultimate and Penultimate
Palps . . .41

Ligature at mid-femur of Pre-penultimate
palp . . 41

Ligature at the tibia-tarsus joint of
Penultimate Palp. . 44

Ligature at mid-femur of Penultimate
Palp . . 44

Regeneration and Autotomy in Legs of L. various 44












Amputation of the Legs ... .

Amputation at the mid-telotarsus .

Amputation at the mid-basitarsus .

Amputation at the mid-tibia and at
the patella-tibia joint .

Amputation at the femur-patella joint

Amputation at mid-femur .

Amputation at the trochanter-femur joint

Amputation at the coxa-trochanter joint


Amputation at the Proximal Margin of


the Coxa. . .

Localized Injury to the Femur ..

Ligature of the Legs . .

Ligature at the mid-basitarsus .

Ligature at the mid-tibia .

Ligature at the patella .. .

Ligature at the mid-femur .

External Force Applied to the Autotomy
Plane of the Leg . .

Summary of Results . .

DISCUSSION . . .

Regeneration . .

Autotomy . . .

APPENDIX 1 Fixation, Dehydration and Embedding

APPENDIX 2 M.allory's Triple Stain Technique

LITERATURE CITED . .

SUPPLEMENTARY BIBLIOGRAPHY . .

BIOGRAPHICAL SKETCH . .


Page

S 44

S 44

S 46


S 46

48

S 48

S 50

50


. 50

. 50

. 54

. 54

. 56

. 56

. 58


. 58

. 58

. 63

. 63

. 68

* 74

S. 76

. 77

. 82

. 85


vii














LIST OF TABLES


Table Page


1 Summary of the amputation experiments performed
on pre-penultimate male L. various palps 38

2 Summary of the amputation experiments performed
on penultimate male L. various palps ..... 43

3 Summary of ligature experiments performed on
the palps of pre-penultimate and penultimate
male L. various . 45

4 Summary of the amputation experiments performed
on the legs of L. various . 53

5 Summary of ligature and external pressure experi-
ments performed on the legs of L. various 60


viii
















LIST OF FIGURES


Figure Page

1 The developmental gradient model for
regeneration and duplication . 8

2 The polar coordinate model for regener-
ation . . 9

3 Schematic diagram of the autotomy mechanism
of spiders. .. . 15

4 Comparative morphology of the spider leg
and palp ...... . .. 15

5 The development of the male palpal organ
and identification of some of the major
parts of the adult organ . 18

6 Restraint apparatus used in amputation and
ligation experiments . ... 23

7 Ligature in place on the leg of L. various 23

8 Pre-penultimate palp of a male L. various 26

9 Early proliferation of pretarsal primordia 26

10 Pretarsal cells at 48 72 hours of develop-
ment . . 27

11 Penultimate palp of a male L. various 27

12 Differentiation of the developing palpal
organ at 24 36 hours into the penultimate
instar ... . 28

13 Differentiation of the developing palpal
organ at 48 72 hours into the penultimate
instar . . .. 28

14 Differentiation of the developing palpal
organ approximately four days prior to the
adult molt . .. .. 29










Figure Page

15 Embolus of the developing palpal organ
visible through the tarsal cuticle 29

16 Results of amputation of pre-penultimate
palp at mid-tarsus . .. 31

17 Results of amputation of the pre-penultimate
palp at the tibia-tarsus joint .. 31

18 Histology of regenerate palp following ampu-
tation at the tibia-tarsus joint 33

19 Results of amputation of the pre-penultimate
palp at the patella-tibia joint 33

20 Histology of regenerate palp following ampu-
tation at the patella-tibia joint .. 34

21 Results of amputation of the pre-penultimate
palp at the femur patella-joint .. 37

22 Results of amputation of the pre-penultimate
palp at the mid-femur and trochanter-femur
joint . . 37

23 Results of amputation of the pre-penultimate
palp at thecoxa-trochanter joint .. 37

24 Results of amputation of the penultimate palp
at the mid-tarsus . .. 40

25 Results of amputation of the penultimate palp
at the tibia-tarsus joint . .. 40

26 Results of damage (puncture) to the tarsus of
the penultimate palp . .. 40

27 Results of amputation of the penultimate palp
at the patella-tibia joint .. 42

28 Results of amputation of the penultimate palp
at the coxa-trochanter joint .. 42

29 Results of amputation of the leg at the mid-
telotarsus . . 47

30 Results of amputation of the leg at the mid-
basitarsus . . 47










Figure


31 Results of amputation of the leg at the mid-
tibia and at the patella-tibia joint 47

32 Results of amputation of the leg at the femur-
patella joint . . 49

33 Results of amputation of the leg at the mid-
femur . .. 49

34 Results of amputation of the leg at the
trochanter-femur joint . 49

35 Results of amputation of the leg at the coxa-
trochanter joint . ... 51

36 Results of amputation of the leg at the
proximal margin of the coxa. ..... .. 51

37 Results of localized injury to the femur of
the leg . . 55

38 Results of ligation of the leg at the mid-
basitarsus . .. 57

39 Results of ligation of the leg at the mid-
tibia .... . .. 57

40 Results of ligation of the leg at the patella 59

41 Results of ligation of the leg at the mid-
femur. .. . .. 59

42 Comparison of the regenerative capacities
of the pre-penultimate and penultimate palps
of the male L. various . 66

43 Healing of the wound produced by amputation of
the pre-penultimate palp at the tibia-tarsus
joint . . 69

44 The open wound of an autotomized leg after
localized injury to the femur of the leg 69

45 A comparison of the autotomy, healing and
regeneration of the legs injured by amputa-
tion and ligation .. . ... 70

46 Histology of the telotarsus of the leg of
L. various showing the leg nerve present. 70


Page















KEY TO SYMBOLS AND ABBREVIATIONS


Al alveolus

btar basitarsus

Cd conductor
Cx coxa
Cx. ms coxal muscle
Cm cymbium

D. Ib dorsal lobe

Em embolus

Fm femur
Fd funds

Haem haematodocha (basal)

Inv invagination

M.a median apophysis

Nv nerve

Pat patella
Ptar pretarsus

R.s receptaculum seminis

Scl sclerite

Tar tarsus
ttar telotarsus
T.a terminal apophysis
Tib tibia
Tr Trochanter

V.lb ventral lobe

amputation


S ligation


xii














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


REGENERATION AND AUTOTOMY IN THE BLACK
WIDOW SPIDER, Latrodectus various
Walckenaer

by

JOHN BROOKS RANDALL

August 1979

Chairman: Dr. Harvey L. Cromroy
Major Department: Entomology and Nematology

The restoration of lost body parts by regeneration has

been extensively investigated in arthropods. Crustaceans,

insects and to a lesser degree arachnids have been utilized

to study this phenomenon. The loss of an appendage most

readily occurs at a predetermined plane of weakness, termed

the autotomy plane, as a mechanism of escape or severence of

a badly damaged limb. The mechanism of autotomy is believed

to be initiated by a nervous reflex. Past researchers state

that the capacity for arthropod regeneration is greatest

at the autotomy plane.

Amputation and ligature of the developing male palpal

organ and the legs were used to determine whether regeneration

occurring in the black widow spider, Latrodectus various

Walckenaer complied with the developmental gradient of regen-

eration. The occurrence of autotomy in this species was

also documented.


xiii









The ability of the pre-penultimate palp to regenerate

and subsequently produce a normal adult palpal organ was

confined to injury to the distal portion of the palp. Ampu-

tation of more proximal segments of the male palp during the

pre-penultimate stage did not result in normal adult palps

two molts later. Amputation of or severe damage to the tibia

and tarsus of the penultimate male palp most often resulted

in the death of the spider. No regeneration occurred follow-

ing amputation or ligature of penultimate palps.

Amputation indicated the most proximal point from which

regeneration of the leg could occur was the mid-point of the

femur. Proximal to that point no regeneration was observed.

Autotomy following amputation was not observed. The regenera-

tion observed in the palps and legs of L. various complied

with the developmental gradient and polar coordinate models

for regeneration.

Ligature of the legs resulted in autotomy when applied

at and proximal to the mid-point of the tibia, increasing in

frequency as more proximal segments were ligatured. Autotomy

always occurred at the coxa-trochanter joint. No regeneration

of the legs occurred following autotomy.

The evidence strongly suggests that autotomy in the legs

was initiated by a wound factor (currently hypothetical)

released after injury and the dose of which may be related to

the size and duration of the wound.


xiv















INTRODUCTION


In their evolution from annelid-like ancestors arthropods

had to sacrifice some advantages in order to become more

specialized and complex. Although their greater complexity

does not allow for the regeneration of large body parts, such

as an entire head, arthropods have retained the ability to

regenerate appendages. When arthropods gained the protection

of an exoskeleton it became necessary for them to molt in

order to grow. Likewise, molting was required for arthropod

regeneration to occur. The rigid exoskeleton, clearly segment-

ed, provides for qualitative and quantitative measurement of

regeneration. For this reason arthropods, especially crusta-

ceans, insects and to a lesser extent the arachnids, have

been the subject of considerable research into the mysteries

of regeneration.

