Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1935 Studies on insect hemolymph, with special reference to some factors influencing mitotically dividing cells Oscar Ernest Tauber Iowa State College

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Recommended Citation Tauber, Oscar Ernest, "Studies on insect hemolymph, with special reference to some factors influencing mitotically dividing cells" (1935). Retrospective Theses and Dissertations. 15215. https://lib.dr.iastate.edu/rtd/15215

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STUDIES OK INSECT HEI»I0LYTJPH. V/ITH SPECIAL REFERENCE TO SOME FACTORS INFLUENCING ICCTOTICALLY DIVIDING CELLS

BY

Oscar Ernest Tauber

A Thesis Submitted to the Graduate Faculty for the Degree of DOCTOR OP PHILOSOPHY Major Subject - Zoology

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Iowa State College 1935 UMI Number: DP13000

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TABLE OP CONTENTS Page I. INTRODUCTION 6 II. LITERATURE SURVEY A. The Cell Theory 10 B. The Protoplasxna Doctrine 29 C. The Organismal Theoiry S3 D. Cell Division S7 E. Insect Hemolymph Cells 1. General Literature 44 2, Classification of Cells 49 a. Table I. Comparison of Nomenclature of Insect Hemolymph Cell Types ... 58 S. Division and Origin of Cells 59 P. Bacteria in Insect Hemolymph 62 1, Table II. Some Papers Listing Bacteria Found in Insect Hemolyn^h 65 G. Factors Influencing Mitosis 74 1, Table III, Some Papers Referring to Con­ ditions or Substances Affecting Mitosis . 77 H, Stimulation of Mitosis of Insect Hemolymph Cells 105 III. MATERIALS 109 IV. METHODS 112 V, EXPIIRIMENTS 116 VI. RESULTS A. Prom Determining Y/hat Nvaaber of Cells Consti­ tute a Pair Sample For Making Bf.D.C. Counts . 120 1, Table IV. Comparison of M.D.C. Counts (^) Based on Different Ntontoers of Cells Counted 122

T5'Z3I 3 - Page B. From Tests to Duplicate Counts £rom Same Sample 123 1. Table V. Duplicate Counts on Same Hemo- iTmph San^le 125 C. Prom Control Animals 124 1, Table VI, Average Mitotic Control Counts . 126 2, Figure I. M.D.C. Counts f3?om Control Animal No,619 129 D. Prom Animals Segregated foi' Sex and Age .... 130 E. Prom Animals Checked for Possible Effects from Changes in Laboratory Temperature 130 1. Figure II, Comparison of M.D.C. Ciirves and Laboratory Temperature Cujrves .... 133 P. Prom Ovipositing Pamales 134 1. Figure III, Composite and Typical Curve from Females During Egg Formation and Deposition 136 2. Table VII. Summary of Data from Oviposit­ ing Females 137 ff. Prom Molting Animals 138 1, Pigvire IV. Composite and Typical Curve from Molting Specimens 140 2, Table VIII, Summary of Data from Molting Animals 141 H, Prom Animals Subjected to Successive Counts Within Short Intervals 142 1. Figure V. Curve of Successive Counts from Animal No.625a 144 I. Prom Animals Subjected to Hi^ or Low Tempera­ ture 145 1, Figure VI. Curve of M.D.C. Counts of Animals Subjected to 37®C 147 2, Figure VII. Curves from Animals Subjected to 5°0. and Room Temperature 149 3, Table IX. Sxjmmary of Data from Animals Subjected to Hi^a and Lov; Temperature . , 150 4 - Page J. Prom Various Experiments Using Different Bac­ teria 151 1. Table X, SmnDiarjr of Data from Experiments "With Bacteria 154 2. Figure VIII. Curves from Animals Inocu­ lated v/lth. Staphylococcus aureus ..... 158 3. Figure IX. Curve from Animals Infected YTlth Coccus Bacteria 160 4. Figure X. Curve from Ani.mals Infected with Rod Bacteria 162 K. From Animals in Certain Pathological Conditions. 1. Table XI. Summary of Data from Animals in Certain Pathological Conditions ..... 163 2, Figure XI. Counts from Anii.mals Afflicted with Paralysis and vAth Abiiorma3.1y Vacuo­ lated Cells 157 L. Prom Injections of Sterile Nutrient Broth, Pep­ tone, and Beef Exti^act 168 1. Table XII. Summary of Data from Injections of Broth, Peptone, and Beef Extract . . . 170 2. Figure XII. Curve from Animal Injected \'Tith nutrient Broth 173 5. Figure XIII. CuxTre from Animal Injected with lOjj Peptone 175 M. From Animals Injected with Chick Embryo Extract, Rat Blood, Hemoglobin, or Egg Albumin . . . . .176 1. Table XIII. Summary of Data from Animals Injected v/ith Embryo Extract, Blood, Hemoglobin, or Albumin 178 K. From the Use of Some Amino Acids, Particularly Cysteine, of Glutathione, and of Insulin . . . 179 1. Table XIV. Summary of Data from Injection of Various Amino Acids, of Glutathione, aiai of Insulin 181 2. Figure XIV. Curves from Animals Injected with Cysteine 184 3. Figure X^r. Ctu?ve from Animal Injected with Glutathione 185 4. Figure XVI. Curve from Animal Injected v/ith Undiluted Insulin 188 - 5 - Page 0. From Injection of Dinitroplienol, Thyroxin, G-lucoso, Load Chloride, or Rotenone 189 1. Table XV. Sutnmary of Data from Animals In­ jected v/lth Dinitx»ophenol, Thyroxin, Glu­ cose, Lead Chloride, or Rotenone 191 P. Total Umber aiid Per Cents of Dividing Cells and Multinucleate Cells Observed 192 1. Table XVI, Total Number and Per Cents of Dividing and Multinucleate Cells Observed. 194 VII, DISCUSSION 195 VIII. CONCLUSIONS 218 IX. SUIffilARY . , 224 X. LITERATURE CITED 230 XI. AClCNiOlVLEDGiasl'ITS . 281 XII. VITA 282 XIII. ILLUSTRATIONS A. Plate I. Sketches of Normal, Dividing, and Other Hemolyraph Cells from Blatta orientalis . 285 B, Plate II. Sketches of Multinucleate Iiemol:ymph Cells from Blatta orientalis 287 - 6 -

I. INTRODUCTION

Although living organisms differ widely in distinguislaing characteristics, all are fiandamentally alike in their \mits of anatomical structure. Plants and animals are built of similar elementary units called cells; and the view that all living things are made of these minute particles is called the cell theory. This belief is considered one of the most important and basic theories of biology. The botanist, the zoologist, the physiologist, and the pathologist study the cell in search of the vital phenomena which occur during health and disease. From this viewpoint, all vital processes of a complex organism appear as the result of the irsiividual vital processes of its functioning cells. Thus, in many respects, the cell is the center arotind which the biological research of the present re­ volves. Experiments included in this dissertation are essential­ ly studies of individual cells in a modified form of a tissue culture; the hemolyraph of a particularly suited living insect (Blatta orientalis) serves as the medium in v/hlch the cells are suspended. Changes in the composition or the reaction of the hemolyraph brought about by pathological or physiological con­ ditions, or by direct injection into the experimental animal are essentially similar to changes v/hich might be obtained in the usual fom of tissue culture by the direct addition to the - 7 - tissue meditxm of toxins, chemicals, or otlaer agents. Use of this living insect in the manner to be described has some advantages over the use of a tissue culture. Keeping and feeding the insect are much more convenient than the daily manipulations demanded by a tis8^XB cultxire. Use of the insect eliminates the daily dlsttirbances to viixich culture cells are subjected when changed from one medium to another. Many more cells are available for study in a sample of hemolymph than can be studied in a piece of growing tissue. Cells in the hemolymph of the living insect probably can be subjected to more vajpied and drastic experiiiiental conditions than can cells in a delicately balanced tissue culture medium. Certain cell changes - such as those occurring in karyoklnesls ~ can be con­ veniently followed in the isolated hemolymph cells of the in­ sect by means of temporary stained mounts, while cells from a tissue culture usually must be prepared by a more complicated histological technique before they can be examined. Changes occurring In experimentally treated cells can be studied in a long series of hemolymph cell seimplea taken from the same source with very short Intervals between samples - a piocedure not possible from the same tissue culture preparation. There are, of course, disadvantages in the use of the in­ sect as described. Since the total hemol^ph volume is not known, the dilution of Injected substances cannot be accxxrate- - 8 - ly determined. Also, since the cells are in an organism pos­ sessed of an excretory system, the concentration of injected materials may bo gradually decreased by a removal of some or all of the substance by way of the excretory mechanisms. It is believed, however, that these disadvantages are outv/eij^ied by the advantages of the insect hemolymph cell method in studies of a general cytological nature. This paper presents the resixlta of a series of studies upon the va3?ious types of clrcuD-ating cells found in the hsmo- lyitiph of the insect, Dlatta orlentalis L,, with particular reference to those cells in different stages of karyokinesis. Mitotic indices for samples of colls from a group of normal in­ sects are given as control counts. Other mitotic indices of cells from individuals serving as e>:perimental aniirals are pre­ sented. The expei-imental studies Include the effect of cer­ tain pathological and physiological conditions, as well as of the injection of various substances, upon the normal mitotic index of this insect. Variations in the mitotic indices of both the normal and experimental individuals d-uring the period of observation are sho-wn by graphs and tables. Investigations Involving some kind of study of mitotic division of cells are numerous. The close relationship between karyokinesia and growth in living forms malces these studies of interest to a great field of scientific workers, if not to all biologists and workers in related sciences. Studies with - 9 - special attention given to factors influencing cell division are of particular interest to many investigators and instruc­ tors in biology, but chiefly to cytologista and pathologists. Any research furnishing new information in regard to these factors, or offering methods and techniques for the further development of the study of these factors should prove of par­ ticular Interest to the latter groups. - 10 -

II. LITERATURE SURVEY

A. TOie Cell Theory

For several reasons ifc is not amiss to include in this dissertation on mitosis a short review of the history of the cell theory and its subsequent modifications. As a structural unit of growth by multiplication, the cell is closely allied vdth the phenomenon of cell division* As a locus of the in­ tracellular activities occurring In karyoklnesls, and as a major contributor to some of tlie structures involved, in mito­ sis, the nucleus of the typical cell plays an important part in cell division. Also, the discovery of the nucleus and the later Interpretations of its significant connection to cell division were additional facts added to the data on which has been founded the so-named cell theory. There is an inclination to accept the cell theory without thouglit of the many investigations and interpretations which had to be made before the status of the cell as a structural unit of tissue had been established. The conception or idea connected with the Y;ord "cell", used scientifically, has been altered considerably since its introduction into biological literature, especially after the proposal of tlie cell theory. Almost tv/o centuries before tlie publications of Matthias Schlelden and Theodore Schwann, pioneers in histological re­ search had made observations on the minute structure of plant - 11 - sold oniriial tissues, and sometimes approached, closely to those conceptions which were fo^anded later. Tliere is no certainty as to wMch early microscopist made the initial observations on either plant or animal cells. In 1665 Robert Hooke (267) presented a publislied description of the cellular organization of plants. The word "cell" was in­ troduced into biological literature in this summary of his v/ork Tidiich included a chapter: "of the schematlsme or texture of cork and the cells and pores of some other such frothy bodies". This is believed to be the first mention of Mstolog- ical cells. Hooke had no distinct notion of the cell contents and in some places va'ites of the cells as "bubbles". His be­ ginning examinations on such dialed materials as charcoal and cork probably led him to believe that cells were only honey­ comb-like cavities. Later, hov/ever, in "Obse3?vation 110.25" on epidermic cells from the under surface of a nettle-leaf, he writes of a "nourisching juice" which ho inferred must pass through pores from one chamber to another. In his illustra­ tions of these cells, something very like a nucleus appears in one of them, but this may be only an artist's or printer's ac­ cident. Between 1661 and 1665 Mar cello Malpighi (355, 336, 337) had seen red blood corpuscles in the oanentum of the hedge-hog, but took them to be fat globules. Ho also published obseinrations upon the minute stmcture of the lungs, kidneys, spleen, liver. - 12 - and membranes of the brain. Most of his discoveries of the var­ ious elements making up the body of the vascular plant are in­ cluded in his Anatome Planatarum (354). He was familiar with individual cells which he called "utriculi", "sacculi", or "globxill". His remarks concerning the Importance of the "utri­ culi" as structural units of the plant are almost a clear fore­ shadowing of the cell theory. Malplghl showed that these units could be isolated and believed them to be independent entities. Anothex' early mlcroscopist wlio studied plant tissues v/as Nehemlah Grew, His investigations began in 1664 and continued •until 1682 when all of them were collected under the title. The Anatomy of Plants (186). Included In this volume was his first in^ortant paper which he read before the Royal Society of London in 1671 (185). I-Ie was strongly impressed by the manner in which cells - called "vesicles" and "bladders" by Grew - appeared to make up the bulk of certain tissues: " the parenchyma of the Barque is much the same thing, as to its conformation, which the froth of beer or eggs is, as a fluid, or a piece of fine Manchet, as a fixed body." (363, p.64). Grew, like Hooke, seemed to give more attention to the walls of his "vesicles" than to their contents, vSiich he leaves un- mentioned. Dxae to an unforttinate error in some early manuscript, Jan Jacobz Swammerdam is often credited with the discovery, in 1658, of the red blood corpuscle of the frog; this, incidental­ - 13 - ly, also credits Mm with the first recorded observation of either a plant or animal cell. The mistake persists into re­ cent literatxire (489, 527), even thougji irdall (363, footnote, p.198) in 1912 had i>ointed out the error:

"The m^ng date of 1658 is assigned to Sv/aittiiordam's discovery of the red blood-corpuscle of the frog by Poster, by Darmstadter, and probably by otlier v/riters. In 1658 Swaimnerdam liad not begun Ms regular anatomical studies; he v/ont to Leyden for ttiis purpose in 1661. No date is assigned, so far as I know, eitlier in the Biblia Haturae. or in Boerhaave's Life prefixed thereto, to the discovery of the red corpuscles Swaminerdam's observations on the red corpuscles of tine frog cannot, therefore, it would seem, be dated at all."

In this connection it is probably significant that Sv/aminerdtam* s inaugural dissertation, Tractatus, Physlco-Anatomico Medicus de Respii'atione usuque Fulmonum, delivered at Gottingen, in 1667, made no mention of red corpuscles (527, p.l65); tMa was nine years after his supposed discovery of red cells. Swamnierdain, however, made many other observations (540, 541, 542) vAiich give Ma v/ork an important place in the Mstory of the biological cell. He made a large number of original, minute, careful ob­ servations. Tlie celliilar structure ("greynkens" or "klootkens") of animal tissues was seen by him in the frog embryo. He also noted the nucleus In the blood corpuscles from a frog, but did not realize the significance of Ms discovery. Prom the view­ point of this thesis it is particularly noteworthy to find that he probably made the first observations, records, and illustra- ~ 14 - tiona of the amebold-like hemolyraph cells from an insect (dog louse)(542, p,31). Although he did not know their exact nature, Sv;ainmordam figured, some of these cells in one of his early papers (542, table II, figure V, 1-5). One of the most prominent names in the early history of microscopy is that of Antony van Leeuwenlioek (119). In the course of his investigations he observed cells ("globules") in the tissues of higher organisms. lie noticed the arrange­ ment of cells in the epidermis of his skin and mistook the nuclei for ducts of glands (317); he called the cells in the tail of a yovmg tadpole "globules" (318). In a letter address­ ed to the Royal Society of London, dated Avigust 15, 1673, Leeuv/eilhoek gave the first accurate published description of red corpuscles from man (S19, Letter No,65). During the same year he also found uncolored corpuscles circulating in small transparent crustaceans (319) and described the nucleus in the frog corpuscle as a "bright oval spot in center" (320), Later he studied the hemolyraph of spiders and wrote several letters about its composition (321). Leeuwenhoek was a keen observer and seems to have believed all tissues to be formed of "globules". Though he designated all his particles by that name, he v/ell realized that they were not spherical, and in some tissues not even round in cross- section. Can it be that he anticipated by more than a century the future proposal of cell structure? - 15 -

After the death of Leeuweiihoek, there ensued a period dur­ ing which the actual Investigation of the structure of organ­ isms remained practically at a standstill, VOien attention v/as again directed to "cells" and "globioles" at tlie middle of the eighteenth century, a number of interesting suggestions were offered regarding the origin and significance of these elements. The first attempt, in 1775, at building up the tissues by an ultimate physical element was that of Albrecht von Haller's (217). He resolved the solid parts of animals and plants into the "fibre" and an "organized concrete". No allusions to the cell, beyond imperfect descriptions of blood corpuscles and spermatozoa, appear to have been made by Hallei>* In spite of the vagueness of its interpretations, the idea that fibers v/ere the groundv;ork of nearly all the tissues continued \antil the latter part of the ei^teenth century, Naturally, it main­ tained itself longest in the case of the fibrous tissues, slme the. appearance of these is that of structures apparently composed of fibers. Wolff's Theorla Generationia (597) vms published in 1759 a few years after Haller's theory was presented. His doctrine as given by Huxley (274) was as follows:

"Every organ is composed, at first, of a mass of clear viscous, nutritive fluid, which possesses no organiza­ tion of any kind, but is at most composed of globules. In this semifluid mass, cavities (Blaschen, Zellen) are now developed; these, if they remain rounded or poly­ - 16 -

gonal, become tiio subaeqiieixt colla, if they elongate, the vessels; and the process is Identically the same, whetlier it is examined in the vegetating point of a plant, or in the young budding organs of an animal In each case they are mere cavities and not ii^dependent entitles; organisation is not effected by them, but they are tlie visible results of the action of the or­ ganizing power inherent in the living mass."

