Table Of ContentANIMAL TOXINS
A collection of papers presented at the First
International Symposium on Animal Toxins
Atlantic City, New Jersey, U.S.A., April 9-11, 1966
Edited by
FINDLAY E. RUSSELL
Director, Laboratory of Neurological Research
Loma Linda University, Los Angeles County
General Hospital, Los Angeles, California, U.S.A,
and
PAUL R. SAUNDERS
Professor and Chairman, Department of Biological
Sciences^ University of Southern California,
Los Angeles, California, U.S.A.
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FOREWORD
SIDNEY R. GALLER
Smithsonian Institution, Washington, D.C.
LADIES AND GENTLEMEN :
I take great pleasure in being with you this morning. In many ways this meeting
represents a capstone to my activities during a long and enjoyable tour of duty with the
United States Navy, Office of Naval Research. One of the foremost problems of a scientific
nature which confronted me when I first arrived in ONR in 1948 was the problem of
icthyosarcotoxism. Some of you may recall that what was popularly but accurately described
as fish poisoning was an important problem confronting the allied forces during World
War II, especially the United States military groups deployed in the Pacific. When viewed
from the sophisticated scientific "heights" of today our initial efforts to cope with the
problem of icthyosarcotoxism were rather naive. Nonetheless, I feel now as I felt then that
the underlying concept which we developed in attempting to solve the problem was a sound
one; namely, to identify scientists with a demonstrated competence and interest in the
general area of marine toxinology, and invite them to join with us in assessing the status of
the problem as well as in establishing a common community of interest.
I must say that our interests ranged far and wide, and I hasten to add that the results
of our first efforts were gratifying indeed, if quite unpredictable at the time. We brought
together scientists, including your conveners: Dr. Russell, Dr. Saunders, Dr. Halstead, as
well as Dr. Banner, Dr. Lane, and a host of others. During the almost two decades since
1948, the modest catalytic sponsorship from the ONR has paid off handsomely—beyond
our fondest expectations. I hasten to add that we still haven't solved the problem; however,
we are in a much better position today to avoid it.
Perhaps far more important are the unanticipated "by products" of our modest program.
This meeting today is singular testimony to the success of the scientific leaders in establishing
an international community of interest in toxinology. This, the largest gathering of
specialists in diverse fields of scientific endeavor, is assurance to me that we can expect
contributions not only to our knowledge of toxinology but in many peripheral fields as well.
The contributions to biochemistry, physiology, and pharmacology are already substantial
and of basic importance. To me the most important product of research is not merely data,
but an interdisciplinary interpretation of biological events. This can come only through
continued close collaboration and constructive criticism among specialists who recognize
no incompatability between their own intellectual interests and the solution of fundamentally
important, as well as practical, problems requiring teamwork.
In closing, let me assure you that I and many of my colleagues at the Smithsonian
Institution are keenly interested in encouraging your research activities. We stand ready to
assist you in every way appropriate. I wish you the fullest success in this International
Symposium on Animal Toxins.
v
PREFACE
In 1954 the First International Conference on Venoms was held in Berkeley, California,
under the auspices of the American Association for the Advancement of Science. Subse-
quently, a number of national and international meetings on venoms and plant poisons
have been held in various cities throughout the world. Perhaps the most important of these
was the Tenth Pacific Science Congress, 1961, sponsored by the United States Academy of
Sciences and the Bernice Pauaki Museum of the University of Hawaii. The success of such
meetings stimulated scholars to organize the First International Symposium on Animal Toxins
for the International Society on Toxinology. From this Symposium, held in Atlantic City,
New Jersey, 1966, came the papers collected in this volume. Taken together, these papers
constitute a compendium of current knowledge on animal toxins.
Los Angeles, California FINDLAY E. RUSSELL
April 12, 1966 PAUL R. SAUNDERS
vii
ACKNOWLEDGEMENTS
No undertaking of the present nature could be accomplished without the assistance
of our friends and colleagues. Although it would be impossible to name the many people
who have assisted in preparing this text, or to note those who helped with the Symposium in
Atlantic City, we wish to thank all of them for their assistance.
In particular, we wish to acknowledge the substantial assistance we have received from
Doctors P. Rosenberg, B. C. Abbott and C. B. Alender; from Mrs. J. L. Russell, Mrs.
Helena Lemp, Mrs. E. E. Buckley, Mrs. U. Smith, Mrs. B. Lane, Mrs. H. Hayes, Mr. L.