Unlike the crustacea that continue to grow and molt after

reaching maturity, insects, once sexually mature, lose the

ability to molt and thereby their ability to regenerate lost

parts. The majority of spiders, like insects, cease molting

after maturity is attained. Exceptions to this include the

mygalomorph spiders (i.e. tarantulas) that continue to molt

after maturation and can live up to 25 years.








The black widow spider has been the subject of much

study in past years. A species of black widow spider,

Latrodectus various Walckenaer, was used in this investiga-

tion to establish the regenerative capacitites of the develop-

ing male palpal organ and legs. The occurrence of autotomy

in this species was also studied. The results establish

three alternatives, besides death, to the injury exhibited

by L_ various; healing with no regeneration, regeneration or

autotomy.

The following literature review provides background

information on arthropod regeneration and autotomy as well

as on the development of the palpal organ of a male black

widow spider.















LITERATURE REVIEW


Regeneration

Regeneration, the restoration of lost parts, has been

described by many authors. Goss (1965) views regeneration

as a physiological process, not simply anatomical growth,

with the primary objective of re-establishing the functional

efficiency of the organism. Thus, regeneration is stimulated

by the physiological demands for increased function caused by

the loss of a body part. Goss defines qualitative regenera-

tion as that process which occurs to replace a lost limb, as

this is the only way efficiency can be restored in a struc-

ture that has a single function. Quantitative regeneration

is the method by which compound organs, such as a liver,

would be restored.

Wolpert (1974) considers regeneration as the re-establish-

ment of the positional field of cells followed by the reinter-

preting of positional values. This can be attained by either

of two methods. (1) Epimorphosis involves growth from the

cut surface of the wound to provide new positional values for

the regenerating portion. (2) Morphallaxis establishes a new

boundary region at the cut surface and new positional values

are assigned within the existing adjacent tissues. Morphall-

axis does not involve growth.








Crustacea

Crustaceans have been utilized extensively in the study

of regeneration (Agar, 1930; Bliss, 1960; Emmel, 1910;

Needham, 1945, 1947, 1949, 1950, 1953; Paul, 1914; Wilson,

1903; Wood and Wood, 1932). Crabs, lobsters and crayfish

have been examined extensively because of their convenient

size, availability, and possession of a preformed breakage

plane (Bliss, 1960). With the possible exception of mouth-

parts, decapods seem to be able to regenerate all types of

appendages. It is believed that in crustacea the peripheral

nerve supply in the region of amputation exerts a local

effect favoring limb regeneration. This, coupled with neuro-

secretory hormones which inhibit the molt-promoting Y-organs

are responsible together for the regenerative capacity in

these animals (Bliss, 1960).


Insecta

Among the insects used in regeneration experimentation,

cockroaches have been studied most extensively. Bohn (1965,

1972, 1974a, 1974b, 1974c) used Leucophaea maderae in experi-

ments which indicated that the integumental tissues that

separate adjacent legs were required for the regeneration of

the leg. Regeneration of a leg occurred only when both the

basal sclerites anterior to and a membraneous area posterior

to the coxa were in contact with each other. Bohn confirmed

these results through transplantation experiments (Bohn, 1974b).

Bohn (1965) found that a V-shaped wedge cut from the tibia of





5



Leucophaea resulted in the next instar developing a lateral

regenerate at the site of injury.

Scientists working on different insects described vari-

ous proximal limits for leg regeneration. Penzlin (1963)

(Periplaneta) reported regeneration after removal of most of

the basal sclerites (episternum and epimeron). Bulliere

(1967) and Urvoy (1963) (Blabera craniifer Burm.) found the

proximal limit of regeneration between the coxa and trochantin

and praecoxa and trochantin respectively.

Luscher (1948) reported regeneration of the leg of

Rhodnius occurring as far back as the coxa-trochanter joint.

In 1933 Bodenstein reported regeneration after removing the

entire leg and surrounding tissues in larval Vanessa urticae

Raupen (Lepidoptera). Bodenstein (1955) not only discovered

that Periplaneta americana could regenerate the entire leg

after amputation at the trochanter-femur joint, but also

found that ecdysone was required for initiating and sustaining

the progression of regeneration. Adult Periplaneta could be

made to regenerate through parabiotic fusion with nymphs and

by transplantation of active prothoracic glands. It was

Bodenstein's feeling that "wound factors" produced at the

site and time of injury played a minor role, if any, in the

initiation of regeneration. Needham (1947) argues that a

wound factor may in fact reduce the regenerative power since

after autotomy of limbs in the crustacean Asellus aquaticus

if the remaining tissues are mechanically damaged there is a

reduction in the animal's regenerative capacity.








O'Farrell and Stock (1953) investigated regeneration of

the metathoracic leg of Blattella germanica, and found that

when the leg was amputated at the proximal autotomy plane

(between the trochanter and femur) either a completely differ-

entiated regenerate or an undifferentiated papilla resulted,

a complete regenerate appeared at the second molt following

amputation. They also described a "critical period" during

the first instar before which amputation resulted in a com-

plete regenerate with a delay in the first ecdysis following

surgery and after which the papilla was produced with no

delay in ecdysis. The ability of B. germanica to regenerate

a complete leg persisted until the last molt. Repeated

regeneration of the same leg prolonged development and

caused additional molts but the adults resulting from such

supermolts were normal in size and appearance. When reared

at 250 C, repeated regeneration of B. germanica initiated

early in development resulted in more supermolts than if

initiated later. Most of the experimental insects reared

at 300 C metamorphosed without supermolts.

Regeneration in insects has also been studied through

experimentation on the imaginal discs of developing larvae.

Bryant (1971) performed in situ experiments bisecting the leg

discs of Drosophila melanogaster. He found that the upper

portion of a bisected disc, still attached to the larval

epidermis, regenerated, whereas the lower half of the disc,

unattached from the larval epidermis, duplicated itself.

Partial bisection of leg discs resulted in branched

legs where one branch was complete and the other branch a








double half. Bryant interpreted these results to mean that

regeneration occurred from one cut edge and duplication from

the other. From this work Bryant proposed a gradient of

developmental capacity and its response to bisection (Fig. 1).

Later Bryant (1975) found that when an imaginal disc was

cut into three pieces those fragments with their cut edge

facing away from the center of the disc underwent regenera-

tion, while fragments with their cut edge facing toward the

center of the disc underwent duplication. The presence of the

center of the disc was not a prerequisite for regeneration.

Fragments with two cut edges on the same side of the center

would exhibit regeneration at one edge and duplication at

the other.

French et al. (1976) proposed the polar coordinate

model for regeneration based on information from cockroach

and amphibian limb regeneration and insect imaginal disc

regeneration. Their model is a two-dimensional system allow-

ing the assignment of specific positional information to an

epimorphic field. One coordinate defines the circumferential

position of a cell by twelve meridinal points numbered clock-

wise one to twelve. Letters A to E define the proximal-

distal position of a cell. Proximal structures are at the

periphery and distal structures are at the center of the

model (Fig. 2).

They also proposed two rules for the behavior of cells

in an epimorphic field. The rule of intercalation states

that when normally non-adjacent positional values in either






















PROX. DISTAL


Bissection


C


I ~,2


Growth


Regeneration Duplication


Fig. 1. The developmental gradient model for regeneration
and duplication (Bryant, 1971).













































Fig. 2. The polar coordinate model for regeneration (French
et al., 1976).








the circular or radial sequence come into contact in a graft

or through wound healing, growth occurs at that junction

until the cells with intermediate positional values have

been intercalated.

The second is the complete circle rule for distal trans-

plantation. The entire circular sequence at a particular

level may undergo distal transplantation to produce cells

with all the more central (distal) positional values. This

rule pertains to Bryant's gradient of developmental capacity

(Fig. 1) and means that when amputation occurs along the

proximal-distal sequence of positional values the proximal

level remaining can regenerate only those positional values

distal to it.


Arachnida

Regeneration in arachnids has been little studied

(Bonnet, 1930; Friedrich, 1906; Schultz, 1898; Vachon, 1941;

Wagner, 1887). The most extensive of these studies was car-

ried out by Bonnet working on Dolomedes fimbriatus (Clerck)

(Pisauridae). Bonnet (1930) reported that D. fimbriatus

could regenerate from one to all eight legs, taking three

molts to re-establish normal size.

Bonnet also performed regeneration experiments on devel-

oping male palps and concluded that if the loss of part or

all of a palp occurred no later than the preantepenultimate

instar (three more molts before maturation) the male could

fully regenerate the palp. Palps injured or lost later than








the preantepenultimate stage would not regenerate completely.