This theory received veiy Httl© notice and probably would have been still less knoivn but for Hvixley's attention to it. It vfill be observed, also, that Wolff's idea involved, the spontaneous origin of cells, that is, independent of any pre­ viously existing cell. Beginning in 1779 a reaction set in against the fiber tlieory, and culminated in the formation of the "globular" theory based partly on the oldei' waitings of Hoolce, Malpighi, Grew, Sv;am!nerdaiii, Leeuwenlaoek, and Wolff (already referred to), and partly on original v/orlc by Prochaska (447), Bichat (62), Pontana (151), Spreiigel (532), Oken (400, 401), Bernhardi (50), and others to be mentioned later. Prochasliai, in 1779, was one of the first to rotuni to the "globular" conception of the composition of cellular material. In that year he described tiie brain and othor nerve tissues as made of globules "eight times smaller than blood-globules". In 1801, BichEt elaborated Ma claasification of tissues and

accepted the globular theory, seemingly v/ithout malcing any orig­ inal investigations in minute structure. Sprengel, in 1802, stated that cells originate iii tlie contents of other cells as - 17 - granules or vesicles v/hich absorb vvater and enlarge. He work­ ed mainly on starchy plant material and It may be that his no­ tion of cell fomation from intracellular "vesicles" was based on the presence of starch grains Via:iich he supposed grov; into nev/ cells. Sprengel's theory was upheld by Treviramis (553) in a v;ork appearing in 1806, and both men fought many years for its support. Treviranus later (33) combined the fiber and globule theories by offering plant and animal elements v/Mch were first a homogeneous, formless matter; second, fibers; and third, globules ("Kugelchen"), In hia earlier v/ork (553) he rendered iirportant service, hoivevor, in proving that vessels or tubes develop from cells by arranging themselves in rows, and transforming into elongated tubes by a breakdown of the partition walls. Kieser (291) further developed Sprengel's theory by explaining tiiat the Intracellular granules were "cell germs" wMch later "hatched" to form new cells. During this period Oken for the first time clearly express­ ed a cellular or vesicular coraposition of animal organisms as well as of vegetables or plants. As early as 1805 (400) he refers to elementary parts as "vesicles". In 1808 (401) he writes: "Animals and plaiats are throughout nothing else than manifoldly divided or repeating vesicles." This most explicit statement seema to have been overlooked by many later %vritera. Hia conception of the "Blaschen" nearly approaches that pro­ posed later for the biological units of the cell theory. - 18 -

For a per3.od of aboxrb thirty years after Okon's early papers, a large number of Inportant cont3?l but ions v;ere added to the information upon which the cell theory was later to be based. Opinions expressed by Link (325) and Rndolphi (496) in 1807 and J. J. P. Moldenlmwer (379) In 1812 gave promise of leading in the riglit direction had their siiggestion been fol­ lowed through, but, unfortunately, their results missed re­ ceiving the attention they deserved. Charles Wenaol and Joseph Wenzel (584), in 1812, confirmed Prochaska's earlier opinion that the brain was composed of small globules. Bauer (33), quoted by Home (266), between 1818 and 1823 described, too, the ultimate particles of the brain tissue as being globular in nature; he also rather clearly discerned the malce- up of muscle fibers. Hensinger's (246, 245) keen interpretations, pixblished in 1822-1824, on the orgaiid-zation of the fiber of the "fibre theory", clearly abolished that view as a suitable explanation of tissue composition. He saw the fiber not as a unit, but as formed by the linear apposition of separate globtiles which he considered the ultimate particles of organj-zation. Interpreted v/ith present day laiov/ledge in mind, Hensinger's views are an approximation of modern conceptions of the cell, but the dis- tortions and twists, given, by o1±Ler contemporary vnriters, as explanations of his conclusions, are sometlinGS quite puzzling. 19 -

For example, part of Henle's (243) account of Honslnger's woi-k reads: *As the result of an equal contest betv/eon contrac­ tion and expansion, there arises the fj-loTatile. of v/hlch all organisms, all orgaiiic parts, are originally com­ posed. By a stronger exercise of power, tliere origi­ nates from the often more homogeneous gloljule, the vesicle. \'7here in an organism ^lohules and foiroless mass are present, the pilobules arrange themselves ac- cording to chemical laws and form fibres. Vfliere vesicles^ arrange themselves, there tirise canals and. vessels."

In addition to Hensinger'a discufJsions, the writlnrjs of Milne-Edv;ards (364, 365) may be looked upon as having givan position and popularity to the globular theory. Betv/een 1823 and 1326 he examined all the principal tissues, and announced that the fibers of fibx-ous tissues, as well as other tissues, were composed of globules. He concluded tliat these corpuscles, by their aggregation, constituted all plant and ani-nal materials. There is little doubt but that Milne~Edwards in­ terpreted tissue cccapositlon in the same fashion as usually accepted today. It must be reusmbered, hov;evor, that he work­ ed vdtli inadequate instruments v/hose magnified viev/s were fiirther distorted by the glare of direct sunlight in which they had to be employed. Bauragartnei* (34) and Arnold (10) have a peculiar place in the history of tlie cell tiioory in that they were advocates of the globular theory, but as late as 1842 (35) contended against the cell doctrine. Baiamgartner, in particular, asserted that - 20 - the globule never divides to form two; in other words, that there is no such tMng as cell division. His contention was that the globules originated spontaneously from a primary form­ less material. Of the major writers vrtiose views received attention dur­ ing this period, only one, Hodgkin (255, 256) did not admit in greater or less degree the importance of the globule as a physical tissue-building element in biology. He did not, how­ ever, offer any other hypothesis, and is not clear in stating what was his real belief. During the years from 1830 to 1840, sudden improvements in optical instruments allowed even more significant advances toward the understanding of tissue makeup. Mirbel (367, 369) in research on Marchantia distinguished three modes of cell multiplication: the formation of cells on tihe surface of other cells; the formation of cells within older cells; and the for­ mation of cells betv/een older cells. The first mode apparent­ ly represented the budding of the germ tube arising frcan the spore, while the second and tMrd methods are the same: the process of cell-multiplication in growing gemmae. Other botanists, including Dumortier (127), Morren (381, 382), Meyen (360, 361), and von Mohl (370), observed the division of cells, chiefly those of certain algae. The work of Hugo von Mohl is particularly noteworthy since it contributed the first carefvil description of cell division. - 21 -

Dr« Robert Bro\m (69) of Edinbiirgli ia iiaually credited witLi the discovery of the nucleus in 1833, althou^^i its presence had been noted before by several other observers. Hooke (267) pi-nDbably sav/ it in plant epidermis; Swaimierdam (542) SBXI it in the frog blood cells; Leeuwexihoek (320) described it in the same source material; Pontana (150) had written of it as early as 1781; Meyen (359) saw the nucleus in Spiropiyra in 1826; and Piarlcinje recognized it in the germ cells of birds. It \ma Brovna, however, v/ho concluded tliat it was a normal constituent of a cell, and who thereby made a most iii^jortant contribution to the anatomy of the cell. After Brom's annoiancement, observations on nuclei in various tissues multiplied rapidly. Animal cells as v/ell as plant cells were closely scmitinized, and as it became evident that cells of widely different types possessed this body, the cell as an indivldml unit began to stand out clearly and alone from the other "fibers", "spirals", "granules", "corpuscles", and "globules" with which It had been confused. The animal and plant were recognized as com.posed of cells, and similar to one another in that respect. Purld.nje (450), Dutrochet (135, 136), "Valentin (561), Muller (385), and Henle (241) were leaders in this particular phase of the history of the cell. To Dutrochet, especially, should go the honor of recognizing the individual­ ity of the cell. He states: -82-

"This astounding organ is truly -Qie fundamental ele­ ment of organization; ©vorything, indeed, in the or­ ganic tissue of plants, is evidently derived from the cell, and observation just proved to us that it is the same with animals,"

This viev; was in contradiction to the doctrine tau^t by La­ marck (311), and expressed by Mirbel (367, 366); both consider­ ed the organism as a cellular v/hole, rather than a composition of single elemental^ organisms. Other contemporary histolo- gists who called attention to the analogy betv/een plant and animal cells were: Raspail (457, 458), Tijrpin (556), Schultz (509), V/agner (575), Quatrefages (452), and Dumortier (126). B\jjmett (74) gives an excellent review of this pcsriod in his essay which was read before the American Medical Association in 1853 at Philadelpliia. Out of this mass of observation, the discovery of cell di­ vision, the presence of a nucleus in a typical cell, and the recognition of cell individuality ~ these three main facts set the stage for the formulation of the cell tiieory. The biolog­ ical tissue unit had not yet been standardized. It v/as then, in 1838-39, that Schwann and Schleiden attempted to frame a really comprehensive cell theory, applicable to all tissues. Although the names of Matthias Schleiden and Theodore Schwann are usually linked together in association with the cell theory. It was really the latter, rather than the former, who did more in formulating its principles. Schleiden (505), - 23 -

In 1838, pointed out the formation of cells in plant tissues as due to a single and uniform method, and from that elaborat­ ed a theory of development in which the cell was the unit. In the opening paragraph of his treatise he states:

" every plant developed in any higher degree is an aggregation of ftilly individualized, independent, separate bei^s, even the cell themselves. Each cell leads a double life: an independent one pertaining to its ovm. development alone; and another incidental in^, so far as it loas become an integral part of a plant,"

Essentially, those words express the principal merit of Schleiden*s work. The greater part of his paper deals v/ith a method of cell multiplication no longer accepted. His idea of cell formation "consists only of a foiroation of cells vd.thin cells." Schleiden did Jiot accept the idea of cell multiplica­ tion by division of previously existing cells. He considered the grovdng across of tiae new partition wall an illusion. For him cell formation was, in the main, an endogenous p3?ocess. S

"A inode of formation of new cells, difforent froia tlae above described (Adaptation of Schleiden's), is ex- Mbited in the multiplication of colls by division of the existing ones; in tiiis case, partition v/alls grow across the old cell; if, as Schleiden supposes, this be not an Illusion, inasmuch as the young cells might es­ cape observation in conseqiience of their transparency, and at a latex* stage, their line of contact would be regarded as the partition wall of the parent cell,"

In evaluating the researches of Schleiden and schvmnn, it is necessai'y to discount the method in vdiich they supposed cells to originate. Spontaneous generation of liviii^ cells is no longer accepted by biologists. Cell multiplication by di­ vision is almost universally upheld. It is also' necessary to realize that tlieir "cell" is not in accordance with the latest expression of the cell doctrine. They both defined the cell as a smaJLl vesicle vAth a firm membrane enclosing fluid con­ tents. To them it was a small chamber, or cell, in the true sense of the word. They considered the cell wall the most im­ portant and essential part of the vesicle, for they believed that its physico-chemical properties regulated the metabolism of the cell. On the other hand, in evaluating that part of the theory whichi still has merit, Schwann's theory as applied to the formation of tissues by differentiation of existing cells is still a fundamental teaching in biology. For that, and for the new direction in the Cell Theory directed cytological research, Schleiden and Schwann will probably al­ ways receive honor. - 26 -

Althou^i tho "Cell Theory" v/as at once vddcly adopted aa a fundamental proposition in biological research, it soon mi- derwent radical reform. It was especially desirable to clear up the matter of cell origin. Botanists of tlrnt period v/ero tho fii^st to extricate themiselves from the eri^ors into vAiich Schleiden had lead them. In 1835, von Molil (370) had already described multiplication of cells by division, but Schleiden had ignored this observation. Meyen (361) two years later made the first attempt to distinguish cell-division from the free cell-formation described by earlier \rorkers, but his con­ clusions had not yet become widely known before Schleiden pub­ lished his theory. Unger (558, 559) and von Mohl (S71, 373, 372, 374) followed Meyen's lead and vdth Kageli (390, 391) completely overthrow Schleiden's conception of free cell-for­ mation. Kageli elaborated a broad tlieory which took into ac- covint all tlie data at hand. He distinctly defined cell divi­ sion and shov/ed that #iat many workers had taken for free cell- fomation was really a special case of division. In consequence of this, a general law was formulated in 1846 for plant cell multiplication; that nev/ plant cells come only from cells al­ ready present, and tliat this occurs in such a manner that the mother cells are divided into two or more dauj^ate:? cells. Progress v/as slower in zoology, mainly because some work­

ers on aniiTial cells continued to modify or elaborate on Schwann's explanation of cell multiplication vdthout realizing that the - 27 -

fundamental foundations of tholr tlaeory v/ore In error. Kar- sten (285, 286, 287) stated that cells originate without a pre-existing nucleus, and arise from free vesicles in pre­ viously existing cells. Paget (404), too, believed a cell might arise in some fashion vrithout the nucleus playing a jpart in the process. Henle (243, 244) accepted Schleiden and Schwania's original idea, but made exception to the universal­ ity of its acceptance by pointing out tv/o other ways by vhlch cells might multiply: by budding ("Sprossen"); and by divi­ sion ("Thellung"). Bergmann (46) and Kollikor (296, 297) be­ lieved that segmentation in the ova of animals was a form of cell division, as suggested originally (1840) by Reichert (466). Bennet (45), as late as 1855, upheld a theory of spon­ taneous origin of aniraal life, including views not essentially different from those of Scliwann. A year later, Todd and Bow­ man (551) still admitted free cell-foiroatlon as one mode of origin, though their spontaneous origin of cells is modified by the explanation that excreted granules first cohere to form a nucleus around i-ihtich the new cell forms. Finally, after many mistakes, the views of Reichert (468, 467), Remalc (479, 480, 478), and Virchow (569, 568) were ac­ cepted by the majority of zoologists. Vlrchov/ in 1858 origi­ nated the "formula", "Omnia cellula e oellula". an application of an earlier maxim, "Omne vlvum ex ova". According to Vir- chow, the cell is the only possible starting-point for all bio­ - 28 - logical doctrinoa. This cell can originate only from a pi-e- viously existing cell, vdiich, in turn, has its primary origin in an ovujn, Ko spontaneous generation of cells occurs either in plants or animals. The many millions of colls v/Mch malce up tlae "body of a vertebrate animal or a tree, for exaanples, liave been produced by the repeated divisions of the one coll in which the life of the animl or plant began. It is not at all strange that Virchov/'s plainly stated facts did not convince all zoologists, Nearly forty years passed before bitter and lengthy controversies concerning Vir­ chov/'s theory began to appear less frequently. Singularly, Carpenter (83), in 1865, continued to adliero to the free eell- forjnation of Schleiden and Scto/ann, but his was a unique po­ sition. ITeai'ly all biologists of that time, as well as of to­ day, accept Virchov/'s dictum with same few sli^t modifications. Occasionally even today, however, one finds papers expressing aljnost complete disagreement to Virchow's idea, even as later modified. The "typical cell" of this period was described as con­ sisting essentially of a cell-v/all, cell-contents, and a nucleus. The nucleolus v/as not considered an essential con­ stituent. A few years later, Virchow concluded that the cell- wall was not an essential part and defined the cell as a "nucleus surrounded by a molecular blastema" (570). In those cells in which a cell-v/all was not clearly defined, its place - 29 - v/as taken by a consolidation of the outer portion of the cell blastema. Presumably, this stnicture corresponds to the mod­ ern "plasma membrane". This later conception (that the cell vmll was not an essential part) of Virchov/'s did much to shift the attention of cytologiats from tlie cell wall to the cell contents, vsith the result that the Cell Theory, as such, under- v/ent radical reform and was superseded in attention and inter­ est by the "Protoplasm Doctrine", associated mainly with the name of Max Schultze.

B. The Pa^otoplasma Doctrine

As early as 1772 Corti (106) had observed movements in the "sap" of Gharaceae and some other plants. This jdienomenon v/as also seen by Fontana (150) in 1781. After being forgotten, it vfas rediscovered by Treviranus (553, 554) in 1807 and 1811, and by Araici (5) in 1819. Just prior to Schleiden's amoxmce- meiat in 1838, Dujardin (125) called the cell contents of lower animals "sarcode" and described it as having the properties of life, schleiden himself noted the delicate, transparent, gran­ ular substance inside cells and called it "Schleim". These notes were, of course, references, in one way or another, to what we now call "protoplasm". To Purlclnje (451) goes the credit of introducing the word protoplasm into biological writings. Etymologic ally, the v/ord - so - has an interesting background. The word "protoplast" was used in theological v/ritings as a reference to Adam ("first formed"). Purklnje, as a student of theology, v/as familiar with this usage and employed the modified term in describing the material of v;hich young animal fflubs^os are foiraed, and also in present­ ing a nev; picture of living plant cell contents. Ho pointed out that this protoplasm completely filled up the interior of young plant cells, and that in older cells it contained large droplets of fluid. Von Mohl (373) adopted Pxirkinje's term of protoplasm In 1846, applying it only to plant cells, Nageli (392) and Payen (429) again pointed out as Virchov/ had done before that the cell content rather tSaan its wall v/as the im­ portant part of the cell, Braim in 1850 (66) contributed a clever observation v/hen he reported that swam-spores, which are cells, consist of naked protoplasm. This stimulated a series of researches on zoological units such as eggs, leuco­ cytes, lymphocytes, chromatophores, etc. Kuhne (308, 309), Reichort (470, 469, 471), Henle (242), Huxley (274), Schultze (511, 512, 514, 515), Muller (385), Siebold (530), Kolliker (298), Leydlg (322), Bischoff (53), and Lieberlcuhn (323) pub­ lished Important papers diAriiig tills period, and, by degrees, came the realization that the "sarcode" of animal cells was identical with the "protoplasm" of plant cells. Cohn (104) in 1850, Prlngsheim (445) in 1854, and Unger (560) in 1855 were among the first to express this opinion. 51 -