Lauritzen, Mr. D. Majerus, Mr. P. Meadows and Dr. B. W. Halstead; and from Mr. James
Gott of the Attending Staff Association of the Los Angeles County General Hospital, and
from his organization.
The Symposium was supported by contract N00014-66-C0068 of the United States
Office of Naval Research, grant AC00273-01 of the Division of Accident Prevention,
Public Health Service, and a grant from the United States Air Force Office of Scientific
Research.
This record of the Symposium received additional support from Dr. Robert K. Cutter,
Cutter Laboratories, Berkeley, California, to whom we are indebted.
Sponsors of the Symposium were the International Society on Toxinology and the
Division of Comparative Physiology of the American Society of Zoologists.
We acknowledge the courtesies of Wyeth Laboratories and Ross Allen's Reptile
Institute.
Finally, we wish to express our particular thanks to the most diligent contributor to the
Symposium and this text, Mrs. Yvonne Majerus, who as Corresponding Secretary for the
International Society on Toxinology has spent unmeasurable time and effort in the prepara-
tion of this book. We are indebted to her for her continued interest and patience.
ix
SECTION I. ARTHROPODS
STRUCTURE OF THE VENOM GLAND OF THE BLACK
WIDOW SPIDER LATRODECTUS MACTANS.
A PRELIMINARY LIGHT AND ELECTRON
MICROSCOPIC STUDY*
DAVID S. SMITH! and FINDLAY E. RUSSELLJ
Laboratory of Neurological Research, Loma Linda University, Los Angeles County General
Hospital, Los Angeles, California, and Department of Biology, University of Virginia,
Charlottesville, Virginia
INTRODUCTION
IN AN EARLY anatomical and histological account, BORDAS [1] noted that the venom glands
of Latrodectus are paired organs, discharging their secretion directly via a duct or canal to the
apex of each chelicera, without the interpolation of a collecting reservoir. He describes the
principal components of the cylindrical glands within which the venom is secreted: a well
defined intrinsic muscular sheath in which the fibers run in a longitudinal-helical fashion, an
underlying "supporting membrane" and a columnar epithelium of secretory cells. Sub-
sequently, the cellular organization of the secretory epithelium of the venom gland of
Latrodectus and other spiders has been investigated by several workers [2-6], The chief aims
of several of these studies have been to establish, firstly, the mode of secretion of venom in
the gland and, secondly, the response of the secretory epithelium following the expulsion
of venom, and along these lines of enquiry, little agreement has been reached. BORDAS [2]
and ANCONA [3] described a holocrine secretory mechanism in Latrodectus and Ctenus, cor-
related with the presence of small basal "replacement cells" amongst the active cells of the
epithelium, while MILLOT [4] reported the occurrence of merocrine secretion in the glands
of Latrodectus. BARTH [6] postulated a more complex and non-regenerative sequence of
secretory events in the venom gland, in which the products produced within the main glandular
cells of the epithelium receive a secretory contribution from small basal "contiguous
cells". These cells appear to correspond to the "replacement cells" of the other authors.
This paper contains a preliminary report of a reinvestigation of the cytological structure
of the components of the venom gland of Lactrodectus mactans, carried out primarily with
the electron microscope. Evidence is presented on the mode of liberation of secreted
products into the glandular lumen in "milked" animals, and of their retention and accumula-
tion during periods when venom is not expelled. Observations on the intracellular synthesis
and sequestration of the various components contributed to the venom by the cells of the
secretory epithelium are at present very incomplete, and it is hoped that these problems may
be considered more fully in a later report.
* Supported by Grant NSF GB-1291 from the National Science Foundation,
f Present address: School of Medicine, University of Miami, Miami, Florida.
J Present address: Laboratory of Neurological Research, University of Southern California, Los Angeles
County General Hospital, Los Angeles, Calfornia.
1
2 DAVID S. SMITH and FINLAY E. RUSSELL
MATERIALS AND METHODS
Spiders used in this work were collected from Altadena, California. Venom glands of
adult individuals of L. mactans, together with the venom ducts and chelicerae, were dissected
out under CO anaesthesia. The material was immediately placed in ice-cold 2-5 per cent
s
glutaraldehyde containing 0-17 M sucrose, maintained at pH 7-4 with 0-05 M cacodylate
buffer. Fixation was continued for 2-3 hr, after which the tissue was passed through several
changes of a washing solution containing 0*34 M sucrose in cacodylate buffer, over a period
of at least 24 hr. In some instances, glands were successfully sent by air from California,
Virginia in glass vials of washing solution packed in crushed ice, at this stage of processing.