When injured at the prepenultimate stage the palps were some-

times perfectly formed at maturation but were too short so

that the animals could not come to normal copulation.

Vachon (1941) reported that the leg segments of the same

regenerating leg were not necessarily all at the same stage,

distal segments being "older" than proximal ones.


Autotomy

Several terms have been used to describe the loss of an

arthropod limb; they include: 1) autotomy, 2) autospasy and

3) autotilly (Bliss, 1960). Autotomy is the ability of an

animal to cast off its own appendage at a pre-determined

breakage plane by a well developed, usually unisegmental

reflex. Autospasy has been defined as the separation of a

limb at a predetermined plane of weakness when the limb is

subjected to force by an outside agent against the resistance

provided by the animal's weight or efforts to escape. Auto-

tilly is the severence of the limb at a predetermined plane

of weakness through use of the mouthparts, claws, or legs of

the animal itself.

The point common to all three definitions is the "pre-

determined plane of weakness," also termed breaking joint,

autotomy plane, plane of least resistance, and locus of weak-

ness or separation (Bliss, 1960).

Injury or amputation distal to the plane of weakness

often causes the entire limb to detach. In most arthropods









no muscles cross the autotomy plane (Needham, 1965). An

exception to this was described by Parry (1957) where the

M. Flexor femoris longus of the spider Tegenaria atrica Koch

(Agelenidae) passes from the coxa, through the trochanter to

attach to the femur. The autotomy plane of T. atrica is at

the coxa-trochanter joint.

Autotomy occurs as the result of a nervous reflex

initiated by injury to the limb (Goss, 1965). Goss also

stated that the capacity for regeneration of lost appendages

in arthropods is greatest at the autotomic breakage plane.


Crustacea

Some crustaceans possess an autotomy plane but no reflex

of autotomy. In others (i.e. Homarus americanus) autotomy

only occurs in the first pair of thoracic legs or chelae.

Autospasy and autotilly may occur in the other limbs as the

plane of weakness exists but the autotomy reflex is absent

in those legs (Wood and Wood, 1932). When Wood and Wood

studied 15 species of crabs they found autotomy exhibited in

all five pairs of legs.

The stimulus for autotomy may occur when injury is sus-

tained to an appendage distal to the plane of weakness.

Hodge (1956) demonstrated in the crab Gecarcinus lateralis

that autotomy never resulted from injury to the dactyl, the

most distal segment of the walking leg, but did occur with

greater frequency as more proximal segments were injured.

This was also demonstrated by Needham (1947) for Asellus

aquaticus and on several species of Brachyura (Wood and Wood,









1932). This may be related to the fact that the leg nerve

does not extend beyond the proximal area of the propus, the

next proximal segment to the dactyl (Bliss, 1960).

Bliss also reported that acetylcholine reduces the fre-

quency of autotomy when injected into a crustacean and that

acetylcholine antagonists (i.e. atropine) facilitate autotomy.


Insecta

O'Farrell and Stock (1953) found regeneration when the

leg of B. germanica was removed at the autotomy plane, between

the trochanter and femur. The same plane of weakness has been

described for mantids, phasmids, and grasshoppers (Bliss, 1960).

A second locus of separation was reported at the tibia-tarsus

joint of B. germanica by Woodruff (1937). A true autotomy

reflex was described for Achaeta domesticus L. by Brousse-

Gaury (1958).

Autotomy is even well developed in Tipulidae and Opiliones

(arachnida) where regeneration is impossible (Needham, 1965).

The ability to escape has value regardless of the ability to

replace the appendage lost in escape.


Arachnida

Autotomy has been examined in spiders (Bonnet, 1930;

Parry, 1957; Wood, 1926). Unlike crustaceans and insects,

spiders autotomize their legs at a functional joint, the coxa-

trochanter joint (Parry, 1957). Bliss (1960) stated that

among the true arachnids, including the spiders, there is

autospasy but not autotomy. Wood and Wood (1932) reported the








absence of a plane of weakness in scorpions, ticks and

Limulus.

Wood (1926) after a detailed morphological study of the

exoskeleton and musculature of scorpions, harvestman, and

twelve species of spiders, reported no autotomizing mechanism

existed in those animals. Severence of the legs did, however,

occur at a point in the limb directly correlated with a

definite structural weakness in the exoskeleton and muscula-

ture. Wood reported that the spider itself removed the injured

leg by grasping it with its mouthparts (autotilly) and conclud-

ed that autotomy as an automatic reflex did not exist in

arachnids. Parry (1957) reported findings contrary to Wood's

1926 report when he described the mechanism by which Tegenaria

atrica autotomizes its legs. Parry found that in T. atrica

the coxal muscles were all inserted onto a ring of sclerites

that fit into a groove in the proximal rim of the trochanter.

The joint fractures when the coxal muscles contract pulling

the articular membrane proximally also causing the sclerites

to converge leaving only a small hole that rapidly seals with.

clotting blood (Fig. 3).


L. various and the Morphology
of Arachnid Legs and Palpal Organ

The biology of the black widow spider has been well

documented (Baerg, 1923; Bhatnagar and Rempel, 1962; Burt,

1935; Chamberlin and Ivie, 1935; Deevey, 1949; Hagstrum, 1968;

Jellison and Philip, 1935; Kaston, 1937, 1954, 1963, 1968,

1970; Lawson, 1933; Levi, 1958; McCrone, 1968; Rempel, 1957;











LEG
INTACT -- --,



Cx. is C Scd Tr F:n



AUTOTOMY


Blood clot



Fig. 3. Schematic diagram of the autotomy mechanism in spiders.








LEG


rsus


PALP


Fig. 4. Comparative morphology of the spider leg and palp.









Ross and Smith, 1979). There are three species of North

American black widow spider: Latrodectus mactans (Fabr.)

and L. various Walckenaer in the eastern U. S. and only

L. hesperus Chamberlin and Ivie in the western U. S.

The adult female L. various has a black cephalothorax

and legs. There is a row or mid-dorsal red spots on the

abdomen and three pairs of diagonal white stripes on each

side with a narrow white stripe encircling the anterior dorsum

of the abdomen. The hourglass mark on the ventarl abdomen is

divided, the two halves separated (Kaston, 1970). In some

cases half or the entire hourglass mark may be completely

absent (Kaston, 1954). The male L. various is colored like

the female but with broader white stripes. Female black

widows may be as much as 160 times larger than the males

by weight (Kaston, 1970).

The range for the number of days spent in each instar

for L. various as reported by Kaston (1970) for instars one

through five are: 1 to 33, 5 to 30, 6 to 48, 6 to 76, and 7

to 76,124 (sic) days respectively.

The pedipalp of a spider is morphologically similar to

the spider leg with the exception that the tarsus of the leg

is subdivided into a long basal part called the basitarsus

(also metatarsus of 1-tar) and a shorter distal part called

the telotarsus (also tarsus or 2-tar) (Fig. 4). The tarsal

subunits of the leg are not true segments as exhibited by the

consistent absence of interconnecting muscles (Snodgrass, 1965)

The development of the male palpal organ of L. curacavien-

sis was described by Bhatnagar and Rempel (1962). Kaston









(1970) disputes the species Bhatnagar and Rempel studied was

L. curacaviensis but was instead L. hesperus.

The pedipalps of newly emerged male and female spiders

are indistinguishable (Fig. 5A). The development of the

papal organ, the male copulatory organ, is first recognizable

when the palpal tarsus becomes slightly swollen (Fig. 5B).

The swelling usually first appears in the antepenultimate

(pre-penultimate) instar but has been observed as early as

the preantepenultimate instar. When the male reaches the

penultimate instar, the palps become very large and bulbous

(Fig. 5C). Inside the bulbous palp the palpal organ is

developing.

Like insect imaginal discs the origin of the palpal organ

is the hypodermis (Bhatnagar and Rempel, 1962).

At the first swelling of the male palp the cells that

give rise to the pretarsus increase in size and number and

form a mass having dorsal and ventral lobes. During this

stage the muscle tendons associated with the tarsal claws

lose their connections and the new claw secreted by hypodermal

cells is immobile and passive. The large number of blood

cells in the tarsus may indicate the swelling was caused by

hydrostatic pressure.

With the next molt the penultimate palp, now extremely

bulbous, continues the internal morphogenesis of the copula-

tory organ. The receptaculum seminis, the sperm storage

tube consisting of the funds, reservoir and ejaculatory duct,

can be seen as an invagination of the ventral lobe. Later in






















I'


4'


\ \


B


/


















is



~C mb 1 rn


Fig. 5. The development of the male palpal organ and identi-
fication of some of the major parts of the adult organ.


M d.


Conductor








the instar the base of the developing organ is joined to the

tarsus by a small neck of cells destined to become the basal

haematodocha. The haematodocha is a folded membrane within

the alveolus of the mature palp that forms an articulation

between the cymbium and the sclerotized portion of the genital

bulb and becomes distended during copulation due to increased

hemolymph pressure. The formation of the small neck of cells

represents the pretarsus becoming withdrawn into the tarsus.