Reitjak (4V9) definitely applied the word protoplasm to tlie sub­ stance of nnimal cells., Subsequently, de Bary (29, 31) and Schultz (513) proved, after careful investigation, that tlos protoplasm of plant and higlier animal cells was the same as ttie substance (for wliich the name "sarcode" had been retained) inside the lower animals such as protozoa. The work of SchTiltse was especially notable in tliat it firmly established the P3?otoplasm Doctrine, namely, that the units of organiza­ tion are masses of protoplasm and that this substance is, in general, similar in all living organisms. The doctrine was famished with a popular expression by Huxley (275) in his es- say. The Physical Basis of Life. Extensive studies on the nature of protoplasm were soon \ioaertal®n by Haeckel (211, 213, 215, 214, 212), Brucke (70), Duffin (123), Remak (482), Cienkov/sky (102, 101), and Hanstein (237)* More prominent v/ere the structui'al theories associated with the names of IQein (288, 289, 290), Plemmlng (144, 142, 139, 140, 145, 143), Altman (4), and Biitschli (76, 77), and Imown, respectively, as the "reticular", "fibrillar", "granu­ lar", and "alveolar" theories. Many pages have been written in support or denunciation of these theories. Cai'Tioy (82) and vail Beneden (43, 41) were particularly active in giving evi­ dence for the reticular theory. Modern researches have demon­ strated, however, that these theories do not liave tloe signifi­ cance attributed to them. It is nov/ generally accepted that - 32 - protoplasm aliows no single imivorsal type of visible structure ajoi such visible structure as it does have, though of much im­ portance, is not ultimate. The identification of protoplasm as the material basis of life processes was one of the most significant scientific events of the nineteenth centtjry. Once again attention had been shifted to the interior of the organised pi-otoplasmic mass. Althougla the use of the word cell was retained, it no longer expressed the conception given it by the originators of the Cell Theory. The cell vmll, upon which the early work­ ers had focussed their attention, turned out to be of second­ ary importance. There were found protozoa which possessed no cell wall. Under certain conditions even plant protoplasmic units v/ere seen to divest themselves of their prominent limit­ ing membranes and svdJn about in water - as in the case of nak­

ed SY/ann-spores. As a result of these later observations, Schultze, v/ho firmly established the importance of protoplasm, described the cell as "a mass of protoplasm containing a nucleus, both nucleus aiad jprotoplasm arising through the divi­ sion of the corresponding elements in a preexisting cell." This definition was accepted by the majority of biologists of that p eri od (1850-1885). - S3 -

0, The Orj^anlsmal Theory

With tho increase of Icnowledge concerning the role of the cell in development and inheritance, "biologists (about 1890) began to realize that certain limits had to be set for the con­ ception that the cell was always the unit of function and or­ ganization, To strict adherents of the Coll Theoi^y, tlie mul­ ticellular plant or aninal appeared as little more than a cell agS3?®Sate, the cells being the ptrimary individualities. The organism, then, v;as considered as something completely depend­ ent upon the cells' activities for all its phenomena, llov/- ever, ov/ing to the liigh degree of physiological differentia­ tion amorig the various tissues and organs, biologists pointed out that all cells were not alite, or as simple as Huxley had implied in his lectures. The cell v/as not Just a "mass of pro­ toplasm". Cells in an organism were not mere independent tuiits but were really integral parts of a higher individual organiza­ tion, and, as such, would be governed in their function to a considerable extent by the organism as a whole. In addition to this line of x'easoning, discoveries in many fields made the cell and protoplasmic theories inadequate as explanations of life phenomena and organizations. Bio­ chemists shov/ed that tlie protoplasms of plants and animals were not the same, though their attributes might be Identical, no matter their soui'ces. Animal protoplasms, themselves, dif­ - 34 - fered: cr-ystals of hemoglobin and hemoglobin derivatives from the blood of one animal v;ere not like the corresponding crystals from others; blood precipitin reactions showed that there were differences; inter- and ii^traspeciea protein sen­ sitivities existed. Physiologists demonstrated how hormones from one small area affected many different types of cells in an organism. Anatomists pointed out how tlie nervous system served as an integrating mechanism for tlie entire body, so that cells v/ere not Isolated units in themselves. Some cytol- ogists made jsreparations showing cells connected by protoplas- niic bridges; others fotmd the Cell Theory lacking in their at­ tempts to make plausible interpretations of plasmodial or coenocytic organisms. Pro10200logists deinonstJTated the complex­ ities of some protozoa in th.elr possession of differentiated areas for quite specialized fianctions. Embryologists noted that an immense amount of development and differentiation oc­ curred in organisms v/ithout any coll formation whatsoever. In eggs of many animals visible regions v^ich later develop into certain organs are set apart before the egg divides, or even before fertilization. As a result of the discovery of these facts, a now con­ ception of multicellular organization was established. ICnown by various names such as "Plasma Theory", "Tissue Thoo3?y", "Plasmodial Theory", or "Organismal Theory", this concept placed emphasis on the living mass as a whole, rather than on - 35 -

Its constituent parts. Ontogenesis was Intoipreted as a fixnc~ tion of the entire organism, end consisted in growth and dif­ ferentiation of a single imit vdth or without septation of the living mass Into subordinate aanl-independent parts, Tlius de­ velopment was not viewed as an association of multiplying un.1.ts to form a new whole, but more as the resolution of one organism into newly formed parts. Development and grov/th v/ere not thought of as a multiplication and cooperation of cells, but rather as a differentiation of pi'otoplasm. Continuity of these differentiated areas was maintaiiiod by actual protoplas­

mic bridges, by ne3?vous connections, by hormonal diffusion, or by some other circulatory mechanism. The living body resorb- ed itself into specialized centers of action by moans of which the physiological division of labor was effected. To discuss fully the history and development of the or- ganlsmal theory does not ccxne within the scope of this paper. However, it might be well to point out that proponents, such as Ritter (486) and others, see in some of Aristotle's (9) vn?lt- ings the beginning of this concept. After tlie Llnnoan era, Guvler (112) and Saint-Hllaire (499) made much of this prin­ ciple. Delage (114) in his book on heredity, 1903, associates Von Baer (17), Bernard (49), Bichat (51), His (5254), and Pfluger (433) with certain embryologlsts vfao found the cell- theory not entirely satisfactory, Proin 1840 to 1890 tlae or- ganlsmal theory lay dormant, but slaortly before the beginning ~ 36 -

of this centxH'y, a revival of its principles v/aa brought about by a number of aoologists, particularly the Americans G. C. Vfloitman (588, 589), E. B. Wilson (593, 594), and P. R. Lillie (324). Rltter (486) also lists Goebel (181), Radl (453), Pfeffer (431), Jost (283), Nusbaum (399), Schultz (510), Rand (454), Holmes (263, 264), and Zeleny (609) as supporters of the reanimated conception of organismal organization. Other v/orkers vAio have had some connection v/ith its reorganization are De Bary (30), Sachs (498), Heitzraann (240), Hertv/ig (250), Sedgi'^ck (518, 519), Heidenhain (238), and Dobell (117, 118). In addition to Ritter (486), the most prolific recent v/rlters in support of the organismal viewpoint have been Rauber (463), Schlater (502), G\arv/it3ch (187, 188), Rohde (492), and Lotsy (329). Rohde, in particular, has made an excellent systematic study of many klMs of plant and animal tissues to collect data to prove the Inadequacy of the cell-theory. His paper. Per plasmodiale Aufbau des Tier- und Pflanzenkorpers« publish­ ed in 1923, contains 111 figures illustrating plasmodial structure of a large number of tissues, both in embryological and aduj-t stages. No very recent comparative evaluation of the elemental and organismal viewpoints has been published, but in viev/ of the arguments that have been presented by followers of the lat­ ter idea, it seems rather clear that the principles of the - 37 - cell-theory, even vrlth its modern interpretation, must be al­ tered somewhat to accommodate the undoubted presence of plas- modial or plasmodia-like arrangements in many adult or growing forma of metazoan life. The close study of iixrotozoa has also shown that each individual protozoan cannot be regarded as a 3l235)le bit of protoplasm vd.th either a definite or a diffiise nucleus. Modern research has demonstrated clear and complex differentiation in certain areas of such "single-cell" animals as Diplodinium. Stylonychia, and the commoxa Paramecium, as v/ell as other protozoa. These conflicts, in both pi*oto2oa and metazoa, with the principles of the cell-theory probably vd.ll someday result in a furtlier change of the conceptions regard­ ing structural, developuental, and physiological units of plant and animal tissue organization,

D. Cell Pivision

I Although other biologists probably observed cell division before 1824, the first v/ritten account of tlie phenomerion ap­ pears in that year in an article by Pr6vost and Dumas (444). Their observations were made during the course of segmentation of an egg. Soon after, in 1827, Brongniart (68) noted the same happening in cells from lower plants. Other early observers were Meyen (360), Hirbel (368), and von Mohl (370), In developing their theories regarding the cell, Schleid- - 38 - on (505) and Schv/ann (516) offered also a tiieory of cell or­ igin which was incorrect, "but due to its association with the cell theory was widely accepted. However, the researches of such zoologists as Kollilcer (295, 296), Remak (481), and Vlr- chow (569) and hotanlsta as von Mohl (S75), Nageli (390, 391), and Unger (558, 559, 560), soon began to point the v/ay to the real significance of cell division and its mechanism. These hioloe'^ists accumulated proof that cells arose only by division of preexisting cells, and that the authors of the cell-theory fell into error v/hen they proposed an independent origin of cells out of a formitlve blastema. It was during this time that Vlrchow (568), in 1855, promulgated Ms famous phrase Omls cellula e cellula. To Kolllker (295 , 296), hov/ever, must go the credit of first concluidlng that cell division extended to both plant and animal cells. His conclusions were adopted first by Remalc (481, 480), and later by Vlrchow (569, 570). Other early investigators who helped establish tiiis idea were Bergmann (46), Blschoff (53), Goodsir (182), and Barry (28), Heider (239) gives an excellent review of tMs particular period. With further vrork it soon became evident that nuclear di­ vision was of tv/o types; direct and indirect. Pleramlng (140, 145, 141) sunnaarized this evidence, and, later. In 1882 (144) suggested the more appropriate terms mitosis (Indirect) and amitosls (direct). About the same time Schleicher (503, 504) - 39 - devised i:.lie word karyokinesis to distinguish the indirect raode of division from the direct. At the present time, mitosis and Jiaryolcineais are used synon^ously. Originally, both of these words referred only to the division of the nucleus, but were soon talcon to mean the division of the entii-e cell. VJhitman (587) introdxiced the tern cytolcinesis in 1887 to designate the associated changes taking place in the cytoplasmic cell-body,

but the Y/ord has not found wide usage. He also suggested other terras to designate the distribution of additional smal­ ler cellular elements during division. For chondriosomea he introduced chrondriold-nesis; for golgi-bodies, dictyokinesis, and so on. To avoid confusiai from the usage of various term­ inologies, Wilson (596) published the following liseful outline:

I. Mitosis (indirect division) 1, Karyolcinesis (nuclear traxxsformation) 2, Cytolcinesis (cytoplasmic changes) a. Cleavage (division of cytosome) b, Chrondrioklneaia, etc. (meristic divi­ sion or distribution) "II, Amitosls (direct division)

Additional notable lilstorical and terminological contri­ butions to the early discussions of cell division were made by Waldeyer (576) in 1870 vdien he gave tlie name cliromosomes to the darkly staining bodies so paraminent during some phases of mitosis J by Reraak (477) who first saw the aster bodies; by von Baer (18), Virchow (569), Kowalev/aky (300), Fol (146, 147, - 40 -

148), and Auei'back (16) who figured and described the changes in the aster "bodies during division. The last tv/o men present­ ed especially iiiportant papers on these bodies: Pol with Ma studies on the eggs of medusae and mollusks; and Auerback on nematods, Fol (149) and Mark (538) also incliaie excellent suimuariea of this period in the study of mitosis. During the histoid of cell division a great deal has been written regax'dii-jg the relative frequency and significance of karyold-netlc and akaryokinetic divisions of cells. Remak (481) and his iimnediate follov;era during the middle of the nineteenth century regarded the latter process the typical mode of cel­ lular multiplication. Van Beneden (40, 42, 44) in 1876 re~ opened the topic, drew a shaip distinction between mitosis and aiiiltosis, and gave his opinion that the latter was atypical and rare, and represented only a nuclear "fragmentation". Pi£m~ vier (455), Arnold (11) and otliers thought true amitosis to be a rare occmn^'enoe. Prom an evolutionary standpoint, Stras- burger (538) and Waldeyer (577, 578) believed amitosis v/as a primitive type of division, from which mitosis had been deriv­ ed. A reverse conclusion v/as reached during the last decade of the late centiAry by Ziegler (611, 612, 613, 614), Flemming (145, 142, 140), and vom Rath (461, 459, 462, 460) who consid­ ered direct division a secondary and simplified type of divi­ sion which acconpanied extreme specialization of cells and vAiich symptomized apioroaching degeneration and death. Plemming - 41 - states: "it (direct division) is a process vailch does not lead to a now production and raultiplication of cells, iDut wherever it occurs represents either a degenera­ tion or an aberration, or perliaps in many cases (as in the formation of multinucleated cells by fragmen­ tation) is tributary to metabolism through the in­ crease of nuclear surface."

In tliis direction Plemming sought an explanation of the fact that leucocytes may divide eitlier mitotically or amitotically* In normal lymph glands, v/here new lymphocytes are formed, mitosis is the prevailing mode, according to Arnold (11, 12, 13, 14, 15). Elsewhei»o, both processes occur. Vom Rath in particular emphasized that cells which divided amitotically were "on the road to ruin". In general, workers of this opinion also held that amitosis in higher foms v/as not a sur­ vival of a primitive process of direct division of the type found in some protozoa. Later views on mitosis versus amitosis turned in another direction. Amitosiii was found to be especially frequent in transitory tissues such as vertebrate decidua, embryonic en­ velopes, periblast of meroblastic ova, accessory nutritive cells connected with developing gem cells, and the like. In some cases it was ^o\m that the two types of division fre­ quently occur normally side by side in some embryonic tissues, Dogiel (121) found a striking example of this in the strati­ fied epittielium from the bladder of the mouse, nuclei of the - 42 - more superficial layers regularly divide by amitosis, givixig riso to large bi- or mulldLnucleate cells v/liich finally degen­ erate and are cast off, while the new cells that talce their place are alv/ays formed by mitotic division of cells of deeper layers. Still later, it was proved that the occurrence of amitot­ ic division of a nucleus by no means precluded a subsequent re- sTjmption of mitosis. Meves (357, 358) found that nuclei of tlie spermatagonia of a salamander may split amitotically at certain seasons of 1±Le year, and, presumably, give rise to nor­ mal sperm. This observation v/as confirmed by McGregor (342) in .Amphiuma, Other examples supporting this conclusion, though not all found in reproductive cells, were seen by Bordeen (237) in cells of regenerating planaria; by Child (97, 91, 92, 93, 94, 95, 96) in spermatagonia of cestodes, developing hydranths of hydroids, regenerating platodes, larvae of amphioxus, and cells of chick embryo; by Patterson (427) in developing pigeon eggsj by Maximow (341) in embryonic tissues of various animals; by Nowikoff (398) in coimective tissue; by Wiemann (592) in early germ cells of a Leptinotarsa beetle; and by Cillends (103) in regenerating uterine epitheliixm of maimnals. Child (96) concluded tliat amitosis is on "important factor in grov/th in many organisms, and in some cases at least, either form of division may be changed into the other by altering the condi­ tions," - 43 -

However, recent examinations have shoxvn tlaat many of the ao-oalled cases of amitotic division were really mitotic divi­ sions in Y/hich the chromosomes liad been distributed along the spindle fibers, or cases in which the mitotic figiires had been very small and overlooked. Indeed, many of the protozoa - which had been pointed to as classic examples of dialect divi­ sion - have been shown to divide mitotically. Pacts now in­ dicate tliat amitosis often means only a fragmentation of the nucleus, usually only temporary in nature. Earely is nuclear fragmentation followed by cleavage. As a result, bi- or mul­ tinucleate cells are formed. These are often seen in cultures of chick embryo tissue. In only few cases, hov/ever, has ami­ totic division of the nucleus been found to be followed by di­ vision of the cytosome. Cytoplasmic cleavage of binucleate cells takes place after a subsequent mitotic nuclear cleavage, or division. Macklln (332) observed such cells and sav/ tliat the two original nuclei fuse together and give rise to a single group of chromosomes associated v/lth a single spindle. At tihe present time, amitotic division Is regarded as oc­ curring much less frequently -Qian it was formerly thought to happen. mm