After washing, material was placed in ice-cold veronal acetate buffered 1 per cent osmium
tetroxide, at pH 7-4, for 90 min, dehydrated in an ethanol series and embedded in Araldite
via propylene oxide. Sections were cut with glass knives on a Huxley microtome, mounted
on collodion-carbon coated grids, and stained first with saturated 50 per cent ethanolic
uranyl acetate (10 min) and then with lead citrate [7] (2 min). Sections were examined in a
Philips EM 200. For light microscopy, the same procedure was followed for fixing and
embedding: 1 \x sections were cut with glass knives, dried and flattened by warming in a
drop of distilled water on a slide, stained in a drop of hot 1 per cent toluidine blue with
1 per cent borax, washed in distilled water and mounted in immersion oil.
RESULTS
The muscular investment
Histological studies mentioned earlier established that the venom glands of Latrodectus
are invested with a compact series of intrinsic muscle fibers, running in a weak helix with
respect to the long axis of the cylindrical gland. Thus, transverse sections of the gland also
display transverse or slightly oblique profiles of the fibers, as is illustrated in the accom-
panying light and electron micrographs (Figs. 3, 1 and 8). These are roughly rectangular in
cross section, ca. 12-20 x 30-50 /x in size, and appear to traverse the length of the gland.
Longitudinal sections of the fibers (Fig. 2) exhibit well defined striations with a sarcomere
repeat of 5-6 /x. BARTH [6] suggested that morphologically distinguishable phasic and tonic
fibers may be present in this sheath, but in the present study only one type has been noted,
in which the contractile system virtually fills the cell, and is poorly demarcated into myo-
fibrils (Fig. 8). As in muscles of other arthropods [8], these fibers receive multiterminal
polyaxonal nerve-muscle junctions, and synaptic close apposition of muscle and nerve is
illustrated in Fig. 8. Further details of the cytological organization of these fibers and their
neuromuscular junctions will be presented separately (SMITH and RUSSELL; in preparation).
The extracellular sheath
A well defined sheath is situated between the muscle fibers of the gland and the basal
surface of the secretory epithelium. This has been described as a "basal or supporting
membrane" [1], a "connective tissue basement membrane" [5] and a "peritoneal membrane"
[6]. This structure, 2-4 /x in thickness, is illustrated in Figs. 1-3 and 8, and will be referred
to as the "extracellular sheath". Electron micrographs (Fig. 8) reveal that this sheath
contains cross-banded closely packed collagen-like fibrils, with a major repeat period of
ca. 660 A, embedded in a sparse matrix. The cellular derivation and embryological origin
of this structure are not known. Both the fibrillar and matrix material extend in thin sheets
between the adjoining muscle fibers, where they may ensure mechanical cohesion between
Black Widow Spider Venom Gland 3
FIG. 1. TRANSVERSELY SECTIONED UNMILKED Latrodectus mactans VENOM GLAND. NOTE THE
MUSCLE SHEATH (Mu) INVESTING THE GLAND AND THE UNDERLYING EXTRACELLULAR SHEATH
(*). THE GLAND IS LARGELY FILLED WITH DROPLETS OF SECRETION OF VARYING DENSITY (S).
CLUSTERS OF SMALL DARKLY STAINING NUCLEI (ARROW) OCCURRING IMMEDIATELY BENEATH THE
EXTRACELLULAR SHEATH ARE BELIEVED TO BE THOSE OF REPLACEMENT CELLS. A PORTION OF THE
LUMEN IS INCLUDED AT Lu. x 500. N.B. THIS LIGHT MICROGRAPH, AND THOSE REPRODUCED
IN FIGS. 2 AND 3, WERE PREPARED FROM MATERIAL FIXED AND EMBEDDED FOR ELECTRON MICRO-
SCOPY. SECTIONS WERE CUT WITH GLASS KNIVES ON AN ULTRAMICROTOME AT A THICKNESS OF
Ca. AND STAINED WITH A HOT SOLUTION CONTAINING 1 PER CENT TOLUIDINE BLUE AND 1 PER
CENT BORAX.