The tarsus will develop into the cymbium which holds the

receptaculum seminis and its accessory sclerties within the

alveolus. Toward the end of the penultimate instar the devel-

oping palpal organ can be seen through the tarsal wall. At

the final molt the adult palpal organ appears (Fig. 5D).

Based on the above information experimental objectives

for the present research were established for investigation

of regeneration and autotomy in the legs and palpal organ of

L. various. Experiments were designed to determine and

compare the regenerative capacities and autotomic responses

of pre-penultimate and penultimate male palps resulting from

injury by amputation and ligation. Another series of experi-

ments was performed to discover the regenerative capacity and

autotomic responses of the legs of immature L. various

resulting from injury by amputation, ligature and local seg-

mental injury. The results of the above experiments would

then allow for the comparison of the regenerative capacities

of the developing palpal organ and legs since these parts of

a spider are morphologically homologous.














METHODS AND MATERIALS


L. various

Latrodectus various was chosen as the subject of experi-

mentation because of its availability, and the fact that of

the three North American species its newly emerged spiderlings

are the largest and they hatch and emerge in the shortest

time (Kaston, 1970). Adult female L. various were acquired

from Tempe, Arizona, and maintained in the laboratory on a

diet of cabbage looper (Trichoplusia ni (Hubner)) larvae.

Egg sacs constructed by the adult females were removed to

separate containers until the emergence of young. The newly

emerged spiderlings, considered to be in the second instar,

were separated into numbered, 35 X 10 mm polystyrene culture

dishes. Spiderlings were maintained at room temperature and

fed adult Drosophila melanogaster. Exuvia were removed from

the rearing containers following each molt thus insuring

against mistakes in instar identification. Pre-penultimate

and penultimate males, third and fourth instar respectively,

identified by the noticable swelling of the palpal tarsus

were separated from the primary rearing colony for experimenta-

tion. Fourth and fifth instar female L. various were used

primarily for leg regeneration experiments.









Histology

Spiders used in histological studies were fixed in

alcoholic Bouin's fixative for 4 to 24 hours before being

transferred to the dehydration series. Dehydration was follow-

ed by infiltration and embedding in paraffin (Appendix 1).

Embedded material was sectioned on a rotary microtome at six

to ten microns, mounted on glass slides and stained using

Mallory's Triple stain technique (Appendix 2). Slides were

then examined and photographed through a compound microscope.


Amputation and Ligature

Amputation and ligature procedures were carried out with

the aid of a dissecting microscope. Due to the possible

effects of anesthesia on the postoperative physiology of the

immature spiders no anesthesia was used. Unanesthetized

spiders were placed in an apparatus I fabricated (Fig. 6)

specifically to restrain while not damaging the spiders for

the duration of the procedures. The apparatus was designed

to hold the spider in a foam rubber sandwich with the appen-

dages to be operated on exposed. The foam rubber allowed

the fragile spiders to be firmly held without injury. From

control studies it was determined that holding the spiders

in the apparatus for as long as three minutes (twice the

upper limit for actual procedures) in no way altered the

development of the young L. various. It took 40 90

seconds for the amputation and ligature procedures, including

the time for removal and return of the spider to its rearing

container.


- J










Pre-penultimate and penultimate male palps were ampu-

tated at various points from the mid-tarsus (most distal)

to the coxa-trochanter joint (most proximal). Likewise,

one of the first pair of legs was cut at various points from

the mid-telotarsus (most distal) to the proximal margin of

the coxa (most proximal). Amputations and other cuts were

made with microscissors with the exception of cuts made at

the proximal margin of the coxa in which case specially

fabricated microscalpels were used.

In all cases only one palp or leg was injured leaving

the corresponding appendage to develop normally to serve as

a standard with which to compare the results of the operations.

Comparisons were made only with the uninjured corresponding

appendage of the same animal.

Wounds made during amputation were not sealed with any

foreign substance; healing was left up to the spiders.

Ligatures were made with sterile, 7-0 Ethicon braided

silk suturing thread. Overhand knots were pre-tied with

forceps leaving a loop approximately one to two millimeters

in diameter. With a spider in the restraint the loop was

slipped over the leg, positioned and tightened (Fig. 7).

The free ends were trimmed close to the knot with micro-

scissors. Ligatures were placed at various points on the leg

from the mid-basitarsus (most distal) to the mid-femur (most

proximal) and at the mid-femur of pre-penultimate and penul-

timate palps and at the tibia-tarsus joint of penultimate

palps.


















Rubber


Petri Dish


Paraffin


Fig. 6. Restraint apparatus used in amputation and ligation
experiments.


Fig. 7. Ligature in place on the leg of L. various.








The possibility existed that the manipulation of the

spiders during amputation and ligature might also cause

injury at other parts of the leg, especially at the plane

of weakness. The legs were pulled with forceps in a pre-

liminary experiment to establish the plane of weakness in

the leg and palps (coxa-trochanter joint) and to simulate

autotomy at that point. An experiment was conducted to tax

the coxa-trochanter joint by pulling steadily on the leg

until the articulating membrane between the coxa and trochan-

ter split releasing some hemolymph. The leak in the membrane

at that point and at no other joint along the leg, indicated

that the autotomy plane had been taxed beyond normal limits

while leaving the leg intact.

Bohn (1965) had removed a V-shaped wedge of tissue from

the tibia of L. maderae resulting in a leg regenerating from

the site of injury. A similar experiment was performed on

L. various by removal of a section of tissue from the femur

of a leg.















RESULTS


ThePalpal Organ of L. various

The pre-penultimate and penultimate palps of L. various

males were sectioned to establish the morphogenesis of

normally developing palps and thereby set a standard with

which to compare the results of subsequent amputation and

regeneration of the palps.


Development of the Palpal Organ

The internal morphogenesis of the palpal organ of L.

various is very similar to that described for L. curacavien-

sis (or L. hesperus) by Bhatnagar and Rempel (1962).

The pre-penultimate palp exhibited a slight swelling,

most pronounced at the tibia-tarsus joint (Fig. 8). At this

stage the palp contains the pretarsal primordia now rapidly

proliferating into the dorsal and ventral lobes of the devel-

oping organ (Fig. 9). The cell mass changes configuration

very little after 48 to 72 hours of development (Fig. 10).

After the next molt the slightly swollen pre-penultimate

palp becomes extremely bulbous (Fig. 11). With the onset of

the penultimate instar morphogenesis increases so that the

developing palpal structures can be identified (Figs. 12 and

13). Near the end of the penultimate instar genital bulb

structures are well differentiated within the palp (Fig. 14)





















Fig. 8. Pre-penultimate palp of a male L. various.


Bl.c


Li


Fig. 9. Early proliferation of pretarsal primordia.


r




27














rFig. 10. Pretarsal cells 48-72 hours of development.

Fig. 10. Pretarsal cells 48-72 hours of development.


IF---


Fig. 11. Penultimate palp of a male L. various.





























Fig. 12. Differentiation of the developing palpal organ at
24-36 hours into the penultimate instar.























.. M .a



Fig. 13. Differentiation of the developing palpal organ at
48-72 hours into the penultimate instar.















Em












Fig. 14. Differentiation of
approximately four days














Tar-


CM







\ I
Cd Tib


the developing palpal organ
prior to the adult molt.








.Tib


Fig. 15. Embolus of the developing palpal organ visible
through the tarsal cuticle.









and can be seen through the cuticle of the tarsus (Fig. 15).

These results form the morphological basis by which

the histology of regenerate palps resulting from the amputa-

tion experiments to follow can be compared.


Amputation of pre-penultimate palps

Pre-penultimate palps were amputated at various points

to discover if palps injured during that stage would regener-

ate to normal penultimate palps and subsequently produce

normal adult palps.


Amputation at the mid-tarsus

In 18 pre-penultimate male L. various a palp was cut

at the mid-point of the tarsus, eliminating the developing

tissue of the developing palpal organ. Amputations were made

when the spiders were an average of 30 days (range = 12-55

days) into the instar. It took an average 15 (range = 9-19)

post-amputation days for the spiders to molt to the next

instar. All of the specimens molted to the next instar and

exhibited normal appearing penultimate palps. At the time of

this report six had molted to the adult stage with normal

adult palps (Fig. 16).


Amputation at the tibia-tarsus joint

The entire tarsus of one palp was removed from each of

18 spiders. The spiders were an average 28 days (range = 3-55

days) into the instar at amputation. The spiders required an

average of 22 (range = 14-39) post-amputation days to molt.














~,~_ it '--'







Fig. 16. Results of amputation of pre-penultimate palp at
mid-tarsus.






.L:. 'molt

























Fig. 17. Results of amputation of the pre-penultimate palp
at the tibia-tarsus joint.