E, Insect Hemolympli Cells

1. General literature. References to iaemolymph cells of insects are rather nm- erous and,have accixmulated rapidly since 1900, particularly in connection with, bacteriological and immunological investiga­ tions, some of v/hlch vriLll be referred to later. For the present, only th.e more important papers dealing with pTOblems relating to the moj^pliology of the cells v/ill be considered. Apparently the first mentioxi or sketches of insect herao- lymph cells were inclxxded in one of Swacmerdani's papers (542) in which he gives a short description of ameTxjid cells from a dog louse. Not until 1326 was the existence of honol^ph cells noted in a regular entomology text, when Kirby and Spence (292) v/rote of "globules" to be found in the body fluid of in­ sects, Tiventy years later Kev/port (394) compared vertebrate blood cells with insect cells, and tv/enty years after that, Weismann (582) made some observations on heraolyraph cells from Mtisca vomit aria and "Sarcophap:a carnaria. During tiie next two years, 1864-65, the Landois brothers (512, 313, 314) published a series of papers on homolyniph cells from a noctuid moth, Ap:rotis seaetum, from Pontia bras3icae« Smerintlius populi. and othor forms; one paper included a short classification of cell types and will be referred to more fully below. In 1871 Gra- ber (183) also attempted to select cell types for description. - 45 - and later (184) v/rote another paper In wMch he discussed the circulation of homolympda cells tiirough the Insect's body. Dur­ ing the next tv;enty year i^eriod Wielovd.ejski (590) reported on the cells of Coretlira plupiicornis; Poulton (442), on cells of some "phytophagous larvae"; Balbiana (SO), on cells from a series of arthropods, including some insects; Wielowiejslci (591) made a more extensive study of cells from bees, wasps, and bum­ blebees; Kowalevsky (301) studied cells from various fly larvae; Miall and Denny (36S) wrote a book about the Oriental coclcroach and mentioned that its hemolymph contained cells; Schaffer (501) observed cells from vaidous insects; and Cu6not (108) began a study of a series of invertebrate heMiolyrnphs. Later in 1091 (109) he contributed the first important clas­ sifications of hemolymph cell types based on examinations of Melolantha vulgaris« Gorabus auratus, Blaps mortisap;a. Dytis- cus marg!;inali a, Acridi-um epiyptim. Libellula de-pressa. Cloe biocialata, Cimex rufipes. Hepa cinerea. Rhizotrogus salstiolis, HydropMlus piceus. Locusta vlridissima, Epliippifyer vitium, Aesclma grandis. Hylotoma roaae, Hotonecta p:lauca. and Bombyx trifolii. Between the time of Cu6not's paper and Hollando's 1909 (257) classification of cells from coleopteran hcaaolymphs, otlaer workers making additional reports v/ere Dvirham (128) on Dytiscus mar/?j.nali3 hemolymph; Kowalevsky (302, 303) on some terrestrial Insects; Cu6not (110, 111) on orthopteran hemo- - 46 lymph; Paclcard (402) on various insects; Koschevnilcov; (299) on honeybee hemolymph; Rossig (493) on hemolymph cells from gall wasps J Metalnikov (346, 347) on cells from Galleria mellonella; V/Qis3onbQr£5 (583) on cells from Tor?7mus nipyicornis; and Koll- man (294) on orthopteran cells v/hich he classified and attempt­ ed to compare vdth vertebrate types, Hollande's cell types v/liich he established in 1909 (257) are baaed on examinations from the CoccinoUidae, Chrj''somelidae, and Cantharidae, and are used as originally defined or in a modified way by many writers at the present time when referring to insect hemolyinph cells. After Hollande's work, there has followed a quarter cen- t\3ry of l:nportant contributions to our Imowledge of hexapodon hemolymph cells. Poyarkoff (443) classified cells from certain Coleoptera; Barrott and Arnold (27) studied the hemolymph from Dytiscus marp:inalia and H:ydrophilu3 piceus; Hollande (258) in 1911 made additional studies on coleoptea?an hemolytaph; Stendoll (535) observed cells from Ephestia Imchiaaella; Deegener (113), from various insects; and Teodoro (548) gave his attention par­ ticularly to the floating fat-cells in the hemolymph, and v/hich he called "cerodecytes". Glaser (171) in 1917 Introduced a new type of study by ^^slns the hemolymph cells fran Malacosoma amorlcanum. Girphis unipuncta, Laphygma fru^iperda. and Fort- he tri a dispar for "in vitro" InvestJ-gations. In his book on "Hie Biology of the Dragonflies**, Tillyard (550) includes somis - 47 - reitiarks on the heinolytnpli from these odonatons. Hxifnagel (271) made a short study of tlie heraolymph elements from Hypanomenta podolla, and attempted a preliminary classification of types. In 1920 appeared another paper f3?om Hollonde's series (259); this one a consideration of "oenocytoids" and "teratocytes" from insect larvae. At about the same time, Palllot (413, 415, 414, 420) put forward a nev/ classification of cells based on types from macrolepidoptera in general, and on Pieris brassicae and Eupx''octis clij^ysorrhea in particular. Kreuscher (307) and Schnella (506) followed soon after with classifications of cells from Dytiscixs marfi:inalis and the honeybee, respectively. Muttkowslci (389) formulated a complicated system of cell clas­ sification in 1924 after finding that other systomu were too restricted for TOSO in many species, Muller (386) in 1925 pub­ lished Ms interpretation of cell types in the honeybee. Apis , melliflca^ after making a rather thorougli study of that insect j in its various stages and under different patliological condi- ; i tions, as v;ell as normal, V/itliin the past ten years, Iwasaki (277, 278) employed hemolymph cells fi-'om Gallerla mellonella larvae for a cytolog- ical problem involving stimulation of mltosla in the insect's body; Haber (206) used cells of the body fluid in some of his physiological researches on Blatella f2:ermanica; Tateivrei (544) suggested changes in the nomenclature for cells from Galleria mellonella; Yeager and Taubor (604) employed hemolymph cell cotints in a fonmla for determining fhe body hemolympli voliime of Peri-jjlaneta fuliji^inoaa; and in the same year, 1933, Yeager, Slmll, and Farrar (603) described the changes tmdergone by cells from Blatta orlentalis dtiring homolymph coagulation. Very roceixt works include a book by Paillot (425) in which he revises Ills previous cell classifications; a terit by V¥eber (581) in which is included a partial review of the more import­ ant papers on insect hemolymph; a text by Ijiiras (276) v/hich ac­ cepts Ilollande's classification of 1911 as satisfactory^ papez's by Yeagor and ICnight (605) who distinguish various types of hemolyiiiph coagulation, depejiding on the part played in the process by the cells, tlie plasma, or a combination of the twoj by Houghton (269) who believes the/t heinolymph cell types or stages from invertebrates correspond to certain cells found in vertebrates, and. that the insect and annelid also contain additional types; by Yeager and Tauber (606) on mitotically dividing colls in the hemolymph of Blatta orlentalis; by dauber and Yeager (545, 546, 547) on total hemolymph cell counts from a largo nxunbor of insects; and by Fisher (138) on the effects of several anticoagulant procedures on total hemolymph cell coimts in Blatta orlentalis. Taylor (547a) used Periplaneta americana for studies on the origin of hemol;ymph cells. 49 -

2, Glaaglficatlon of cells. Aa early as 1345, Newport (394) made the first attempt to olasaify invertebrate liemolyinph cells, including some from in­ sects. In svmcnarizing his v;ork, he decided that there v/ere at least four types of corpuscles, some of which he compared to the corpiiscles of human and otlier vertebrate blood. His des­ criptions, for the most part, are iimdequate for obtaining a clear conception of his typos. The first ho called a "molecule' the second, a "nucleated or oat-slaaped corpuscle" which he con­ sidered analogous to vertebrate v/Mte cells; the thii^i, a "spherule" wliich he thought was a free nuclooltis released from a disintegrating corptiscle; and fourtli, a "disc" which he be­ lieved finalogous to the vertebrate red corpuscle. Cu6not (108, 109) in 1891 made the soconcl important attempt to classify in­ sect hemolymph cells, vdiich he called "amibocytes". He con­ cluded from Ms studies that insects could be sepai-ated into two categories depending on the typos of cells found in the body fluid. The first group included lopidopteran larvae vdiose amebocytes had been transformed into reserve cells; the second v/ao made of all otiier insects, larvae or adiilts, that possessed typical araebocytes. Using the cells of Bombyx tri- folii as chai-acteristic of insects in tlae first group, Cu6not described and figured three cell types: (1) Sroall normal amebo­ cytes vd.th fine refractive granules;(2^ Large reserve amebo­ cytes with large granules; (S) Small non-ameboid cells contain­ - 50 ing needle-shaped ci^-atalSk The typical amebocyte found in other insects of the second categoiy was a cell from 10 to 20ii,, vrlth a few larger in the Orthoptera^ It contained small re­ fractive granules, especially numerous around the nucleus; its sliape was either spherical or fusiform; and when degenerating became vacuolated* Gufenot apparently soon realized that this simple classification was inadequate for many insects and of^- ferod another (110) grouping of cells based on or'thopteran hemolymph elements. Pour types were recognized; (1) Young amebocytes; (2) Phagocytic amebocytes; (3) Acidophilic amebo- cytes, or corpuscles with small acidophilic granules and which are undergoing initial stages of degeneration; (4) Degenerating amehocytes. These four forma were interpreted as representing distinct stages in the evolution of a single initial type. In 1908 Killman (294) accepted in general this latter clas­ sification of Cu6not's, but considered the cells called oeno- cytes by YJielowijalci (591) as a form of hemol^ph cell, v^eroas they actually are a fixed cell and are not normal elements of insect hemolymph. This mistake made by Kollman and others has resulted in much confusion in cell type nomenclature. Metalnikov (347) based Ms classification on cells from the larvae of Qalleria mellonella^ in v4iich he found foijr types of cells: (1) Small leucocytes; (2) Large phagocytic leuco­ cytes, p3?obably an older stage of the aniall leucocyte; (3) Large granular leucocyte with vacuolated cytoplasm; (4) Large leuco­ - 51 - cyte, rare, with a homogeneous cytoplasm, Tlie first classification of dlpteran hemol^nnph cells was furnished by Berlese (47) in 1910. Based primarily on Muscidae it is as follows; (1) Amsbocytes, of embryonic origin; (2) Splanchnocytes; (3) Sarcocytesj (4) Steatocytes, or cells vjhich detach themselves from the fat deposits of the body. In the same year, Poyarkoff (443) made an examination of coleopteran body fluid at the time of metamorphosis and found six Idnds of cells, excluding any wandering "fixed" cells. The types were: (1) Cells vdth "spherules"; (2) Phagocytic leucocytes; (3) Young leucocytes; (4) Crescent-shaped cells; (5) Myoblasts; (6) "Oenocytoldes". Two other coleopterans, Dytiscus marginalis and Hydrophilus piceus. were studied by Barrott and Arnold (27) v/ho believe only two types of cells are present in the hemolymph. These are the phagocytes (17 to 19ii in D. marglnalis, and 15 to SOti in H. pioeus), and the "small round oella" which are less numerous than the phagocytes. During this same period Ilollande published several papers on the hemolyinph of Coleoptera. In 1909 (257) he suggested a simple grouping of cells into (1) Lymphocytes, or large cells v;ith strongly acidophilic cytoplasm and not phagocytic; (2) Leucocytes, or smaller, spindle-shaped cells, phagocytic, and inclined to syncytium formation; (3) Cells of a size varying from 5 to S2ji with inclusions, sometimes yellow. Two years later (258) Ilollande completely revised his classification to - 52 - such an extent that the tv/o arrangements of types must be con­ sidered separately. Five kinds of colls were recognized In the revlsionj (1) Proleixcocytes, or yotmg leucocytes which give rise to other types; (2) Phagocytes, or spindle-shaped cellsJ (3) Granular leucocytes, or cells with either acido­ philic or "basophilic granules; (4) "Oenocytoldes", or rounded, non-phagocytic cells, imrkedly acidophilic; (5) "Spherule" cells, or cells wMcsh are not constsaitly present, but appear in the hemolymph picture under abnormal conditions. Althou^ Tillyard (550) made only v/hat appear to be pre­ liminary observations on dragonfly hemolymph cells, they are important because they seem to be one of the few studies on odonatan body fluid elements. He distinguishes tv/o kinds of corpuscles: miocytes and amebocytes. Miccytes are described as oat-shaped; amebocytea as Irregulai' in shape. He states, however, that "imder certain circumstances the shape of the elongated cell is known to alter"; and, "it seems possible that the oat-shaped coipuscles and the amebocytes are only var­ iations of a single form of corpuscle". Hufnagel (271) has carefully worked out the hemolymph picture of a lepidopteran, Hyponomenta podella. and finds five varieties of corpuscles: (1) Proleucocytes; (2) Young leuco­ cytes, somewhat phagocytic; (5) Old leucocytes, a much advanced stage of the young leucocytes; (4) Cells with fat inclusions, abundant at emergence and probably a modified leucocyte; (5) 53 -

Granular leucocyte. Beginnings In 1919 Pail lot started a study of heinolympli cells v/Mch has continued to the present time. His first im­ portant systematic paper (413) was en the hemolymph of Macro- lepidoptera larvae. In general he accepted Hollande's scheme, but proposed specific descriptive names for the cell types, of which he recogniised five: (1) Mcronucleocytes, phagocytic and slightly hasolphillc cytoplasm; (g) Micronucleocytes, with eosinophilic granules; (3) Macronucleocytes, (a class to in~ elude both the phagocytes and proleucocytes of Hollande) divid­ ed into three subgroups of small macronucleocytes, fusiform macronucleocytes, and large macronucleocytes; (4) Cells with "sphj&rules"; (5) "Oenocytea". Other investigations of addition­ al groups of insects by Paillot will be referred to in their chronological order. Amoiig the more recent classifications has been that of Muttkov/ski's (389) who believed a revision was necessary be­ cause previous systematic schemes had been based "unfortunate­ ly on what is foiond in a single genus or femily and less on comparative studies". His arrangement, presented in 1924, is rather complex in its subdivisions due to an attempt to make the scheme fit all the different types of cells fotind in many genera such as Aeshna, Libellula, Enallapjna among Odonataj Dytiacus, Hydrophilus« Leptinotaraa. Prlonus and other Coleop- P5.eri3 and other Lepidoptera; Dlssosteria^ Melanoplus and 54 - various other Locustidae; Beloatoma, Motonecta among Hemiptera; Leptocercua and Phryf^anea aiaong Tricoptera; Sta^atiOEiyia. Odontomyia, muscoid larvae, etc. among Diptera; smd various sav;fli0s and bees among Hyinonoptera. Two types of leucocytes v/ore found by Muttkowalci to be prominent in adults of the above forms, and were recognizable in the various species by both tlieir size and staining reactions with aniline dyes. The first type of chromophil leucocyte is a small rounded corpuscle with a marked cytoplasmic affinity for aniline dyes; the second type or amebocyte is a largei', spindle-shaped corpuscle v;hich

reacts faintly V7ith the same dyes. The latter amebocytes orig­ inate from the chroraophil leucocytes; are highly ameboid; and have a small nucleus. The chroraophil leucocytes are embryonic in natiire; have short pseudopods; and a large nucleus. Various types can be recognized according to activity: (a) Secreting chromophils v/ith a "terminal mass" of "cytoplasmic bodies" at one end; (b) Transporting chromophils with numerous external fat bubbles, especially during feeding periods; (c) Phagocytic chromophils which are of two types, small phagocytes, and larger granular spheres derived from the small phagocyte; (d) Splanchnocytes or corpuscles which penetrate tlae intestinal coat to replace the intestinal epithelium; (e) Degenerating leucocytes v/ith acidophilic granules. In addition to the above, Muttkowski also finds a number of other tissue cells or pro­ ducts in the body fluid, but these are not true hemolymph cells - 55 and will not be considered here. Uains the components of the hemolyraph of a Gerrld, Limno- trechus» for a type, Polsson (437) described seven kinds of cells: (1) Proleucocyte; (2) Youiig leucocyte; (3) Old leuco- C7/te; (4) Leucocytea containing fat globules - a cell type ?/hich Poisson states he saw transform into adipocytes of the fat body; (5) Leucocytes vdth granules - like No.4, but vrlth granules;(6) Leucocytoid ocnocytes, rare in adults but abun­ dant in older nyiuphs; (7) Seleniform elements, which are some­ what like Hollaixle's embryonic forms. Another recent classificatioix came out of Muller's (386) careful examimtion of the homolymph elements of five hundred honeybees (Apia mellifica L.). He finds that only three types of corpuscles are present; (1) Leucocytes, or gran-ular, phagocytic, ameboid cells wliich may be round, oval, spindle- or egg-shaped; (2) Embryonic or formative cells, ("Bildungs- zellen"), or non-granular, large-nucleated corpuscles which are young leucocytes; (3) Small rouiod cells, or tiny, rare cor­ puscles v/ith constant round shape. LaiTvao of Gallerla me Hone 11a have been used as a source of hemolyraph for many studies of corpuscles in insects. In preparation for a series of experiments involving the analysis of shifts In the hemolymph picture after subjecting the larvae to different conditions, Iwasalci (277) deTlsed his ovm clasai- fication of cells based on exajnj.nations of fresh and stained — 56 -

preparations. Ho describes, "but does not name, four cells; (1) Rotmded, non-granular, large-nucleated corpuscles; (2) Rounded, granular, sniall-nucleated corpuscles; (3) Like the second type with retictilar cytoplasm and small acidophilic granxiles; (4) Ellipsoidal, non-granular, small-nucleated cor- ptiscles. In 1931 Hamilton (236) follov/ed Poisson's classification of 19S4 in describing cells from the wator-scorpion, Kepa cinerea. He finds: (1) Proleucocyte; (2) Young leucocyte; (3) Old leucocytes; (4) Iieucocytes v/ith granules; (5) Seleni- form elements. The latest classification and sumsimry of Insect hemolymph corpuscle types v/as made by Paillot (425) in 1933. He points out that neai'ly all authors agree that most of the cell types in hexapodan hemolymph are stages in the development of an in­ itial element characterized by a nucleus and a small periphery of cytoplasm, such as the proleucocyte of Hollande. After studying homolymphs of many forms, particularly in Macr'o- lepidoptera, Coleoptera, and Hymenoptera, and evaluating the published accounts of other workers, Paillot concludes that there are two general types of corpuscles v/hose size may vary according to species, but v/hose morphological characteristics are constant. These types are (1) Mlcronucleocyte, a phago­ cytic corpuscle v/ith a small nucleus and cytoplasm which stains a faint blue with Giemaa's stain, and which may contain reserve - 57 - inclusions or fino eosinophilic granules; (S) Macronucloocyte, or non-phagocytic cells with a large nucleus and a cytoplasm which stains a deep blue v/ith Giemsa's stains. All other cells are considered hy Paillot aa bein^ modifications of one or the other of these types. The only exceptions are cells he calls "Oenocytoides" wMch appear normally in the hemolymph of all larvae, make up ahout five per cent of all cells, and wMch are round and contain a large amount of cytoplasm. In suimnarizins the literature on liemolymph cell classifi­ cation, the difficulties of nomenclature v/liich present them­ selves while working in this field may best be stovni by means of a chart, as given below (Table I). In the horizontal spaces (I) are placed the names, reference numbers, and piiblication dates of the various authors; in the next spaces (XI) are given the source material studied; in (III), (IV), (V), (VI), and (VII) are placed the synonymous names of the various cell types, determined as closely as possible from published descriptions; in (VIII) ai'e listed additional cell types which, for the most part, have questionable or no synonyms; in (IX) are placed any notes, regarding cell types, v/hich couM not easily be includ­ ed in other spaces in the table. I ; Wewport Cu^bt (108, Cu^not (110) Metaliiiltov Hollande : ; (394) 1845 109) 1891 1895 (347) 1908 (257) 1909 : II : Invert0- InsectB in Orthoptera Larvae ot Goleoptera ; • bratos In general, with in general Galleria in general ; tim general partlculai' mellonolla 3 reference to larvae of Bont- byx trifolll III 1. ? : 1. Small hor- 1. Ifbvihg 1, Small 1, Lyin- : (Moleciile) lajal amebocyte amebocyte leucocyte phocyte ; IV 2. Kuelo- 2, Large 6. Phagocytic 2. Large 2, Leuco- : ated, or reserve amebocyte phagoQytic cyto : oat-shaped amebocyte leucocyte corpuscle V 3, Acidophil­ 3. Large 3. Cell : ic amebocyte grantilar with in- ; leucocyte clus ions :