FIG. 2. LONGITUDINALLY SECTIONED UNMILKED Latrodectus mactans VENOM GLAND. NOTE THE
STRIATED MUSCLE FIBERS LIMITING THE GLAND (Mu) AND THE UNDERLYING EXTRACELLULAR
SHEATH (*). LARGE BLOCKS OF SECRETION PRODUCT (S) VIRTUALLY FILL THE SECRETORY EPI-
THELIUM. THE TRANSVERSE STRIATIONS IN THE EXTRACELLULAR SHEATH ARE AN ARTIFACT OF
SECTIONING. THE LUMEN IS INCLUDED AT LU. X 500.
FIG. 3. LIGHT MICROGRAPH OF A VENOM GLAND OF Latrodectus mactans FIXED WITHIN AN HOUR
AFTER EXPULSION OF THE VENOM, AND SECTIONED TRANSVERSELY. NOTE THE MUSCLE FIBERS
(Mu) AND THE EXTRACELLULAR SHEATH (*). THIS FIELD ILLUSTRATES AN ACTIVELY SECRETING
EPITHELIUM CONTAINING SECRETORY DROPLETS (S) SITUATED AT VARIOUS LEVELS WITHIN THE
CELLS, AT THE BORDER OF THE LUMEN (Lu) AND LYING FREE WITHIN THE LATTER. CELL DIS-
SOLUTION DURING THE PROCESS OF RELEASE OF SECRETION IS SUGGESTED BY THE PRESENCE OF
FREE NUCLEI WITHIN THE LUMEN OF THE GLAND AT n. NOTE THE SMALL DENSE NUCLEI SITUATED
IMMEDIATELY BENEATH THE EXTRACELLULAR SHEATH (ARROWS; CF. FIG. 1)*. THESE ARE THOUGHT
TO REPRESENT THE NUCLEI OF UNDIFFERENTIATED "REPLACEMENT CELLS" THAT BECOME ACTIVE
TO TAKE THE PLACE OF EPITHELIAL CELLS LOST AS A RESULT OF HOLOCRINE SECRETION. X 700.
FIG. 4. SURVEY ELECTRON MICROGRAPH OF THE BASAL AND CENTRAL AREAS OF SECRETORY CELLS
IN A MILKED GLAND. A NUCLEUS, CLOSELY ADJOINING THE BASAL SURFACE, IS INCLUDED AT
UPPER RIGHT (N) (CF. FIG. 3). THE EPITHELIAL CELLS HAVE A HIGHLY DISSECTED FORM, AND THIS
MICROGRAPH REVEALS THE CHARACTERISTIC CLOSELY APPOSED TO INTERDIGITATING CELL
PROCESSES; SOME OF THE CELL MEMBRANES LIMITING THESE ARE INDICATED WITH ARROWS. NOTE
THE NUMEROUS PROFILES OF SPHERICAL SECRETION DROPLETS CONTAINED WITHIN THE CELL
PROCESSES (S); THESE VARY BOTH IN SIZE AND ELECTRON DENSITY OR OSMIOPHILIA. X 7000.
FIG. 5. A FIELD WITHIN MILKED GLAND, ILLUSTRATING A REGION OF APICAL SECRETORY CYTO-
PLASM BORDERING THE LUMEN (Lu). IN THIS INSTANCE, THE CYTOPLASM (Cy) IS LARGELY FILLED
WITH VARIOUS TYPES OF SECRETION DROPLETS (S), AND WHILE IN PLACES (ARROWS) THESE ADJOIN
AN INTACT CELL MEMBRANE SURFACE, ELSEWHERE (*) THE LATTER APPEARS TO BE UNDERGOING
FRAGMENTATION, WHICH PROCESS WILL ULTIMATELY RELEASE THE SECRETED MATERIALS AND CELL
DEBRIS INTO THE LUMEN OF THE GLAND (CF. FIG. 6). X 17,000.
FIG. 6. ELECTRON MICROGRAPH FURTHER ILLUSTRATING THE APICAL DISINTEGRATION OF THE
SECRETORY EPITHELIUM IN A MILKED VENOM GLAND OF LatmdeCtUS mactans. NOTE THE NARROW
INTERDIGITATING CELL PROCESSES (BLACK ARROWS) CONTAINING A FEW SECRETION DROPLETS (S).
THESE PROCESSES APPEAR TO UNDERGO DISPERSION INTO SPHERICAL OR IRREGULAR FRAGMENTS (*)
WHICH RETAIN THEIR INTEGRITY AS THEY PASS INTO THE LUMEN (WHITE ARROWS) (CF. FIG. 7).
x 8000.
Figs. 1, 2, & 3.