One specimen displayed no regeneration after the post-amputa-

tion molt having only the coxa of the injured palp remaining.

It is interesting that this spider spent the longest post-

amputation period (39 days) before molting yet did not regen-

erate.

The 17 remaining spiders exhibited imperfect regeneration

in several modifications of the same general regenerate;

the tarsus had regenerated much smaller than the normal

corresponding penultimate palp. Examples of the different

regenerates are shown in Figure 17. Histological examination

of the regenerates indicated that the pretarsal primordia

had been re-established and morphogenesis of the palpal organ

was occurring but on a much smaller scale (Fig. 18). None

of the eight spiders molting to the adult stage displayed

any further regeneration of the injured palp.


Amputation at the patella-tibia joint

By amputating at the patella-tibia joint the entire

tibia and tarsus were removed from one palp of 25 pre-penulti-

mate male L. various an average of 23 days (range = 2-63 days)

into the instar. These spiders took an average of nine (range

5-16) post-amputation days to molt to the penultimate instar.

The regenerates from this experiment were similar in appear-

ance to those of the preceding experiment. In 18 cases the

regenerate appeared as a miniature penultimate palp one-third

to one-fourth the size of the corresponding normal palp (Fig.

19). In six cases the regenerate was a small bud distal to

















Si.c-


-. D.lb


a em l
iHaefT VJlb


Fig. 18. Histology of regenerate palp following amputation
at the tibia-tarsus joint.


Fig. 19. Results of amputation of the pre-penultimate palp
at the patella-tibia joint.














Haem


"-- BLc


Fig. 20. Histology of regenerate palp following amputation
at the patella-tibia joint.









the palpal patella. One spider exhibited no regeneration

having only the coxa of the injured palp remaining.

One specimen exhibiting the distal bud regenerate

molted to a second penultimate stage where the uninjured

palp remained unchanged and the regenerate palp had shrunk

in size from the previous penultimate instar.

None of the eight spiders molting to the adult stage

exhibited a normal palp following amputation of the tibia

and tarsus during the pre-penultimate stage.

Again, histological examination of the regenerated

palps indicated that morphogenesis of the palpal organ had

been re-established but the developing organ was much smaller

than the normal palp of the same age (Fig. 20).


Amputation at the femur-patella joint

In twelve spiders the palp was amputated at the femur-

patella joint at an average of 37 (range = 1-47) days into

the instar. The spiders molted to the next instar in an

average of 20 (18-21) days following amputation. Nine

specimens displayed only healing of the femur. The remaining

three spiders exhibited regeneration in the form of a small

bulb (Fig. 21) attached to the distal end of the femur.


Amputation at the mid-femur and Trochanter-femur joint

In 14 pre-penultimate male L. various the palpal femur

was severed at its mid-point. In ten other specimens of the

same stage one palp was amputated at the trochanter-femur









joint. One specimen from the group cut at the mid-femur

died three days after amputation without molting. Amputations

were made an average of 24 days (range = 5-55 days) into the

instar and the 23 survivors took an average 20 (range = 5-47)

post-amputation days to molt to the penultimate instar.

Only three of the spiders (two from the trochanter-

femur cut and one from the mid-femur cut) displayed any

regeneration. The regenerates consisted of only the coxa,

trochanter and femur. The regenerate femurs were one-third

the size of their normal counterparts (Fig. 22). The remain-

ing 20 specimens, upon molting to the penultimate stage, had

only the coxa and trochanter of the injured palp present and

a normal palp in the corresponding position.


Amputation at the coxa-trochanter joint

Of the 16 pre-penultimate male L. various used in this

experiment ten had one palp cut at the coxa-trochanter joint

and six had both palps removed by pulling with foreceps.

The palps removed by pulling all severed at the coxa-trochanter

joint, establishing the plane of weakness for the palp.

All sixteen specimens molted to the next instar with no

regeneration of injured palps. Only the coxa of each injured

palp, whether cut or pulled remained (Fig. 23). Five spiders

molted to the adult stage with only the palpal coxae present.

The amputation experiments performed on pre-penultimate

male L. various palps are summarized in Table 1.
















i ---7


/ I:
~ffiI~J


Fig. 21. Results of amputation of the pre-penultimate
palp at the femur-patella joint.


ii=


N /i

'.1 ,
Ai


/,


Fig. 22. Results of amputation of the pre-penultimate
palp at the mid-femur and trochanter-femur joint.





7 ~^-: .










Fig. 23. Results of amputation of the pre-penultimate
palp at the coxa-trochanter joint.














0 0 C 0 0








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Amputation of penultimate palps

As in the previous series of experiments, it was desir-

able to discover the regenerative capacity of the penultimate

palps. According to Bonnet's work on Dolomedes (1930) it

would not be possible for either pre-penultimate or penulti-

mate palps to produce normal adult structures after injury.


Amputation at the mid-point of the tarsus

In twelve penultimate male L. various the distal half

of the tarsus was removed from one palp an average of 17

days (range = 9-25 days) into the instar. Eight spiders died

shortly after amputation without molting. The remaining four

spiders molted to the adult instar in an average of twelve

days (range = 4-17 days) with no regeneration occurring.

Coxae of the injured palps were the only structures present

(Fig. 24).


Amputation at the tibia-tarsus joint

A palp of each of ten penultimate males was amputated

at the tibia-tarsus joint an average of five days (range = 2-

16 days) into the instar. All of the specimens died within

two days of amputation without molting (Fig. 25).


Damage to the palpal tarsus

The tarsus of one palp of five penultimate males was

damaged by puncture an average of twelve days (range = 1-25

days) into the instar. The only spider to survive molted

to the adult instar 19 days after injury without any regener-

ation (Fig. 26).











/i

I'
,* \


1DEAT -no olt
6 o


33"/o


Fig. 24. Results of amputation of the penultimate palp at
the mid-tarsus.


I'


DE AT'N -no molt


Fig. 25. Results of amputation of the penultimate palp at
the tibia-tarsus joint.






/\. DEATH-no molt
/ /








200/0



the penultimate palp.








Amputation at the patella-tibia joint

The tibia and tarsus from each of ten penultimate males

was amputated an average of two days (range = 1-2 days) into

instar. Two spiders died without molting. The remaining

eight specimens molted to the adult instar an average of 41

(range = 30-61) post-amputation days with no regeneration

displayed by any spider. The injured palps appeared in the

adult stage with a coxa, trochanter, femur and patella.

The distal margin of the patella was healed over (Fig. 27).


Amputation at the coxa-trochanter joint

In twelve penultimate males a palp was amputated at the

coxa-trochanter joint; eleven of which survived to the adult

stage in an average of 15 days (range = 4-27) after amputation.

No regeneration of the injured palps was observed (Fig. 28).

The amputation experiments performed on the palps of

penultimate male L. various are summarized in Table 2.


Ligature of Pre-penultimate and Penultimate Palps

Ligation of pre-penultimate and penultimate palps was

performed to discover if this type of injury would result in

the autotomy of the palps.


Ligature at mid-point of pre-penultimate palp

In ten pre-penultimate males the mid-point of the palpal

femur was ligated an average of nine days (range = 1-32) into

the instar. Two spiders died one day after ligation. Four of

the remaining spiders apparently autotomized their injured

palp one day after ligation.























Fig. 27. Results of amputation of the penultimate palp
at the patella-tibia joint.


molt


Fig. 28. Results of amputation of the penultimate palp
at the coxa-trochanter joint.











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Ligature at the tibia-tarsus joint of penultimate palp

In five penultimate males, an average of 14 days

(range = 1-20) into the instar, the tibia-tarsus joint of a

palp was ligated. One spider died a day after ligation. One

of the surviving four specimens exhibited the apparent autot-

omy of the palp two days after the application of the liga-

ture.


Ligature at the mid-femur of the penultimate palp

In eight penultimate males the mid-femur of the palp was

ligated an average of 48 days (range = 12-129) into the

instar. Six specimens died within two days of ligation. No

autotomy was observed in the remaining two spiders.

The ligation experiments performed on the palps of pre-

penultimate and penultimate male L. various are summarized

in Table 3.


Regeneration and Autotomy in the Legs of L. various

Regeneration and autotomy in the legs of L. various were

studied using two types of injury, amputation and ligation.

Amputation left an open wound requiring healing whereas

ligation did not. The first leg on the left side of fourth

and fifth instar female L. various was amputated at differ-

ent points in an effort to determine the most proximal point

from which amputation resulted in regeneration of the limb.


Amputation at the mid-point of the telotarsus

In six spiders the distal half of the telotarsus was

removed two days into the instar. With the first post-ampu-


















CO






0
4,-



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d
OE
.


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03












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station molt the injured telotarsi had regenerated to approx-

imately 50% the size of the normal corresponding structure

(Fig. 29). The tarsal claws were present and normal in

appearance. With the second post-injury molt all injured

legs were normal in size and appearance.