VI 4, "Spher­ ule" cell

VII 5. Large non-gran- ular : leucocyte

VIII 3'. "Spher­ 3, Small non- 4« Degene­ ule" J araeboid cell rating 4. "Disc" amebocyte :

IX No.2 above Wo,3 above Includes also in­ llos.2, 3, 4 cludes Ko.4 of Gu^not, of Cu^not, 1895 1895

r •

- 58 -

Table I

Oompariaon of Komenclaturo of Insect Ilemolymph Cell Typea

>v Hollande Berlese (47) Poyarkoff Barrott, Ar­ Hollande Tillyard )8 (257) 1909 1910 (443) 1910 nold (27) 1910 (258) 1911 (550) 191 li" Coleoptera Diptera, witia Coleoptera £iy-feiacua Coleoptera Odonata in general particular in general marginalis in (ijeneral in genera ja reference to the Muacidae

1. Lym­ 1. Splanch- i, Yotaig 1. "Sinall 1. r'roleu- 1, Amebo- ;e phocyte nocyte leucocyte roiand cell" cocyte cyte 2. Leuco­ 2, i\inobocyte 2, Phago­ 2. Phagocyte 2. ].'hago- 2, Miocyt ;ic cyte cytic cyto :e leucocyte

5. Cell 3, Sarcocyte 3. Cell 3, Granular « with in- with loueocyte ;e cluaions "Sphe'riiles

4. ''opher- LI \ac" cell

5« "Oeno-

1 cytoide" O

4. Steato- 4. Crescent cyte aliaped eel], 5. Myoblast 6. "Oenocy- toide"

/e No,6 above No.4 above No.l aboi i* also in­ also in­ also in­ 0.4 cluded in cluded in cludes N >t, No.l of No.3 of of HcQlffl Hollande, Hollande, 1911; ai 1909 1909 No.5 of Hufnagel 1 1918

ph Cell Types

Tillyard. Hiifnagel Paillot Muttkowaki Poisson {437) Miiller (386) 1 (550) 1917 (871) 1918 (413) 1919 (389) 1924 1924 1925 a Odoriafca Lepidoptera^ Macrolep- Insects in Aquatic Rhiyn- Hoheytjee, 1 in general with partlc-' idoptera general chota, with Apis \ilar refer­ in general particular mellifica ence to reference to Hyponomenta Limnotrechua p^islla «• 1. Amebo- 1, Proleu- 1. Micro- « 1. -i\mebo- 1. Proleuco- 1, i'oiTnative cyte cocyte nucleocyte cyte cyte cell 2, Miocyto 2, Old 2, Pusi- 2. Phago­ 2, Old leu­ 2, Leucocyte leucocyte form macro-' cytes of the cocyte nucleocyte ChromophilL leucocytes ar 3, Granular 3, Large 3, Degene­ 3. Leucocyte e leucocyte macronuc- rating leu-- v/ith granu­ loocyte oocyte lar inclu­ sions 4. Cell 4. "Spher­ 4. Leucocyte 1 with fat ule" cell containing inclusion fat globules 5, "Oeno- 5. Leuoocy- cytoide" toid oeno- cytes

, 5. Young 6. Eosino­ 4, Splttnch- 6, Young 3. Small leucocyte philic mi- nocyte leucocyte; round coll oronucleo- 7. Seleni- cyte; form 7. Small elements macronucleo- cyte /e No.l above No.l above No,7 above '^''Also in­ Ho,6 above is « also In- also in­ same as cludes a. young form Ln cltides No.3 cludes No.7 No,4 of Secreting, •of 130,2 above; of Hollsiid© of Paillot, Muttkowski b. Trans­ NCB,3 and 4 5, 1911; and 1919 1924 porting above are No,5 of Chroaiophil same type H\jfnagel, leucooyteojs with differ- 1918 No,3 also : ont inclu- includoa ; 3ions Ho,4 of • Bamot, 1919• i>olsson (437) MiAller (386) Iwasakl Hamilton Paillot (425) 1924 1925 (227) 1925 (236) 1931 1933 Aquatic Rhyn- Honeybee, Larvae of Water- Many forms, ohota, with Apis Callerla scorpion particularly particular mellifica mellonella Nepa Macrolopido- reference to clnerea ptera, Cole- LlranotrechuB optera, and Hymenoptera 1. Prolouco- 1. Poamiatlve 1. Rouiicl, 1. Pro- 1, tilacro- oyte cell small nucleus leucocyte nucleocyte 2» Old leu­ 2. Leucocyte 2. Romd, 2. Old 2, Mlcro- cocyte non-graiau- leucocyte nucloocyte lar, large nucleus 3. Leucocyte 3. Reticular 3. Leuco­ .3, Macro- v/ith granu­ cytoplasm, cyte with' nucleocyte lar inclu- acldoflllc granules v/ith "Sphe'r- Biona granules ules" 4. Leucocyte containing fat globules Leucocy- 4. Ellip­ 4, "Oeno- toid oeno- soidal , cytolcle" oytes non-granu- lar, small nucleus Yoking 3. Small 4. YoTAng leucocyte j round cell leucocyte J 7. Seleni- 5. Seleni- fona form elements elements

No.6 above is No.4 above young fom is young •of No,2 abovej forfii of HOB,3 and 4 Ho.2 above,

above are - Nb.5 same same type as Mo.7 of with DIFFER- Poisson, entj inclu- 1924 alona

- 59 -

S. Division and orlf^in of cella. Division - either kaiTyokinetic or akaryokinetic - is generally recognized as occurring among those corpuscles v/Mch moke up the circulating hemolymph cella of insects. Opinion is not unanimous, hov^ever, as to the part played by these di­ visions in replacing cells lost through age, disease, cytol- ysis, or other means. Those investigators who believe tliere are definite cell-forming organs which supplement those di­ visions taking place in the body fluid in maintaining the hemo­ lymph cell population, assign this function to various tissues, usually the pericardial cells. The raa,^orlty of workers in this field believe, though, that these cells are mainly ex­ cretory in nature, as shown by Kowalevsky (301) and others. Most researchers who have v/orked on the classification of insect hemolymph corpuscles (See E,S of Literature Siarvey) record observations of cell division. Among these are Cu6not who noted both mitotic and amitotic divisionsj Metalnikov, mitosis; Poyarkoff who sav/ only the indirect process; Barrott and Arnold who forind atnitosia to occur "comparitively rarely" and mitosis, "fairly common"; Hollande observed karyokinesis; Tillyard noted cells undergoing binary division; and Paillot who saw both methods of division, as did Muller, Muttkowalci, and Iv/asaki, Others mentioned previously (See E,1 of Litera­ ture Survey) who have seen division of on© forin or the other include Kollman, Landois, Miall and Denny, Weber, Yeager and - 60 -

Taubor, Tauber and Yeager, and Yeager, Shull, and Parrar. Taylor (547a) saw mitoses In smears of horaolyiixph from Perl- planeta amerioana and in cultures of heraolympli cells wMcli he kept for several weeks or longer, Haber, alone, reports negatively, on Blatella ^ermanica: "During my investigations I have not seen mitosis although I have rather closely examin­ ed several hiuidred smears." Fevi reports on the quantitative values for tlie number of dividing cells in insect hemolymph have been made. Paillot (420) found tliat injections of Bacillus melolanthae non lique- faciens into Euproctis chrysorrhoea raised the normal mitotic- ally dividing cell rate from three to four per thousand cells to thirty to forty per thousand cells. Iwaaaki (277), using larvae of Galleria mellonella, reports that the normal rate of one to two mitotically dividing cells per thousand cells counted, can be increased (maximum) to six, seventeen, and 156 cells per thousand by injections of peptone, albumin, and bac­ terial eimilsions, respectively. Yeager and Tauber (606), in a prelimina3?y report on mitotically dividing cells in Peri- planeta orientalia published in 1933, state tiae average M.D.C. (Mitotically Dividing Cell) count was found to be 0«51^j, or approximately five dividing cells per thouaand cells counted. Although Cu^not, Kollman, Metalnikov, Hufnagel, Paillot, Deegener, Miiller, Tillyard, Hollande, and Poyarkoff have stat­ ed that some of the hemolymph cells are undoubtedly stages in - G1 - tho devolopTiiciit of a formative cell of some kind, only a few have vontux'-od to express an opinion aa to tho actual origin of these cells, llslson (395), in his study of the cmbryolocy of the heo, states that the corpu-sclos develop from the mesoderm of tho embryo and :miltlply by fission; there being no other method of formation. An^ilaa (439) thinlcs that in the pupa of tho bee aojiie cells are formed from other sotirces, Cu6not and Paillot bollQVO that after the cells have once been released into the body fluid during the emb3?yonic growth of the insect, tlrie hcmolytnph coll population is maintained by div3.sion of tho cells in the hemolymph. Paillot states in 19S3; "v/e think, as Gu6not and more recent authors, that the renewal and multi­ plication of blood elements OCCUJ? by Icaryokinetic division and not by a functioning of a corpuscle fonning organ". Contrary to this statement, Kollmon believes there are definite cell fontiag organs in certain Orthoptera and larvae of Lepidoptera; Kowalevslcy states that the corpuscles come from small "nests" or "islands" of cells near the heart; Balbiana believes they come frora the pericardial cella; and Schaffer, from fat bodies. Yoagor arid Taubor (607) are inclined td believe that most di­ visions occur in the free homolymph cells, Taylor (547a) states "Mitoses occur too rarely in the blood of mature roaches to ac­ count for tho rapid increaaea of aBiebocytes to occtir v;hen these animals are subjected to uiifavorable conditions". Ho believes that Muttlcov/ski'a aniebocyte is the primary cell element and - 6S - gives rise to others "by a secondary cell transformation. The Qitiehocytes, In turn, are furnished by some outside tissue, Snodgrass (530a) suggests that the free cells in adult hemo- lymph perhaps are descendants of the embryonic vmndering vitel- lophags (yolk-eating cells). It is apparent that more investi­ gations are necessary to settle this question.

F, Bacteria in Insect Hemolymph

Pasteur's discovery (426) that the ralcrosporidlan, Moaeroa bombycis. v/as the causative agent for a silkworm disease, pebrine, set in motion a large number of investigations v/hlch have shown the presence of bacteria as well as other parasites in the bodies of many insects. Even v/hen restricted to those bacterial forms which are fouisd in the hemolymph of the hexa- pod host, the list of symbionts and parasites is quite lengthy. In fact, Pospolov (440) is of the opinion that every insect species has its specific Coccobacillus. So far as they concern man's welfare, either directly or indirectly, those bacteria found in insect body fluids con be divided into two classes: those that produce diseases affect­ ing insects of economic importance, such as silkwoms and bees; and those that tend to originate oplzootlcs among injur­ ious insects. In both classes, but particularly in the latter, it has been shown that the infecting bacteria are often normal - 63 -

ayinbionta :ln the hemoljmph of the Insects concerned, "but that •under certain conditions these symblonts change their beliavlor, become very numerous,, and transform into parasites. Such bac­ teria, normally., are believed by some authors to perform use­ ful functions. Mereskovsky (345), Pospolov (440), and others think these symbiotic foims may help to liquefy the connective tissues in periods of ecdyais; or to assist in the accumula­ tion of urates in the fat-bodies and inalphlgian tubes; or even to serve as food reserves when they lose their vitality and cytolyae. Historically, the study of bacterial ontomophytes can be separated into tv/o periods: that from about 1879 to 1911, and from 1911 to the present. Only a few papers appeared during the first period. At its beginning Metchnikoff (356) reported that he believed a disease of the larvae of Anlsoplia austrlaca was due to Bacillus aalutarius. A few years later Forbes (152, 153, 154, 155, 156, 157, 158, 159, 160) recognized a disease of the chinch bug, Bliasus leuconterus, caused by a micrococ­ cus and made additional notes on other contagious diseases. Bloclimann (6S) found bacteria In the tissues of Perlplaneta niTieri'cana and Blatta orlentalls. Krassilstchick (304), work­ ing on the larvae of Galleria mellonella found tv;o bacilli: B. tracheitis sive t^raphitoaia and B. aepticus insectorum. In 1911 tb.e second period in the investigation of entomo- phytes Y/as begun v/ith a paper by d'Herelle (S47) v/ho noted that - 64 - a bacterial disease caused by Coecdbacill'as acrldiorum v/as re­ sponsible for the death of Schistocerca pallens in a certain region in Mexico. He siiggestetl and experimented v/ith the pro­ duction of artificial epidemics as a control for locust plajjues. The su^estion was put into practice over a period of four years, 1913-1916, in Algeria by Sorgent (521, 522) and Sergent and Lh6ritler (520) against Schistocerca perefzylna. V61u and Ban3.n (562, 565, 564, 565) attecmpted the same biological con­ trol methods in Morocco. Du Porte and Vanderleck (439) used a cvO.tore of 0. acridiorum for experimental pm-'poses on a grass­ hopper infestation in Canada. Paillot (425) used bacterial parasites to control several insects, among them being Gal- leria raellonella and Pieris brassica. Artificial dissemina­ tion of bactei^ial disease was tried as a control more recently against the corn-borer by Metalnikoff and Chorine (354, 355), Husz (273), and Chorine (99). Results of these field tests, hov/ever, have not been in agreement as to their vwrth in fi^^t- 3.ng insects. Some of the additional bacterial species which have been recorded from insect hemolymph have been recorded chronological­ ly in Table II. Tablo II

Some Papers Listing Bacteria Pound in Insect Heraolymph

Reference Autiior Date Number Host Bacterium Metohnikoff 1879 356 Aniaoplia Bacillus austriaca salutarius Poroes 1891 158 Chinch **A micrococcus" Krassilatchlclc 1893 304 Gallorla Bac, tracheitis mellonella sive i2;raphitosi3 and Bac, septicus inaector'um d*Herelle 1911 247 Schistocerca C'occobacillus pallens acridiorxam Gallardo 1912 166 Grasshopper "natural ^bacterial enemies" Picard and 1913 434 Arctia ca.ia Coccobacillus Blanc larvae ca.lae Picard and 191S 435 LOTJantria Bacillus Blanc diapar l:73iiantria0 la3?vae Picard and 1913 435 Cholonla B. ca.lae Blanc ca.ja Cliatton 1913 89 Galleria Bacillus mellonella melolahthae Cliatton 1913 89 Sillcworm B. bombycis Paillot 1913 405 Gortyim B, Kort^mae orchracoea Paillot 1913 405 Pyrameis B. pryameis I and cardui II Loionsbury 1913 330 Grasshopper "Bacterial disease - 66 -

Table II (continued)

Reference Author Date Nutnber Host Bacterium Glaser 1914 170 "Caterpillar" "Several un- noEied" d'Herello 1914 248 Grasshopper "Coccobacilli" Mereskov/slcy 1914 344 Grasshopper Coccobacillus ("Wanderheu- acridiorum schreclcen") Wortlirup 1914 396 White grubs Micrococcus niRrofaciens Velu and 1915 562 Schistocercus Coccobacillus Bsuiin porefiirina acridiorum Aokl and 1915 7 Silkwonn "Sotto-Bacillen" Ghlgasalci Barber and 1915 84 Grasshopper Coccobacillus Jones acridiorum Paillot 1916 408 Galleria "Several varie­ mello'nella ties and races of coccobacillj Paillot 1916 407 Macrolepi- "Coccobacillus" doptera larvae B6guet 1916 36 Schistocerca Coccobacillus •Derigrina acridiorum Itcaus 1916 305 Grasshopper Coccobacillus acridiorm Sergent 1916 522 Schistocerca Coccobacillus perif3:rina acridionjin Du Porte and 1917 439 Grasshoppers Coccobacillus Vandorleck acridiorum Paillot 1917 409 L:piantrla "parasitic^^ dlspar microbes" - 67 -

Table II (continued)

A . Reference Author Date dumber Host Bacterium Palllot 1917 410 Vaneaaa urticae Parasitic iii5cibtx«3 Barbara 1917 23 Grasshoppers Coccobacillua acridlorum Chorine 1917 98 Galleria 'pathogenic mellonella microbes" Grlaser 1918 172 Gipsy moth *Nev/ bacterium" larvae Glaser 1918 173 Melanoplus B« poncei femur-rubmjm Glaser 1918 172 Lachnoaterna Streptococcus "digpar disparis Dufresnoy 1919 124 Cuethooampa Baot» pityocampe, pityocajgpa Strept» pit;yo~ caxapae, and St. pityocampae

Hollande and 19S0 260 Malaoosoma Coccobacillus in- Vernier caatrenais sectorum var. malacosomae Metalnikov 1921 349 Honeybee B. paratyphosus and GasChen ^vei Metalnikov 1921 349 SilkwoiBx Streptococcus and Gaschen bombyoia Rouband and 1923 495 'Caramon fly" Bact. delendae Descazeaux muacae V/hite 1923 586 Sphingidea?phing3 Bac. sphinp:idis larvae V/hite 1923 585 Honeybee B, larvae and B« plu^n - 68 -

Table II (continued)

... ^ Refferenco Author Date Host Bacterlm Metalnikov, 1924 552 Galleria Baot, Kostrlslcy and mellonella tumefaciens Touraanoff Glaser 1924 175 Apirotis B. nocturuffl aquiliiia Glaser 1924 175 Slllcworm "New bacterium" Glaser 1924 176 Adult house '*Wev7 bacteritim" fly Mereshkovsky 1925 345 Schistoceroa Coccobacillus amegicajia ~'acri'cLi'oruni Pospolov 1926 440 Schlstooerca Coccobacillus amierloana! acrldiorum Hubault 1928 270 Daaclly5.ra "Parasitic^^ TDudilDtmS'a bacillus" larvae Metalnilcov 1928 354 Corn borer 'Large number of aiid Chorine pathogenic bac­ teria Chorlnfl 1928 99 Corn borer Bac, canadensis. B. pjLbsoni. and S. ontarionl Husz 1929 273 Corn borer Bac, thurin- giensis Pfelffer and 1930 432 Mamestra Bact. laaemo" Staranier oleracea phosphorevim larvae Paillot 1933 425 Various in­ Bact, neurotomae, sects Bact« pieris liquefaciens, ^cb. melo-ianthae lique'fccieiis. and Bty-fa« iyartttrlcola adipOBUs - 69

Table II (ccaitinuod)

„ . Refereaico Host Baoterimi Author Number Paillot 1933 425 Galleria "Eigjat species of mellonella Goccobacillus, a 'bacille aporule', a KiBm"'"Baaillu3. and a p;rain.+Di- plococcus" Paillot 1933 425 L?mantria "Tliree cocco- gispar bacilli" Paillot 1933 425 Piei-ia "six coccobaciUi, a braaaicae gr am+M^lococciis, two grSFbaciSl, and a Vibrion" Paillot 1933 425 Silkworm "several kinds of bacteria" Paillot 1933 425 Meurotoma "a gram-bacillus, noaoralia and^ a grani-coc- larvae cus" Tauber and 1935 546 Udeopaylla "Bacteria in herao- Yeagor robuata lymph" Tauiber and 1935 546 Blatta "Bacteria in hemo- Yeager oriental!s lymph" Taubor and 1935 547 Photinua sp.? "Bacteria in hemo- Yoager l^ph" Tauber and 1935 547 Phyllophaga "Bacteria in hemo- Yeager sp.? lynQ)li" Tauber and 1935 547 Heleuoanla "Bacteria in hemo- Yoager albllinea l^ph" Tauber and 1935 547 Heliotlxia "Bacteria in hemo- Yeager obaoleia lymph" Tauber and 1935 547 Meodlprion "Bacteria in herao- Yeager iecontel lymph" - 70 -

Other v/orkers who Imve employed bacterial entoinophytes for "biological control of injurious insects, or for experi­ mental ptirposos of various kinds, are numerous. Some of the more important references include Aold. and Chigasalci (8) vjiio v/orked on the silkworm parasites; Barbara (22) v/ho tested bio­ logical control of grasshoppers v/ith Coccobacillus acrldiorum in Argentine; Barber and Jones (24) who did the same in the Philippine Islands; B6guet (37) and B6guet, Musao and Sergent (38) tried it in Algeria; Belanovalcy (39) studied the trans­ mitting agents in epidemics; Berliner (48) isolated the bac­ terium responsible for "schlaffsucht" of insects; Carle (280) attempted biological control with bacteria in grasshoppers of Madagascar; Chatton (90) investigated the pathogenicity of several coccobacilli from Galleria mellonella, on the silkworm, and other insects; Chorine (99) listed the bacteria he fotind In the hemolymph of G. mellonella; Glaser (173) made a system­ atic study of the organisms distributed under the name Cocco­ bacillus acridioriAm d'Herelle aM decided that several species were carrying tliat name; he also experimented v/ith a bacterixuii causing a disease in the adult house fly (179) and made other studies on specificity of bacterial diseases of insects (178) and on acquired Immunity in silkworms (177). D'Herelle (249) gave an account of the procedure for the destruction of locusts by "seeding" bacteria in the affected area; Hollande (262) found that Injected bacteria were phago- - 71 - cytosed by corpuscles of the hemolympli, and he and Moreau (261) again suggGsted tliat symblonts lai^t transform into parasites; Lalnes (510) used bacteria to conibat grasshoppers in Ilondurus and termed the method the "most effective raeana"; McKillop and Gaugh (343) used biological control in Egypt; Mereslcovslcy (345) showed that Coccobacillua aoridioruni. a normal aymbiont of Schlstocerca amerlcana changed to a parasite under certain con­ ditions of temperature and humidity. Metalnllcov (346 , 347 , 348, 351) and later with Chorine (355, 353) and ICitajxma (350) began Ms studies of hemolymph bacteria as early as 1907 when he used Galleria mellonella larvae for iminuxiity st^udiesj, a bi-anch of investigation vrith v/hich he has continued to be mainly concerned, althougji he has also headed investigations in using bacteria for control of the corn-borer, Musso (388) participated in a series of experiments in Algeria when Schlstocerca perepirina v/as attacked by parasitic bacteria. Muttlcowski (389) Included bacteria among the "other bodies" wMch he listed as having fouaid in the hemolymph of the series of insects vfhich he studied. Korthrup (395) found a bacterial disease in the larvae of Jime-beetles, and saw that it was transmitted to the adult, throu^^i the pupae. Among all the literature pertaining to bacteria found in insect hemolymph, tlie name of Paillot appears most often. He has made a great number of contributions (406, 411, 412, 414, - 72 -

416, 41V, 418, 419, 420, 421, 422, 424, 423) on various phases of taie subject. In 1933 he summarized the results of these papers, itlong v/lth much additional data, in a book, "L'infec­ tion che2 les insectes". Paillot has discovered several new species of bacteria in insect hemolymph; he has made many im­ munity and specificity studies usii^ these and other entoiao- phytes; he has conducted "in vitro" studies v/ith iiTsect hemo- lymiih and bacterial entoiuophytes; and has studied the effect on the hemolymph of the presence of bacteria. Bergent and Lii6ritier (520) and Ser^jent (521) also aided i-ji experiments conducted in Algeria v/hen bacteria were tried as a metliod of control against £p?asshoppers. Tangl (643), Tateiwa (544), and Toumanoff (552) have contributed articles in connection with bacterial diseases of Galleria mellonella. V61u (564 , 565) and ivith Banin (563) v/ere associated v/ith at­ tempts to control grasshopper plagues in Moroccso by use of biological (bacteria) methods. Ze3moff (610) made an inter­ esting report on the bacteriolysins fourd in insect body fluids, lilvaluation of attempts so far made in the employment of artificial spread of bacterial diseases as a control measiare for insects, is difficult becaiise results have been very con­ tradictory and only partially successful* From accounts given, the technique is quite simple. Various reseai'ches have shovm that the Infections can be produced by feeding, and in those forms which are likely to b© camibalistic, the infection caia - 73 - be transmitted because of this tendency. Infection may also occTir by "puncture inoculation" - that is, through breaks or abrasions in the body covering. One of the greatest difficul­ ties seems to be the selection of the specific bacterium to produce tlie desired control. In many cases immunities are es­ tablished against normal symbionts vtiich might prove injur­ ious if transformed to parasites. On the other hand, Metalnl- kov found that larvae of the bee-motii I'rere very susceptible to small doses of Bacterium coll. Bacillus subtilis, and other very common bacteria, and it may be that use of these bacteria, of others not commonly found in the Insect may prove of value. In many cases of infection, symptoms include general body v;eak- ness, paralysis, refusal to eat, and sometimes diarrhoea. Pal Hot (425) believes that tv7o factors need to be con­ sidered in the examination of the vimlence of a bacterium for an insect: (a) The bacterial factor, v/hich he defines as the ability of the bacterium to multiply in the Insect; (b) The host factor, which he explains as tho resistance of tho host to the Invading parasite. Deatii of tlie Insect is due, he be­ lieves, less to any toxins that might be produced, but more to excessive multiplication of bacteria in the heciolymph and other tissues. He says, "Death results from a general suppression of the vital functions of the host". Age of tiie insect in some cases is an important factor in regulating susceptibility. Larvae and nymphs are more easily Irllled by infection than are - 74 - adults, and moulting pex-iods seem particularly vulnerable. The BQQ factor is believed to be correlated with a decrease in the number of hemol2/iuph cells, phagocytes included, as the insect becomes older. Mot a great deal has been doxie, hov/- ever, in investigating these control factors and mechanisms; the field for research is crav/ded with many problenis to be solved before a real decision can be made in regax*d to the po­ tential value of hemolymph-infecting bacteria as a biological control for insect plagues.

(j. Factors Infltiencin^g Mitosia

Numerous investigations dealing v/ith various conditions or substances affectinf^ laitoais have been reported upon during the past half centuiy. A great nuraber of agents, both chem­ ical and pliysical, have been found to atirnulate, retard, or even stop division of cells. Among the earliest papers is tliat of Yung's (G08) who found, in 1881, that snail eggs (Limnaea) under violet light hatched in seventeen days, whereas otiier eggs under rod li^t did not hatch imtil thirty-four days had elapsed. Later, De- moor (115), using plant matez>ial (stamen hairs of Tradescantla), saw that the absence of oxygen in the environment of the plant stopped mitosis; and that specimens placed in hydrogen posses­ sed cells v;hose divisions were completed but vihose cell walls - 75 - did not fom. In the aame year of 1895, Loeb (326) noted the effect on cleavage of echinodem, annelid, and fish eggs plac­ ed in oxygen—free water. This set of experiments led to a long series of papers "by Loeb (327) and his followers on meth­ ods of artificial parthenogenesis, an investigation in which many coiatributlons have been offered. Tiiese paper's, however, v/ill not be reviewed here. Table III sununarizes the results from some of the more important researches carried on in the restricted field as con­ sidered in this paper. Examination of the table shows that many combinations of materials and agents have been employed in these studies. Experimental plant and animal tissues and reproductive bodies in which karyoklnesis has been examined are very diverse, and conditions and substances used to affect mitosis either positively or negatively are even more diverse, including light of various v/ave lengths, gases, acids, allcalies, heat, cold, drugs, extracts of tissues, salts, fats, alcohols, protein derivatives, dyes, mitogenetic rays of different types from many different sources, growing cultures of yeast, bac­ teria, protozoa, and fly larvae, minerals, etc. V/ithin recent years two groups of investigators have been particularly active in gathering data to support their respective theories as to the manner in v/liich the mechanism of mitosis can be controlled. One of these headed by Alexander Gurwitsch (191, 200, 193, 190, 194, 195, 192, 201, 196) believes that the stimulus which sets - 76 -

In itiotion the procoss of karyoMnesis is a pliysical one con­ sisting of "mitogonotic rays of a definite v/ave-length emanat­ ing from many soxjrces"; the other band of workers is led by

Frederick S, Hairanett (220, 221, 223, 227, 228) who believes the essential stimulus is chemical - specifically, that the activator is the sulfhydryl (-SH) radical. In view of their prominence and present day notice, these two theories v/ill be examined more closely. Table III

Some Papers Referring to Conditions or Substances Affecting fiitosis

Author atid. Ref­ Date Exnerimental Agent Effect on Division erence Niuriber Material Yxmg (608) 1881 Limnaea eggs Light of various Eggs in violet hatch in wave lengths 17 days; in red, 36 days Demoor (115) 1895 Stamen hairs of Absence of oocy- Hot couple ted Ta^adescantia gen Demoor (115) 1695 Stamen hairs of In hydrc^en Completed but cell wall Tradescantia not formed <3 Loeb (326) 1895 Echinoderm eggs Absence of free Not completed oxygen Loeb (326) 1895 Gtenolabrus eggs Absence of free Prevented oxygen Loeb (326) 1895 Fundtilus eggs Absence of free None for many divisions oxygen Loeb (327) 1898 Arbacia eggs 0.006 to 0.008?^ Accelerated NaOH in sea HgO Hertwig (251) 1898 Rana fusca and Various toapera- Retarded at low; in­ R» esculenta tures creased to a maximum

Mathews (339) 1901 Asterias and Pilocarpine Accelerated Arbacia eggs Mathews (339) 1901 Asterias and Atropine Slowed Arbacia eggs Table III (continued)

Autlior and Ref- erence Kxanber Date Exse: Agent Effect on Division Godlewski (108) 1901 Rana temporaria Pvire oxygen In 47 hours reached eggs normal 73 hour stage Calkins and 1902 Paramecii;ai 1 to 2,500 Accelerated Lieb (78) Caudaturn parts alcohol Lyon (331) 1902 Arbacia eggs Oxygen-free sea Stopped water Lyon (351) 1902 Arbacia eggs Hydrogen Stopped Lyon (331) 1902 Arbacia eggs Sea water contain­ Stopped ing potassium cyanide Bohn (63) 1903 Sea-urchin eggs Radiusi 40 minute exposure accelerated; longer, retarded Spaulding (531) 1903 Arbacia eggs EthOT Stopped Gilman and 1904 Chicken eggs X-rays daily for Accelerated develop­ Baetjer (169) 10 minutes ment during first 36 hours; then retarded and deformed VYoodiniff (598) 1905 Oxytricha fallox Dibasic potasaiuni Increased, then de­ phosphate; K sul­ pressed fate; K bromide Mathews (339) 1907 Starfish eggs '<2uinine sulfate 0..00l^ retarded; h3gja- er concentration killed Table III (continued)

Autlios? and Ref- Date lerim^nijal Agent Effect on Division erence Nuniber TflailSl Mathews (339) 1907 Starfish eggs Atropine sulfate 0.02;i retarded; higher, Id-lled Mathews (339) 1907 Starfish eggs Caffeine hydro­ Retarded chloride Mathews (339) 1907 Starfish eggs Cocaine hydro­ Retarded chloride Mathews (339) 1907 Starfish eggs Strychnine sul­ 0*01/i retarded; fate higher, killed

Mathews (339) 1907 Starfish eggs Veratrine Retarded ohlo3rf.de Maiiiews (339) 1907 Starfish eggs Sea water at 2®C. No division until warmed ITowikoff (397) 1908 Paramecium Thyroid extract Increased Woodruff (599) 1908 Paramecium and Small amounts Increased, then Stylonychia of alcohol slowed Haaland (205) 1910 Mouse tumor Disintegrated Treated tumor Qvevi carcinoma tis­ more rapidly than sue control Higuchi (253) 1912 Mouse tumor Macerated mouse Accelerated growth embryo skin in treated animals Lazarus-Barlow 1913 Ascaris ova 5x10 _7mgr. radium, Accelerated; longer and Becton (315) 30 hours, 0°G, expos^lre retarded arable III (continued)

Author and Ref­ Date Experiaentintal Agent Effect on Division erence ITuiriber f^atsrial Mottram (385) 1913 Ascaris ova Beta and gasma Dividing cells 8 tiirieg radium rays as vulnerable as resting Carrel (84) 1913 Chick fibroblast Extract of em­ Increased 2 to 40 culture bryo and lymph­ times oid tissue Richards (484) 1914 Planorbia eggs X-rays for 10 Developed as far in minutes 10 minutes as nor­ mally in 60 Buddington and 1915 Pararaecium Thyroid Increased Harvey (73) Paclcard (403) 1916 Arbacia eggs Radium Short time stimulat­ ed; longer, retarded Shmaway (562) 1917 Peramecium Thyroid emul­ Increased 65)5 aurelia and sion P. Caudaturn Paul and 1919 Moulting cyus- Phospholipin Increased speed of Sloarp (428) tacea (lecithin) mitosis Chambers (88) 1919 Paramecium Ground yeast; Increased divisions caudatum pituitar^r prepaa?- ations; supra­ renal solutions Haberlandt 1921 Plant tissues "y/vindhornione" Accelerated (209) Talale III (continued)

Autlior and Ref­ erence Huidtier Date Agent Effect on Division Dustin (130) 1S21 Thymus of mouse Intraperitoneally Increased nuiaber of injected human dividing cells and horse sera Dustin (1S9) 1921 Thytnua, bone mr- Injected huiaan and Increased number of rowj spleen, other sera mitoses Peyer's patches, lymph nodes of mouse Dustin (132) 1922 G}hynius, sp le en, Nine injections Stimulated, then lymph nodes, of li5 c*c* 6/0 decreased peptone Peyer's patches CD of mouse H Clssmbers, Scott 1922 Rata vfith tianors Irradiated Jen- Decreased tumor and Russ (85) sen's rat scrconia growth Dustin and 1922 Thymus, Peyer's 1,0 c.c. 5yj pep- Sttcralated mitoses Chapeauville pat Che s ^ lymph tone (131); ajid nodes of mouse Dustin (133) Haberlandt (210)1922 Leavesi stems, V/undhormone Accelerated roots of var­ ious plants Richards (485) 1922 Haminea 0^004 to 0i009>3 Accelerated; higher Yirescens eggs IJaOH concentrations re­ tarded Table III (continued)

Author and iie^ Date Exnerimental Agent Effect on Division erence Number Matsrial Richards (485) 1922 Hamxnea IIH4OH Slightly accelerated virescens e^aQs Richards (485) 1922 Heitiinea KOH Slightly accelerated virescens eggs Richards (485) 1922 Haminea Ba(OH)B or IJone virescens eggs Cr(0H)3 Richards (485) 1922 Haminea Thyroid Accelerated virescens eggs Richards (485) 1922 Haminea Pilocarpine Accelerated virescens eggs hydrocioloride Richards (485) 1922 Haminea Pilocarpine Hone virescens eggs nitrate Gtirwitsch (191) 1923 . Onion 3?oot Mitogenetic rays Increased number of from tadpole tail mitoses Drew (122) 1923 Tissue cultvires Saline extract of Accelerated malignant tumors Robertson (487) 1923 Infusoria Cholesterol and Accelerated tothelin Robertson (487) 1923 Infusoria Yeast extract Accelerated Robertson (487) 1923 Infusoria Tissue extract Accelerated Robertson (487) 1923 Infusoria Fhenyluretliane Suppressed Table III (continued)

Author and Ref­ erence Ntnnber Date Agent Effect on Division Robertson (487) 192S Glandular tissues Starvation Retarded Robertson (487) 1923 Fish embryos Magnesium salts Differentially re­ tarded Robertson (487) 1923 Sea-urchin embryos 0.17/i lecithin Inhibited Robertson (487) 1923 Rat carcinoma Injected lecithin Retarded growth Robertson (487) 1923 Rat tumor Injected Accelerated cholesterol