Amputation at the mid-point of the basitarsus

In ten immature female L. various a leg was amputated

at the mid-point of the basitarsus an average of three days

(range = 1-11) into the instar. Following the first post-

amputation molt all of the spiders exhibited regeneration

where the basitarsus was 33-100%, and the telotarsus 25-33%

normal by comparison to corresponding structures. By the

second molt four had regained normal appearing legs. After

the third post-injury molt all specimens displayed normal

legs (Fig. 30).


Amputation at the mid-point of the tibia and at the patella-
tibia joint

In 14 specimens a leg was amputated at the mid-point of

the tibia and ten other spiders had a leg amputated at the

patella-tibia joint. Amputations were made when the spiders

were an average of nine days (range = 1-45 days) into the in-

star. All exhibited regeneration at the first post-amputation

molt an average of 17 (range = 7-45) days later with the tibia

33-50%, basitarsus 20-50% and telotarsus 20-50% normal by

comparison with normal corresponding structures (Fig. 31).

Two spiders had regenerated a normal leg by their second post-

injury molt and twelve had normal legs by the third molt

following amputation.


















~.1


//


Fig. 29. Results of amputation of the leg at the mid-telo-
tarsus.


- /7


Fig. 30. Results of amputation of the leg at the mid-
basitarsus.


*1~


Fig. 31. Results of amputation of the leg at the mid-
tibia and at the patella-tibia joint.









Amputation at the femur-patella joint

In each of six spiders a leg was cut at the femur-patella

joint one day into the instar. One specimen died two days

after amputation without molting. Four of the remaining

spiders molted in an average 25 (range = 18-39) days later and

regenerated the patella 50-100%, tibia 50-75%, basitarsus

33-50% and telotarsus 20-33% normal by comparison (Fig. 32).

No spider had regenerated a normal leg by the second and only

two had regenerated normal legs by the third post-amputation

molt. One spider had only the coxa of the injured leg

evident after two post-injury molts.


Amputation at the mid-point of the femur

In 20 spiders a leg was amputated at the mid-point of

the femur an average of 18 (range = 1-35) days into the instar.

One spider died without molting. The first post-amputation

molt came an average of 38 (range = 16-58) days later with

70% of the survivors regenerating the femur 33-75%, patella

33-50%, tibia 20-50%, basitarsus 20-33% and telotarsus 20-25%

by comparison to normal structures (Fig. 33). None of the

above had regenerated a normal leg by the second and only one

spider had regenerated a normal leg after the third post-

amputation molt. The remaining 30% of the survivors display-

ed wound healing at the site of amputation with no subsequent

regeneration.





















Fig. 32. Results of amputation of the leg at the femur-
patella joint.


I 70% 30%


Fig. 33.


Results of amputation of the leg at the mid-femur.


Fig. 34. Results of amputation of the leg at the trochanter-
femur joint.









Amputation at the trochanter-femur joint

In ten spiders a leg was amputated at the trochanter-

femur joint an average of 27 (range = 5-48) days into the

instar. No specimen exhibited regeneration at any post-

amputation molts. In every case the trochanter had healed

over (Fig. 34).


Amputation at the coxa-trochanter joint

A leg from each of 15 spiders was removed at the coxa-

trochanter joint by pulling on the leg with forceps. Ampu-

tation occurred an average of 12 (range = 1-39) days into the

instar. No regeneration occurred at any subsequent molts

leaving only the coxae of injured legs (Fig. 35).


Amputation at the proximal margin of the coxa

In ten spiders one of the second pair of legs was removed

by cutting around the proximal margin of the coxa. Only two

spiders survived the injury and exhibited no regeneration of

any leg structures. The wound healed over completely in the

two survivors (Fig. 36).

The amputation experiments performed on the legs of L.

various are summarized in Table 4.


Localized Injury to the Femur

This experiment was performed to discover if L. various

had the ability to regenerate a leg from a local injury to

the femur. Bohn (1965) inflicted local injury to the tibia

of a cockroach (L. maderae) by removing a V-shaped section of
















MO


Fig. 35. Results of amputation of the leg at the coxa-
trochanter joint.


Fig. 36. Results of amputation of the leg at the proximal
margin of the coxa.



































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tissue. This resulted in a lateral regenerate, in the form

of a leg, at the site of injury.

From each of 15 spiders a section of tissue was cut

from the femur. One spider died without molting. Six

specimens autotomized the injured limb at the coxa-trochanter

joint in an average of four (range = 1-7) days after injury

(Fig. 37). Autotomized limbs did not regenerate with only

the coxa remaining following subsequent molts. These

results are comparable to those obtained by mechanical removal

of the leg at the autotomy plane.

The other eight spiders exhibited healing of the wound

following injury and a concave scar following the post-injury

molt (Fig. 37). The scar area was characterized by a lack

of setae. No lateral regenerates resulted from this type of

injury to L. various.


Ligature of the Legs

Ligature was used to inflict injury to the leg without

resulting in an open wound. Such an injury, however, is

sustained for longer periods of time since the ligature is

in place until discarded by some mechanical means or at the

post-ligature ecdysis.


Ligature at the mid-point of the basitarsus

Ten spiders were ligatured at the mid-point of the

basitarsus an average of 49 (range = 36-67) days into the

instar. One day after ligature four spiders had lost,




































AUTOTOMY 400io


Fig. 37. Results of localized injury to the femur of the
leg.


Y




U U


apparently by a mechanical means, the portion of the injured

leg distal to the ligature. The effect therefore was of

amputation at that point. No autotomy was observed. All of

the spiders regenerated at the next molt, an average of 20

(range = 17-40) days later, with the basitarsus 75-100% and

telotarsus 50% normal (Fig. 38). Fifty percent of the

spiders had regenerated a normal leg by the second molt.


Ligature at the mid-point of the tibia

A leg from each of 20 spiders was ligatured at the mid-

point of the tibia an average of 19 (range = 16-22) days

into the instar. Fourteen specimens autotomized the injured

limb at the coxa-trochanter joint in an average seven (range

1-14) days after ligature. No post-autotomy regeneration

was exhibited, leaving only the coxa of the ligatured/autot-

omized limb in evidence.

The six spiders that did not autotomize the ligatured

limb did, however, remove the leg tissue distal to the

ligature as described above in the preceding experiment in

2 to 23 days. These spiders did regenerate the tibia 50-67%,

basitarsus and telotarsus 25-33% at the first post-ligature

ecdysis (Fig. 39).


Ligature at the patella

Six spiders were ligatured at the patella of one leg.

Five spiders autotomized the injured leg in an average of

seven (range = 1-12) days after injury. The remaining specimen

molted 20 days after ligation with the patella 50%, tibia 50%,

basitarsus 33% and telotarsus 20% normal by comparison (Fig. 40).












MI
/7 "molIt








Fig. 38. Results of ligation of the leg at the mid-
basitarsus.








/ \

AUTOTOMY



7 030/o


70%


Fig. 39. Results of ligation of the leg at the mid-tibia.









Ligature at the mid-point of the femur

One leg of each of ten spiders was ligatured at the mid-

point 6f the femur an average of 38 (range = 1-59) days into

the instar. All ten specimens autotomized the ligatured

limb at the coxa-trochanter joint within one day of injury.

No regeneration of the limbs was observed. Only the coxa

of ligatured/autotomized legs remained after subsequent

molts (Fig. 41).


External Force Applied at Autotomy Plane

This experiment was performed to determine if an injury

applied to the plane of weakness powerful enough to produce

bleeding but mild enough not to cause severence of the limb,

would result in autotomy. It was necessary to perform this

experiment since the possibility existed that this type of

injury may occur as a result of the manipulation of spiders

for any of the previously described experiments.

In the 20 spiders tested in this manner at an average

of 18 (range = 5-55) days into the instar no autotomy result-

ed and all specimens molted to the next instar with no

morphological anomalies observed.

The ligature and external force experiments performed

on the legs of L. various are summarized in Table 5.


Summary of Results

Latrodectus various has the capacity to regenerate a

normal adult male palp only if the injury occurs distal to

the mid-point of the tarsus during or before the pre-penulti-













t// _


AUTOTOMI 830/o
!


Fig. 40. Results of ligation of the leg at the patella.


AUTOTOMY


mo it

V


'Fig. 41. Results of ligation of the leg at the mid-femur.


17o/o

















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mate instar, thus allowing at least two more molts before

maturation. Injury to the palp sustained proximal to the

mid-point of the tarsus during the pre-penultimate instar

did not result in normal regenerates. Ligation at the mid-

femur of the pre-penultimate palp resulted in apparent

autotomy 40% of the time and in death 20% of the time.

Injury to the penultimate palps by ligation or ampu-

tation did not result in regeneration. Amputation of the

tibia and/or tarsus of the penultimate palp frequently

resulted in the death of the spider. Ligation at the tibia-

tarsus joint of the palp resulted in apparent autotomy in

20% of the cases and in death in 20% of the cases.