Robertson (487) 1923 Amphibia larvae Anterior Rapid growth a> pituitary OS Robertson (487) 1923 Protozoa Anterior Accelerated pituitary Robertson (487) 1923 Rat y;ith car­ 30 mgm. per day Accelerated tumor cinoma of tethelin growth Robertson (487) 1923 • Amphibia Thyroxin Accelerated meta­ morphosis Robertson (487) 1923 Salamandra Iodine or io­ Accelerated meta- dides moiphosia Smith and 1924 Asterias forbesii Increased GOg Decreased; prevent­ Clowes (528) and Arbacia tension ed at tension of punctulata 3.8^^ Table III (continued)

Author and Ref- erenc© llu33ber Date al Agent Effect on Division Chambers and 1924 Rats VTith tumors Breakdown products Treated rats grew tu- Scott (86) of tumor tissue mors with volume of 34i4 c.c, Ts^ile controls grev/ 14,6 c.c. Rusinoff (497) 1925 Onion root Rays from tad­ Increased in onion pole tail or tadpole emulsion Fischer (137) 1925 Tissue culture Di sintegrating Promoted cells cells

Fischer (137) 1925 Tissue culture Liver extract Inhibited 03 cells Fischer (137) 1925 Tissue culture Pus extract Depressed cells Fischer (137) 1925 Tissue culture Thyroid extract Increased cells Fischer (137) 1925 Tissue culture Dog muscle ex­ Increased cells tract Robertson (480) 1925 Euchelys cul­ 1/20,000 to Retailed ture 1/200,000 acri- flavine Robertson (480) 1925 Euchelys cul­ Janus green Retarded ture Robertson (480) 1925 Euchelys cul­ Proflaviiae Retarded ture Table III (continued)

Author and Ref­ Date ExBeyiiapntal Agent Effect on Division erence Ktmber ^aateriaj. Terao (549) 1925 Eggs of Ostrea Uraniiim rays Arrested or changed f^igas, 0. apinosa, 0, dens elamello sa Ball (21) 1925 Paramecium Thyroid Increased Ball (21) 1925 ParameciTita Liver and hy- None significant pophysi s Ball (21) 1925 Paramecium Glycogen Decreased in strong concentration Denny (116) 1926 Potato Thixorea Stimulated sprouting CD cn Baron (25) 1926 Onion root Yeast cultures Increased in onion Gurwitsch and 1926 Onion root Rays from blood Increased Gtirwitscli flowing in ab­ (196) dominal vein of frog Prank and 1926 Onion root Sunflower root Increased in onion Salkind (162) tips Camot and 1926 Cutaneous wounds Extracts of pro­ Accelerated healing Terris (81) liferating skin Crowther (107) 1926 Colpidium col- 1 to 2,5 micro- Treated ones divided pooa coulombs of x-ray and grew to nearly twice size of controls Table III (continued)

Autiior and Kef- Date Agent Effect on Division erence Number Ganti and Don- 1926 Sclerotic, choroid,Radium Stopped aldson <79) and cartilage tis­ sue culture from 6 to 8 days chick Strangeways aM 1926 Tissue cultures X-rays Decreased; stopped Hopwood (537) from chick em­ with doses of 60 ' e bryo or more Mottram, Scott, 1926 Rats with Jen­ Beta rays from Stopped by lethal and Russ (384) sen' s sarcoma radium dose (1 hour) Burrows (75) 1926 Skin area of rat Injection of ex- Stimulated locally tract of grow­ ing tissues Wrigjit (600) 1926 Skin area of rat Injection of ex- Stimulated locally tract of yolk of incubated hen egg Chambers and 1926 Tissue culture Extract from auto- Stimulated Scott (87) lyzing tumor tis­ sue in K/20 HCl Dobrovolskaia- 1927 Mouse testes X-rays Cells disintegrate Zavadskaia (120) during mitosis Magrou and 1927 Onion root Bac. tumefaciens Increased in onion Magrou (333) Table III (continued)

Author and Ref­ Date Experimental Agent Effect on Division erence Xlumher SauiHal slx&Iv**Stat" 1927 Onion root S'onfloT/er seed­ Increased in onion kowitsch (293) ling leaves; potato V/agner (574) 1927 Onion root Vicia faba root Increased in onion tips Easamett (220) 1928 Plant roots Pb-ion Retarded Hajamett and 1923 Chick embryo Pb-ion Iniiibited differ- Wallace (221) entially 3aron (26) 1928 Onion root Bac, autracoides; Increased in onion Sarclna flava; Bac. co31 conmnme Schwarz (517) 1928 Oaiion. root Onion root Increased in **de- tector" Petri (4S0) 1928 Onion and germ~ Onion and germin­ Increased inatin^ Zea mays ating^ Zea maYS Reiter and 1928 Onion 3?oot Onion mash and Increased GdlDor (476) roots; tadpole heads; ttimor tissue Bucciante (72) 1929 Tissue cultures Various tempera­ Stopped at 1 -1" tures \7ulzen (601) 1929 Planaria maculata Chicken egg fed Promoted and P. apiilia Table III (continued)

Author and Est- Date ExTjeidmental Agent Effect on. Division erence Nttmber T)Tatf?vlaj Hammett (223) 1929 Onion and corn Pb( 1103)3 Retarded roots Haimiett and 1929 Skin injury on 1 to 10,000 thio- Accelerated healing Reimann (227) rats glucos e Hammett (228) 1929 Paramecium Thio-glycollic Stimulated caudattm acid; Na-dithio- diglycollate; cysteine;cystin; glutathione; ergo- thioneine; insulin 00 Hananett (228) 1929 Zea mays 2?ootL9ts Reduced glutathione None CO Baker (19) 1929 Sarcomatous fib- Glutathione Stimulated 3?obla3t cultiire Reimann and 1929 Ulcers on man 1 to 10,000 thio- Sti:3iulated healing Hammett (472) glucose Reimann (473) 1930 Skin of rats aiid 3;2 thiocresol in Increased mitoses mice 95^"^ alcohol Slia3?pe (525) 1930 Paramecium HoS solution di- Stiitsulated caudatum luted to Sxl0"9-S- in culture fluid Sharpe (525) 1930 ParameciTim IlaoS Stimulated caudatum Table III (contiiiued)

SubSf' ^KlSfei^ ^sent affect on Elvlsion Voegtlin and 1930 Amoeba proteus I;V^100,000 gluta­ Accelerated GhaUaej (572) thione for 4 days Gigon and Ko- 1930 Onion root Carcinomatous tis­ Increased iia onion verraz (168) sue from hunian prostate> liver, colon, ovary Hammett (229) 1930 Hermit crab Siilfhydryl com­ Stimulated; entire pounds claw regenerated in 10 days Blacher and 1930 Onion root Me t amor pho sing Increased in onion Holzmann (58) ampliibia Blacher and 19.30 Oixion root Regenerating tail Inci'eased Holziaann (56) of Bombina bom- bina Blacher and 1930 Onion root Lie tamorpho sing Increased Holzmann (59) Ambystoma ti^^ri- nun and Pleuro- dele waltii Blacher and 1930 Cnion root Regenei'ating tail Increased Broialey (57) of upodeles Holziaann (265) 1930 Onion root Transformation of Increased in onion Drosophila melano- gaster larvae to pupae and pupae to adults Ta'ole III (continiied)

Author Eiiid FhL- Date erence lluniber Agent Effect Oil Division Elaclierj Woron- 1930 Tail and intes­ Pcreles of Rana Increased grov/th aowa, etc< tine of metamoi*- temporaria {61) phosizya; Rana ridibunda Borodin (65) 1930 Onion root Hyacinth root Increased in onion meristem Borodin (65) 1930 Onion root Willov/ root Inci'eased in onion meristesi

Borodin (65) 1950 12 hour CTilture Bacillus anthra-* Increased in yeast of Saccharontrees coides cerevisiae Borodin (65) 1930 12 hour culture Onilon root Increased in yeast of Saccharomyces cerevisiae Gaunt (167) 1931 Physa hetero- Cysteine solti- "Comparative accel­ stropha and tions eration" LrTsmacea oolum- ella eggs Gaunt (167) 1931 Physa hetero- Tlai o-gly c olli c lone stropha and acid Lymnacea ccQum- ella eg^^s Haumett and 19S1 Paf?aru3 lonsi- -SH as 5*2x10"'' Stiuiulated regener­ Smith (251) carpua sulfur from ation p-thiocresol Table III (continued)

Aut3ior and Ref­ Date Agent Effect on Division erence Ntiiriber Eanmett and 1931 Pa/^arus lonfyj- Unoxidiaed sxilto'. Retarded Smith (231) carpus S=0 as 6.4x10-^ sulfur f3?om di~ phenyl- sulfoxide Hammett (232) 1931 Mouse skin 5/0 henzyljaercap- Stimulated young tan applied daily cells for 4-1/2 to 6 months Morgulis and 1931 Podarka obscura P-iaiiocresol, Regeneration not Green (380) thiophenol, iiiio- stimulated glycollic acid, cystine Reimann (474) 1931 Mouse skin 5% benzylmercap- Stimulated tan applied 3 times weekly for 6 months Reimann (475) 1931 Leg ulcers (Man) l/l0,000 thiocre- Stimulated skin sol solution; formation 0^25^ thiocresol in fat Moissejewa (376)1931 Onion or Tulipa Onion root Increased in de­ 3?oots tector" VYassermann 1931 Isolated onion Onion root Increased (579) root lable III (continued)

Author and Ref- Date Agent Effect on Division erence Number Prank and 1931 Onion root Tetanized frog Increased in onion Rodionov (164) miscle; beating frog heart Sewertzova 1931 Bac« mesentericua Yeast cultures Increased in bac- (523) fuscus ; Bac. (Hadso^a); teta- teria atherim; 8ac« nized frog n2iscl3; lactis aeropienes; live frog heart Bac^ pyocaneuin Steepell and 1931 Various "receptca^s'Ordon piilp, onion Increased Romberg (534) root mash> onion embryo mash, bac­ teria, potato, turnip inash, yeast culturesj hydra, annelids, Daphnia, Cyclopa, Coretbra and Chironomous larvae, tadpoles Hamnett (233) 1932 Obelia eammis- Suboxidized cys­ Retarded growth surallg tine about 30>o Hainmett and 1932 Pa/^arus loivsicar- Compounds con- Stimulated differ­ Eanmett (234) PU3 chelae taining -SH ential growtli Hammett and 1932 Pa^arus longicar- Compounds con- Retarded Hamaett (234) pus chelae taining partial­ ly oxidized S Table III (continued)

Author and Bef- Agent Effect on Division erence Ntmiber Birribaum (55) 1932 Wounds and skin Thiocresol Promoted more rapid grafts (Man) healing Moissejewa 1952 Onion or Tulipa Yeast cultures; Increased in onion (377) roots blood of rat and Tulipa 2?oots and man Hamraett and 1932 Eggs of Lophnis Glutathione Stimulated embryonic HanEEett (218) piscatorius; Pap;- growth arus longicarpus; PTirpura lapillus; Doris bilaaeHate.; Polynices; Grepi- dula Hammett and 1932 Eggs of Lophnis Dittoenyl-sulfox- Retarded emb2?yonic Hammett (218) piscatorius; Pag- ide grov/th arus lonpiicarpus; Purpura lapillus; Doris bilamellata; Polynices; Crepi- dula Jahn (280) 1932 Chilomonas para- 0«0015}'& Increased as much mecium culture fluid as 100^ Coldwater (105) 1933 Planaria macu- M/100,000 gluta- Stimulated (?) re­ lata thione generation Coldwater (105) 1933 Procotyla fluv- U/100,000 gluta- None iatilis tiiione Tattle III (continued)

Author and Ref­ Agent erence ilumber Date Effect on Division Coldwater (105) 1935 Tubifex txibifex IThioglycolli c Stimulated; treated acid and glu- aniir^als regenerated thione nearly 5 times as mucla as controls in same time Brunsting and 1933 Cutaneous ulcers Cysteine solu­ Stimulated healing Simonsen (71) (Man) tions Jahn (281) 1933 Chilomonas Para­ Solutions con­ Accelerated mecium taining -SH Hasamett (235) 1934 Obelia ^enicu- Glutathione Accelerated growth lata Voegtlin (573) 1934 Ameba proteus Minute amounts Inhibited nuclear of copper salts g3?ov/th Sutton (539) 1935 Wounds, particu­ 1/5,000 thio- Increased healing larly burns glycerol (Man) - 95

Gurwltacli'3 experiments began in 1922 and first resixlts were published (191) tile following year. Tills was soon after Haberlaiidt (207, 208) had offered his theories of "cell-divi­ sion hormones", "wound-hormones", "necro-hormones", and "lepto- hormones". All of these are based on a purely biochemical ex­ planation of some cellTilar process. Gimirltsch's explanation, hov/ever, is entii'ely biophysical, as already stated. At first, ta?ial materials xised by Gianvitsch were confined to rapidly grov;ing plant tissues, particularly onion rootlets. Later a great many other plant and animal iziaterials served as sources of Ms mitosis-stimulating factor. TMs factor was found to pierce certain light filters and thicknesses of quartz and led Gurvd.tsch to postulate that the stimulus v/as a light ray of some kind. Generally it has been referred to as the "mitogenetic ray". In addition to Gurvritsch's vjork, very many other inves­ tigations have been carried out to support or disprove the ex­ istence of a mitosis-stimulating ray. Some of these papers are smnmrized in Table III. Reiter and Gdbor (476) believe the mitogenetic rays have a v;ave lengtti from 280 to S40niJ.. Blacher and his associates (56, 59, 57, 61) state that differ­ ent regions of metamorphosing amphibians give off different length ana. strength rays over various periods of time. Holz- raann (265) finds that Insects in different stages and types of transforation are a source of these rays. Borodin (65) dls- - 96 - covered that the induction of the stimilatiiag ray is not lim­ ited to the action of a species or variety upon itself, but that different biological sources may be used as senders for a given detector of the rays. Prank and Popoff (163) measured the rays they used and found tiiey were associated vAth the ultra-violet rays of the spectrum in tiie region of 2,000 Ang­ stroms. Many data have been published in substantiation of Gur- wltsch's technique as applied to Ms theory. Among those of­ fered Imve been the results of Prank and SaHcind (162), Baron (25, 26), ItLslialc-Statkowitsch (293), Magrou and Magrou (333), Rawin (464), IVagner (574), Loos (328), B'xunlc and Rodionov (164), Maxia (S40), Gigon and Noverraz (168), Siebert (529), Prank (161), Blacher and sonierajew (60), Ghrustschoff (100), Jaeger (279), Pototsky, Salkxnd, and Zoglina (441), Prinzes- sin- Ypsilanti (446), and Wassiliew, Pranic, and Goldenberg (580). An idea of the extremely varied "induction" sources of the stimulating rays, as well as the "receptors" of the mito­ sis factor can be gained by an examination of Table III. Some results, such as those of Petri (430) and Y/assermann (579) are difficult to Interpret and liard to place either as supporting or disproving Gurv/itsch's theory. Adverse criticisms of the theory are becoming corataon as the technique la beconili-ig more widely used. Rossman (494) and von Guttenberg (203, 204) do not find evidence for mitogenetic - 97 - rays. 'J?he former believes that many errors in interpretation have resulted, particsularly in rootlets, from a chance exami­ nation of treated material when its cells happened to be un­ dergoing a "wave" of divisions; the latter fiMs results are too vai-iable and inconsistent in that effects "nnnifest them­ selves in diverse v/ays". Frederickse (165) and Schwars (517) were mmble to confirm results from Gurwitsch's experiments. Moissejevm (376, 377, 378) adJiored closely to Gur\'iritsch*s publislied methods and got negative results, "iithin the last year Kreuclien and Bateraan (30G) have made tMs statement af­ ter £m ana3.y3is of xarmj investigations: "'liiere is little Jus tification to be found for the viev; that anyone has succeeded in producing a mitogenetic effect by artificial radiations". Schreiber (507) supports this conclusion. Hafftier (216), in referring to the many statements of stimulating effects of ultra-violet radiation witi). reference to mitogenetic problems suspects tMt resiilts are, in many cases, merely "the second­ ary effects of cell destruction". Batemon (32) in 1955 has made an excellent review and criticism of published articles on mitogenetic rays and summarizes Ms findings with:

"The unsatisfactory state of mitogenetic literatiire malces it advisable to regard all that has been wit ten in support of the ©xistence of mitogenetic radiation with the greatest scepticism. The existence of a mito genetic effect has been neither finally proved nor finally disproved; the evidence against it, though ex­ tensive, is as yet insufficient. But supposing that a - 98 -

mitogenotic effect does exist, it is Mglily improbable tliat it has anything to do with ultra-violet radia­ tion."