Amputation of the legs of immature L. various resulted in

either regeneration or healing of the wound with no subse-

quent regeneration. Regeneration resulted when amputation

occurred at or distal to the mid-point of the femur. Amputa-

tion at points proximal to the femoral mid-point to the

proximal margin of the coxa resulted in the healing of the

wound. Penultimate males (15) had legs amputated at various

points and all exhibited regeneration or healing as described

for immature females. Amputation of a leg of a penultimate

male in no way interferred with palpal development.

Autotomy of the leg was first observed resulting from

localized injury to the femur. Removal of a section of

tissue from the femur resulted in either healing of the wound

at the first post-injury molt or autotomy of the entire limb

at the coxa-trochanter joint with no subsequent regeneration.




62



Ligation at various points of the leg of L. various

resulted in either regeneration or autotomy. The more

proximal the ligation the greater the frequency and earlier

was the onset of autotomy. Ligature of the basitarsus never

resulted in autotomy. Autotomy of the legs always occurred

at the coxa-trochanter joint.















DISCUSSION


Regeneration

This investigation has established the regenerative

capacities for the legs and developing palpal organ of L.

various. The occurrence of autotomy in L. various was

documented and compared to regeneration and healing as

alternative responses to injury by amputation and ligature.

This research has demonstrated that like other arthropods,

L. various has the capacity to regenerate limbs injured by

amputation or ligation. However, the black widow spider,

L. various does not have the ability to regenerate a leg or

palp following autotomy. This completely contradicts state-

ments made by Goss (1969) and Needham (1965). Goss stated

that the capacity for regeneration in arthropods was greatest

at the autotomic breakage plane. Needham remarked that the

specific rate of regeneration is greatest when amputation

occurs at the autotomy plane. The evidence strongly suggests

the capacity for regeneration is non-existent at the autotomic

breakage plane of L. various. In fact, the capacity for

regeneration in the leg does not become apparent until some

distance distal to the autotomy plane, at about the mid-

point of the femur.

The palps of pre-penultimate male L. various can regen-

erate to normal penultimate and subsequent adult palps if









the injury involves the loss of less than the distal half of

the tarsus. When loss of more than the distal half of the

pre-penultimate tarsus is sustained, there is tissue regen-

eration but insufficient to produce normal penultimate and

adult palpal structures.

These results help to confirm the suspicions of

Chrysanthus (1955) and Kaston (1963, 1968) that some malforma-

tions of penultimate and adult male spider palps could be

attributed to imperfect regeneration following injury.

Kaston (1968) reported a deformity in a penultimate palp of

L. hesperus where one palpal tarsus was only half the size of

the corresponding normal palp. This was observed in experi-

mentation with L. various when the tibia and tarsus of the

pre-penultimate male palps were amputated.

In the current research amputations of penultimate palps

never resulted in regeneration. Death occurred soon after

the amputation of the distal half of the tarsus in 60% of the

cases and in 100% of cases where the entire tarsus had been

removed. Death did not occur when amputations were made at

or proximal to the patella-tibia joint. In those cases the

wound healed and the subsequent molt to the adult instar result-

ed in no regeneration.

Amputation and ligation of penultimate palps resulted

only in the healing of the remaining portions of the palp or

the death of the spider. Once the palp has reached the level

of development seen in the penultimate palp damage is either

fatal or repaired by healing since the evidence suggests that








the tissue of the penultimate palp has lost the capacity to

regenerate. The pre-penultimate palp retains the ability to

regenerate the palpal organ as the histological examination

of regenerate penultimate palps indicated. The regenerate

cells in such a palp still form a developing palpal organ,

although smaller than its normal counterpart, but has apparent-

ly lost the ability to react to the developmental hormones

at the final molt. Palps injured during the pre-penultimate

stage that regenerated a small penultimate palp never develop-

ed beyond that point even when the spiders involved molted

to the adult stage.

A comparison of the regenerative capacities of pre-

penultimate and penultimate palps is presented in Figure 42.

Ligation of the femur of pre-penultimate palps resulted

in death in 20% and apparent autotomy in 40% of the cases.

Ligation at the tibia-tarsus joint and mid-femur of penulti-

mate palps resulted in death 20% and 75% and apparent autotomy

in 20% and 0% of the cases respectively. Due to the size and

delicacy of the palps and the extreme difficulty in performing

the ligation procedures I consider the data on ligation of the

palps to be less than totally reliable. Further sophistica-

tion of the techniques for palpal ligation may yield more

satisfactory results.

There seems a strong possibility that deaths resulting

from amputation through the large cross-sectional areas of

the penultimate palp may be related to the size of the wound.

Cuts made through the bulbous portion leave a much greater





















PRE-PENULTIMATE


j-Healing --- Regeneration-o-I


Death --


PENULTIMATE


Fig. 42. Comparison of the regenerative capacities of the
pre-penultimate and penultimate palps of the male
L. various.









wound than do cross-sectional cuts through the more proximal

segments of the palp. Harvey and Williams (1961) reported

that the "injury factor" in diapausing cecropia seemed to be

released until the wound was sealed by blood cells. Amputa-

tion through the largest cross-sectional area of the pre-

penultimate palp, at the tibia-tarsus joint, is sealed by

the healing process (Fig. 43) and the spiders survive to molt

again. However, injury at the same point in the penultimate

palp results in the death of the animal. The only perceivable

difference is the size of the wound. Since the palps did

not exhibit autotomy in response to amputation it may be

possible that a larger amount of wound factor released from

the larger wound, a wound that was not sealed by blood cells,

may have resulted in the death of the spiders.

Amputation indicated that the regenerative capacity of

the legs is greatest in the more distal segments. However,

amputations as high on the leg as the patella-tibia joint

resulted in some regeneration 100% of the time. No leg

injured by amputation or ligature regenerated completely

at the first post-injury molt. It is from the mid-point of

the femur (30% of the time) to the proximal margin of the

coxa that healing of the wound occurs with no subsequent

regeneration.

The regeneration observed in the legs and palps of L.

various complies with the developmental gradient model for

regeneration set forth by Bryant (Fig. 1). Proximal struc-

tures left after amputation or ligature regenerate those

portions lower (more distal) on the developmental gradient.








Autotomy

Autotomy was first observed in the experiment where

local injury of the leg resulted from removal of a section

of tissue (Fig. 37) from the femur. Forty percent of the

spiders thus injured exhibited autotomy of the entire limb

at the coxa-trochanter joint not followed by any regeneration

of the lost limb. A possible agent involved in the physio-

logical "choice" between healing and autotomy may be the size

of the wound and the corresponding release of wound factor.

Autotomized legs in this experiment showed no signs of heal-

ing (Fig. 44) at the time the leg was released from the body,

possibly indicating a wound too large to be sealed. Wound

factor would then be released until the threshold for autot-

omy was reached, after which the leg would be severed from

the body by the mechanism described by Parry (1957).

Autotomy of the legs was a frequent result of ligation.

A comparison of the autotomy, healing and regeneration of

legs injured by amputation or ligature is presented in Figure

45. Ligature resulted in the autotomy of the entire leg 70%

of the time when applied to the mid-point of the tibia.

Amputation at the same point never resulted in autotomy.

Autotomic reactions increased in frequency as more proximal

segments of the leg were ligatured.

Ligation distal to the mid-point of the tibia (at the

mid-point of the basitarsus) did not result in autotomy. The

lack of autotomy following injury to the dactyl of crustacea

has been attributed to the fact that the leg nerve of those

animals does not extend into that segment (Hodge, 1956;











Healed
C wound


Fig. 43. Healing of the wound produced by amputation of
the pre-penultimate palp at the tibia-tarsus joint.


V


Open wound






S*' -;7


Fig. 44. The open wound of an autotomized leg after
localized injury to the femur of the leg.


-Kf- .1











Ligation



Amputation


-l Autotomy ------Regeneration--



Healing -- Regeneration -----]


Fig. 45. A comparison of the autotomy, healing and
regeneration of the legs injured by amputation
and ligation.


S 9 1


Fig. 46. Histology of the telotarsus of the leg of
L. various showing the leg nerve present.









Needham, 1947; and Wood, et al., 1932). No autotomy occurs

when injury, either by amputation or ligation, is applied to

the distal segments of the leg of L. various even though

the leg nerve is present to the tip of the telotarsus (Fig. 46).

This fact argues against nervous reflex as a cause.oof autotomy,

at least in the distal segments of the leg, in L. various.

It seems logical at this point to assume that ligation

resulted in autotomy whereas amputation did not because of

the greater duration of injury sustained with ligation. A

cross-sectional cut of a leg leaves a wound capable of healing

before the hypothetical wound factor threshold for autotomy

is reached, thus no autotomy due to amputation was observed.

Ligation is applied for longer periods of time, either until

its mechanical removal or the first post-ligature molt. The

greater duration of injury may result in wound factor pro-

duction long enough to reach the autotomy threshold. This

theory presupposes that wound factor is released in the

absence of an open wound.

The argument that a wound factor contributes to the

initiation of autotomy becomes stronger when the experiment

taxing the autotomy plane is considered. Following the: split

of the articulating membrane at the coxa-trochanter joint

caused by pulling, the release of hemolymph is proof the

autotomy plane has been directly damaged. The pressure was

not great enough to cause the direct loss of the leg.

According to Harvey and Williams (1961) injury factor would

have been released from such an injury but because the wound









was quickly sealed when the split edges of the membrane

came back into contact with each other the dose of wound

factor would have been relatively small. Although the

plane of weakness was injured directly and wound factor

supposedly released, autotomy did not occur, presumably

because the dose of wound factor did not reach the threshold

and no reflex in response to the pressure applied caused

the loss of the leg.

Autotomy has been demonstrated following injury to

points on the leg distal to the plane of weakness where

the injury was sustained for a longer period of time, either

by duration of application (ligature) or by the failure of

the wound to heal in some reasonable amount of time (local

femoral injury). It seems possible that the duration and/or

size of the injury, both related to the dose of wound factor

released, contributes to the initiation of autotomy.

Autotomy is a costly alternative to regeneration in

L. various since once a limb is autotomized regeneration of

that limb is impossible. The adaptive advantage to a plane

of weakness in a spider appendage is great. It is more

advantageous for an animal to sacrifice a limb in order to

escape than to be killed or fatally wounded in an encounter

with a predator. Loss of a single leg to a web-dwelling

spider may be of little consequence although no investigation

of that phenomenon has been performed. In the black widow

spider, L. various, the ability to escape or discard a badly

injured limb outweighs the advantage of regenerating those

limbs.









The difference in the regenerative capacities of the

legs and palpal organ may be related to the degrees of com-

plexity of the two morphologically homologous appendages.

The loss of one leg may be of little consequence since

there are seven remaining. The palps of the male are at a

relative premium since there are but two. Although only

one palp is required for successful copulation, two palps

would enhance the chances that a male spider's genes would

be transmitted tothe next generation.

A physiological response gradient has been established

for the legs and developing palpal organ of L. various dis-

tinguishing between regeneration, healing, autotomy and death

as responses to injury by amputation and ligation (Figs. 42

and 45).

In answering the questions posed earlier concerning the

regenerative capacities of the legs and palps of L. various

a major question has been reopened. What is the "wound

factor" and how is it related to regeneration, healing,

autotomy and death, the alternative responses to injury? Is

wound factor a universal agent found in all organisms in the

same form or is it unique to each species?

The answers to those and other questions await discovery

and discoverers.














APPENDIX 1


Fixation, Dehydration and Embedding Protocol

From fixation in alcoholic Bouin's fixative:

70% EtOH 5-15 min.

70% EtOH 5-15 min.

30% EtOH 5-10 min.

50% EtOH 5-10 min.

70% EtOH 5-10 min.

80% EtOH 5-10 min.

90% EtOH 5-10 min.

95% EtOH 5-10 min.

100% EtOH 5-10 min.

1:1, 100% EtOH:Acetone 5-15 min.

Acetone 5-10 min.

1:1, Acetone:Terpineol 10-15 min.

Terpineol 4 hours to overnight
(can be stored in terpineol)

Benzene 5 min.

Benzene 5 min.

1:1, Benzene:Paraplast
@ 55-600 15-30 min.

Paraplast @ 55-600 15-45 min.

Embed in Paraplast





75



Paraffin blocks were trimmed and sectioned in rotary
microtome.

Sections were mounted on standard glass microscope
slides.















APPENDIX 2

Mallory's Triple Stain Technique

Xylene 2-5 min.

Xylene 2-5 min.

100% EtOH 2-5 min.

95% EtOH 2-5 min.

80% EtOH 2-5 min.

70% EtOH 2-5 min.

50% EtOH 2-5 min.

30% EtOH 2-5 min.

Water 2-5 min.

Stain in 1% acid fuchsin for 2-5 min. (time not
critical)

Rinse in water for 1 min.

Transfer to 1% phosphotungstic acid for 2 min.
(time not critical)

Dip twice in water

Transfer to second staining solution (100 ml water,
0.5g methylene blue, 2g orange G and 2g oxalic
acid) for 5 min. (no more than 8 min.)

Wash in water two 1 min. washes

Transfer to 100% alcohol for 1 min. time is critical

Place in xylene can stay in xylene until ready to mount

Pro-Tex or Permount mounting medium are both good
mountants.















LITERATURE CITED


Agar, W. E. 1930. A statistical study of regeneration in
two species of Crustacea. J. Expt. Biol. 7:349-369.

Baerg, W. J. 1923. The black widow: Its life history and
effects of its poison. Sci. Monthly 17:535-547.

Bhatnagar, R. D. S. and J. G. Rempel. 1962. The structure,
function and post-embryonic development of the male and
female copulatory organs of the black widow spider
Latrodectus curacaviensis (Muller). Can. J. Zool. 40:465-
510.

Bliss, D. E. 1960. Autotomy and regeneration. Pages 561-589
in T. H. Waterman, ed. The physiology of crustacea.
Academic Press, N. Y.

Bodenstein, D. 1933. Beintransplantationen an lepidopter-
enraupen II. Zur analyse der regeneration der brust-
beine von Vanessa urticae Raupen. Wilhelm Roux
Arch. EntwMech. Org. 165:303-341.

Bodenstein, D. 1955. Contributions to the problem of regen-
eration in insects. J. Expt. Zool. 129:209-224.

Bohn, H. 1965. Analyse der regenerationsfahigkeit der
insektenextremitat durch amputations und transplanta-
tionsversuche an laven der Afrikanischen schabe Leuco-
phaea maderae Fabr. (Blattaria). II. Achsendetermin-
ation. Wilhelm Roux Arch. EntwMech. Org. 156:449-503.

Bohn, H. 1972. The origin of the epidermis in the super-
numerary regenerates of triple legs in cockroaches
(Blattaria). J. Embryol. exp. Morph. 32(1):81-98.

Bohn, H. 1974a. Extent and properties of the regeneration
field in the larval legs of cockroaches (Leucophaea
maderae). I. Extirpation experiments. J. Embryol. exp.
Morph. 31(3):557-572.

Bohn, H. 1974b. Extent and properties of the regeneration
field in the larval legs of cockroaches (Leucophaea
maderae). II. Confirmation by transplantation experi-
ments. J. Embryol. exp. Morph. 32(1):69-79.









Bohn, H. 1974c. Extent and properties of the regeneration
field in the larval legs of cockroaches (Leucophaea
maderae). III. Origin of the tissues and determination
of symmetry properties in the regenerates. J. Embryol.
exp. Morph. 32(1):81-98.

Bonnet, P. 1930. La mue, l'autotomie, et le regeneration
chez les Araignees. Bull. soc. hist. nat. Toulouse
59(2):613-939.

Brousse-Gaury, P. 1958. Contribution a l'etude de l'autot-
omie chez Acheta domestic L. Bull. biol. France et
Belg. 92:55-85.

Bryant, P. J. 1971. Regeneration and duplication following
operations in situ on imaginal discs of Drosphila
melanogaster. Devel. Biol. 26:606-615.

Bryant, P. J. 1975. Pattern formation in the imaginal wing
disc of Drosophila melanogaster: Fate map, regeneration
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BIOGRAPHICAL SKETCH


John Brookes Randall was born in Ft. Wayne, Indiana,

on April 7, 1949. He moved to Connecticut a year and a

half later, then to Maryland at age four. He attended high

school in Severna Park, Maryland graduating in 1967. In

September of the same year he entered Maryville College,

Maryville, Tennessee, and received the Bachelor of Arts

degree in Biology from that institution in 1971.

For nearly two years after graduating from college he

worked as a physician's assistant in clinical research at

Johns Hopkins School of Medicine.

In September of 1973 he began graduate studies in

Entomology at the University of Florida under the direction

of Dr. Willard H. Whitcomb, during which time he served as

a graduate research and teaching assistant. He was awarded

a Visiting Graduate Student Fellowship to the Smithsonian

Institute in 1974 to study scientific illustration in that

museum's Department of Entomology. He received the Master

of Science degree from the University of Florida in June,

1976.

He continued graduate work for the doctoral degree under

the direction of Dr. Harvey L. Cromroy. He has recently

accepted a post-doctoral position at the State University









of New York at Buffalo where he will be investigating the

regeneration of insect nerve cells.

He holds membership in the Society of Sigma Xi, The

Entomological Society of America, The American Arachnological

Society, The Cambridge Entomological Club, The Florida Ento-

mological Society, The Guild of Natural Science Illustrators

and the International Society of Artists.

He has been married to his wife Carol for eight years

and they have a three-year-old daughter, Brooke Kathryn.




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