A complete bibliography to the end of 1930 of papers dealing vdth luitosenetic rays has been prepared by schreiber and Limtz (508), Altiiou^ its papers were not yet available at this writing, th.e photochemical syiaposium at Gold Spriiog Harbor Biological Laboratory during the past aummer probably will also aewQ as an excellent source of latest literattaro on tills subject. (See Science, 1935, 81, 311.) V/itb.ln the past five years increasing attention has been given, partiCTolarly by American and English researchers, to Hamiiiett^s theory, already referred to, that the sulfhydryl radical (-SH) is the essential stimulating factor governing cell division. The theory has been found interesting not only tiieore tic ally but also practically because the application of sulfhyd3?yl containing corapourids has been found to promote healing of btirns, ulcers, and other wounds. Its possibilities in cancer studies have not yet been explored, but a start has been made and it may be that future research will uncover in­ formation beneficial in man's fight against benign and malig­ nant growths. Hararaett's theory has had an interesting development, grow­ ing from a series of researches (222, 219, 220, 223, 224, 225, 221) Y&ich he began on the "Biology of Metals". Starting v/ith 99 - lead, he fomid that onion rootlets grovm in solutions of that metal's salts v;ould concentrate the absorbed salts in the re­ gions of rapid growth. Next he saw that rootlets in solutions of lead salts grow more slowly than control rootlets, aiid that this was due to a retardation of cell proliferation. Later he discovered that itdLtotic nuclei Imd on especial affinity for lead, Vflay? And in what form was this metal? Analysis of the lead containing deposit in the rootlets indicated that the pre- ciiDitate was a combination of lead and an organic sulfhydryl compound analagous to, if not glutathione itself. With these facts as a begiiming, Hansiiett started an intei'- prstation of malignant grov/th based on the chanistry of cell dJ.vision, referring pointedly to his data. He atteinpted first (226) to coi'relate clismical data from the field of malignancy vd-th his hypothesis that nuclear activity of cell division was stimulated by sulfhydryl. He believed tliat Kahn and Goodr-idge's (284) results, indicatina thi3.t volatile sulfides, cysteine, cystine, and other suboxldlzed forms are increased in patients witli 3iiallgnant growths, were consistent with his belief that malignancy mj.^it be related to a distortion of sulfliydryl equi- llbrlvun. Further, Roffo (490) reported an increase of reducing power of blood serum from animals v/ith tumors, TMs property, Hamraett believed, was probably due to an increase of sulfhydryl containing compounds, as indicated in a paper by Hunter and Eagles (272). Voegtlin and Thompson (571) showed that the llv~ 100 - lug part of tumor tissue contained a high concentration of sulf- hydryl, 'Eiis was confirmed by Lecloux, Vivario, and Pirket (516). Roffo and Correa (491) also isolated an insulinoid sub­ stance from malignant grovrtihs - a fact that llsrunett believed aigpificant since Vigneaud (576) had fovind suboxidized sulfur in Insulin and Aldersberg and Peruta (3) discovered tliat ap­ plications of insulin promoted cell proliferation in old ul­ cers of the leg. In continuing Ms d3.scussions, Hammett pointed out tliat malignant growth seemed abnormally sensitive to sulflaydryl, a sensitivity he suggested as being constitutionally determined. Thus he remarked fiirther on Bierich and Kalle's (54) analyses showing that tratuna liberates sulfhydryl as tissue autolyses and tliat trauma is frequently productive of malignancy. In tliis comiection it is fitting to point out that Reiche (465) found that tissue autolysates favor mitosis; that Haberlandt (210) and Hammett (225) observed stimulation of cell division after injury; and tixai Carnot (81) got healing of cutaneous wounds by applying extracts of proliferating skin as dressings. Hamnxett, himself, pointed out that malignant cell prolifera­ tion can. be initiated by chemical substances, particularly those related to tarry by-products of the distillation of coal and petroleum. These by-products, as well as oils, contain significant amounts of mercaptans and sulfides. Other experimental work (230) followed. Hammett reasoned - 101 - that if local concentration of siiLfhydryl was favorable to malignant grpv/th, then wealc concentrations migjit prove of help in the healing of vrounds. Baker (19) reported at this time that addition of glutathione to fibroblast tissue cultures re­ sulted in more rapid growtli. Ackroyd (2) had shown some yeai's previously that cystine (a sulfur containing amino acid) vms essential to growth, Haimnett interpreted these results as supporting his general hypothesis. The practical application to human medicine of Hammett's theory was first attempted by Riemann and Hammett (472) in a sex'ies of cases of leg or foot ulcers. With each patient the effects of thioglucose treatments were beneficial aiid healing began within tv/enty~four to forty-eight hours. One varicose vein ulcer had existed for ten years v/ithout healing after various and numerous treatments. An objection to the use of thioglucose arose in that bacterial grov/th was likev/ise stimu­ lated in the wounds. The use of thiocresol or thiophenol was suggested to overcome this difficulty. Following this suggestion, Heimann tried thiocresol on skin of rats and found that applications caused a thickening of the epithelium and an appearance of "nuclear activity". Haramett (232) also tried benzyl-mercaptan on skin of mice and got the same results. Reimann (474) confirmed Hsimmett's re­ sults witli benzyl-mercaptan dissolved in various agents such as ethyl alcohol, glycerin, and liquid petrolatum. Later, - 102 -

Relmaim (475) was again tiho first to use tMocresol in wound healing on hummi "beings. Leg ulcers were treated v;ith l/l0,000 solution or vrf.th Q»25% in lanolin. Within three weeks the ul­ cers were healed. Other materials have heen used to show the effect of var­ ious sulfur compounds on the division mechanism. Sharp (525) found that vevj dilute water solutions of HsS or Na^S were stlniulatoi»y to the reproductive rate of Paramecium caudatum. Voegtlln and Chalkley (572) exposed amebae to M/100,000 gluta­ thione vrf.th the result that many polynucleate cells farmed. Gaunt (167) experimented with cystein solutions on snail eggs, but did not get consistent results. Haminett and Smith (231), Hammett (229), and Hammett and Haimnett (234) used the regen­ erating claw of a hermit crab to meaaxire the stimulating ef­ fect of p-thlocresol, as well as the retarding effect of di- phenyl sulfoxide. Morgulis and Green (380) concluded that sulfhydryl compo\inds were not stimulatory to mitoses after making a number of investigations on regeneration in the poly- chaete worm, Podarke obsoura. Ilammett (253) treated Obelia commlaatiralls with suboxidized cystine and concluded that sul- fer in that form was retardative to cell multiplication. He suggested tliat the control of neoplastic growths might even­ tually be possible tlarough chemical means. Hammett and Ham­ mett (218) made an interesting group of investigations with larvae of a nuiriber of marine forms. Those exposed to gluta- - 105

thlone gi»0w more rapidly than the controls which were not sub­ jected to the glutathione, Jahn (280, 281) found that -SH ac­ celerated divisions in cxjlturea of Chilomonas Paramecium as imich as one hundred per cent. Ooldwater (105) has reviewed the literature concerning glutathione and its connection with regenerative grovirth. He refers to Hopkins (268) who first isolated the compomid; to White (586) who shov/ed tliat growing areas of plant and animal tissues have hi^ior -SH contents than corresponding adult tissues; to Murray (387) who found tlmt the glutathione content of chick embryos was in inverse proportion to ago; to RapldLn (456) who detected increased glutathione in echinoderm eggs just preceding first cleavage; and to many others, including the Hammetts, who have employed glutathione in mitosis-stimulating researches. Ooldv/ater's investigations had to do with the effect of ^utathione in speeding up regeneration in several species of marine v/orras. Yaol (602) determined that glutathione content of the rat was at its maximum in the early embi>yo stages and decreased with age. That -SH compouj3ds are of practical value in healing wotinds has beeia shown by others than Reimann. Bimbaum (55) confirmed Reimann's v/ork by using thiocresol to assist in the healing of skin grafts, Brunsting and Simonaen (71) reported favorably on more than two hundred clinical cases of cutaneous ulcers treated at the Mayo Clinic with cystein solutions. The - 104 - latest report haa been given, in 1935, by Sutton (539) vrtio found that thioglycerol was satisfactory and bonsficial in pro­

moting Y/orund healing. His opinions are based on 264 clinical cases. Ko entirely satisfactory theory has yet been offered to explain how aulfhydryl compounds are a stimulating factor for karyoldnesis. The nearest Haminett has come to an explanation is contained in a paper (235) written in 1934 in vvhich he gives results from a set of experiments with various c\iltures of Obelia, using the three amino acids v/hich make up sl^'^^^^'^Jiione: namely, glycine, glutamic acid, and cystine. He concluded that glycine "stimulated that part of regeneration concerned with protein reconatitution"; tiiat glutamic acid "accelerates progress of selective building up of the protein molecule"j and cystine "accelerates cell proliferation". Jahn (281) be­ lieves the acceleration of divisions may be explained on the basis of the oxidation-reduction potentials involved, Voegt^ lin (573) thinks that glutathione activates intracellular en­ zymes such as proteinase which causes partial hydrolysis of genuine protein. Gurwltsch has attempted to correlate his results with those obtained by the tise of sulfhydryl compounds. To him tliree possibilities of interrelationsMp exist:

"1. Mitogenetic rays are the primary and direct cause of mitosis, and Mgh concentrations of sulfhydryl in re­ - 105 -

gions of active cell multiplicatioia are a result and not a cause of the divisional activity. **2, Sialfhydryl is the direct stiraulus to growth by increase in cell niimber and mitogenetic rays serve as accessory factors enliancing that of the naturally occiir- ring enz^pnea in tlie liberation of -SH in the free and active form from its previously bound state in the pro­ toplasmic protein. "S. Mitogenetic rays are not the cause but the result of the chemical and physical changes incident to mito­ sis, and that their presence assists the process either in the manner described in the preceding paragraph, or by helping to keep the sulfur in the reduced or -SH form necessary for the exhibition of its stlimilatliis property,"

In ansv/er, Hammett rejects Gur^vitsch's first intea^rela- tionship on the grounds that the experimental increase in -SH concentration alone accelerates proliferations; the second he accepts in so far as It has not been proved tliat the rays are essential to division, irahereas that has been proved for -SH; the third he is in agreement v/lth so far as the mitogenetic rays are secoiidajTy and subae3?vient to the chemical processes. At the date of this writing no other explanations or cor­ relations have come to the writer's attention.

Stimulation of Mitoais of Insect Hemolymph Cells

Only four references to stimulation of mitosis of insect hemolymph cells have been found available. Palllot (420) ap­ parently was the first to attempt it in 1924, Larvae of Euproo- - 106 - tia clipysorrhoea were xnjecfced with eimilslons of Bacillus melolanthae non liquefaciens vrith tilie result that the dividing cells in the honolymph rose from throo or four per thousand oel3,s to thirty or forty cells. Tlie reaction, v;hioh he called the "oaryocin.6to30", he "believed was due to the affect of anti­ bodies which the bacteria produced. A year later, Iwasaki (277) tised larvae of Galleeria mel- lonella to study the effects of various injections, inclxjding bacteria, normally, the mitotic count ranged from one or two per thousand cells, but after injections of 1/60 c.c. of five per cent peptone, tlie comt reached a mximimi of six within four days. Two larvae injected with l/70 c.c, of five per cent albumin had inax:lmum covuits of three and five mitotic figures per thousand cells during a five day counting period. Larvae injected with bacterial eBiulsions reached raaxiraxam counts of 136 dividing cells per thousand within 2,75 days in one case, and forty-five within twenty~fotxr hours in anotlier case. To test tlie itifluence of temperature, Iwaaald. used lots of five to eif^it larvae at three different temperattu?es. Those at 25° to 28° had a ma^iinrum covmt of tliirty-four mitotic cells per thousand vAtMn tliirty hours; at 20° to 23°, 15 on the third day; at 17° to 18°, 10 on the fourth day. Lots of six larvae injected with vaseline emulsions in various alcohols, xylene, or turpentine also showed increases in mitotic divisions of hemolymph cells. He continued these injection studies in an­ - 107 - other report (278) to see If the introduction of foreign aub- stojices would provoke mitoses In other tissues than the hemo- lymph elements. He fo\ind no evidence tlaat any other cells save the hcmolymph cells vsrere affected. In 1933, Paillot (425) wrote another report on results of in.lections of B, melolantha non liquefaciens into larvae of E. chrysorrhoea, but this time analyzed the counts to show what types of cells v;ere concerned in the increased number of divi­ sions. He fOTond that the increase i2i mitotically dividing cells was mainly in the type he calls macronucleocytes. In­ jections of "bacteria into lasrvae of M. braasicae also caused a mitotic reaction v/ithin 15 to 20 hours, of tv/o to three days dijiration. Heatii-jg to "boiling or filtering did not destroy the emulsion's property of acting on macronucleocytes. Heating above 100°C. destroyed it very readily, hov/ever. Paillot "believes that the karyokinetic reaction is con­ nected in some v/ay with the establishment of an inraimlty prin­ ciple. Insects, such as Vanessa urticae. Erio/^aster lanestris, Pleria brassicae, or Pieris rapae. are not a"ble to produce this rise in mitosis and are extremely sensitive to bacterial in­ fections; phagocytosis is particixlarly active in these sensi­ tive species. He thinks that macronucleocytes secrete anti- "bodies antagonistic to the development of bacteria, and, there­ fore, increase to reinforce the imniunity mechanism. Paillot found, too, that the injection of albuminoid sub­ - 108 ~ stances such as QIKS wlilto, blood of vertebrates^ and sera of vertebrate bloods also caused a rise in mitotic figures. In tlolo connection ho refers to the experiments of Achard (1) and Dustin (129, 130, 151, 132, 133, 134) who brouf^t about in­ creased mitoses in cells of certain organs and tissues of rab­ bits and mice by injections of proteins or protein derivatives. Altliough Tateiv/a (544) did not record sMfts in mitotic indices after injections, he found, as did Paillot, that there vjere changes in the relative numbers of different typos of cells. - 109 -

III. MATERIALS

Specimens of Blatta orientalia used for this series of in­ vestigations v;ere collected either in Ames, Iowa, or were ship­ ped in from collectiiig localities in Kansas or Mississippi, No attempt was made to keep the different groups separate; all insects captured or sent to Amos wore placed together in large stock cages. Fresh water and food, in the form of bread and well-ripened banaiaa, v/ere supplied regularly. Before being used for expea^imental purposes, all specimens were allowed to remain in the stock cages for several weeks of acclimatization. Cultures of Serratla niarcescens. Bacillus subtilis. and Bacterium coll were furnished by the Bacteriology Department of Iowa state College. The culture of Staphylococcus aureus was "Test organism Ho.208" used by the Pood and Drug Division of the United States Department of Health for tests of disinfect­ ants. Nutrient broth employed in several experiments was pre­ pared accoiHfling to the standardized formula for bacteriological investigations, namely: three grams of meat extract, five grams of peptone, five grama of sodium chloride, and distilled water to make a liter of solution. V/itte's peptone was used for the peptone solution of various concentmtions. Beef ex­ tract solutions v/ere made from the extract manufactured by "Difco", These preparations v;ere sterilized before injection. - 110 -

Thyroxin tablets and solutions of insulin Virero secured f3?om Lilly; rotenone from McLau£^lin Gormley ICing; hemoglobin ciystala from Pfanstiehl; glutathione and various amino acids from Eastman; dinitrophenol from Eimer and Amend; and other chemicals from Balcer. Defibrinated rat blood was prepared in tlie customary man­ ner by v/hipping freshly drawn whole blood vdth a smll bundle of slender v/ooden sticks to remove the fibrin. The e::ctract3 of chick ejnbryos v/ere made by washing fresh 4a-hovir etaibryos in several changes of Hobson's solution to re­ move any adhering yolk or albumin, after v/hich they were mascer- ated with mortar and pestle and extracted with a small amount of Hobson's solution. The extracting material vras allovired to stand for at least twenty-four hours in a refrigerator before use. The preparation was not filtered before injection. Hobson^s solution consists of titie following: 0.161 M NaCl, 0.003 M KCJ., 0.002 M OaCla. All preparations - solutions, dilutions, extracts, etc, - were made \7lth the usual precautions to avoid bacterial con­ taminations. For staining the hemolymph cells in the te^^orary mounts prepared for the mitotic counts, the following stock solution was made; To one liter of distilled water were added 4.7 grams sodium chloride, 0.11 grams potassium chloride, 0.11 grams cal­ cium chloride, and 0.005 grams gential violet. This was heat­ - Ill - ed gently until the dye completely dissolved. When prepared for use, five drops of 10 per cent acetic acid v/ero added to 20 c.c, of the stock solution. To allov; use of oil immersion lens and to prevent drying of the diluted hemolymph moxmt, the clear mineral oil, laiown commercially as "Nujol", was used. To inject some of the experimental animals, 0.5 c.c. Luer tuberculin syringes witii 26 gauge needles were employed. These were sterilized "by boiling before each injection. - 112 -

IV. METHODS

Insects, large njmplis or adults, male and female. Intend­ ed for experimental use v/ere selected randomly from the stock colony, placed in a glass jar in the laboratory, and kept un­ der close observation for ten to fourteen days while given access to the tisual stock diet. All injured or slxiggish in­ dividuals were removed, A second period of observation fol­ lowed when individual specimens were isolated in 125 c.c. Erlenmeyer flasks and observed for another week before actml experimentation began. Small amounts of the customary food were supplied to each aniiml in its Individual container. Since no separate source of v/ater was available, the pieces of bread were v/ell moistened before being placed into the flasks. The mouth of each container was covered with a small piece of cheese-cloth, held in place by a inibber band. Each insect was transferred daily to a clean flask and a new supply of food. To prevent too rapid drying of food, the small flasks were kept in a loosely covered glass aquarium v/hich contained a large uncovered beaker of water. Ov/ing to their condition - such as obvioTis illness, re­ cent ecdysis, or v/hile carrying an egg-case - some animals did not undergo the second preliminary period of observation in the Erlenmeyer flasks, but v/ere placed immediately into the in­ dividual containers and tised for counts. This was done in an - 113 - effort to learn if their peculiar condition v/aa reflected in a deviation from the normal mitotic count. V/ith the exception of the specimens mentioned in the above paragraph, all apparently normal experimental animals under- Tsrent an additional check-control period of five days during which three mitotic counts v/ere made, before subjected to any type of treatment. If any abnoimality in the hemolymph pic­ ture appeared during tiiese five days, the animal was discarded or kept for a study of its own peculiarity. Injections of bacterial culttares, emulsions, extracts, and other fluid substances were made by means of a small syringe with the hollow needle inserted through the conjunc­ tival fold of the coxa-femur joint of either the ri^t or left metathoracic leg* In some cases slight leakage of body fluids would occur on witlidrawl of the needle but the chances of los­ ing the nev/ly injected material were minimized as much as pos­ sible by leaving the needle in position for about a zninute af­ ter the injection in order to allow the injected fluid to mix with the body hemolyraph and circulate through some of the hemo­ lymph spaces# Usually about l/20 c.c. (approximately 10 per cent of the average body wei^t) was injected. Heedle inocula­ tions of bacteria were made by inserting a solid dissecting needle, wet with the bacterial culture, into the body cavity through tlae conjunctival folds between the tergal plates of the abdomen. The puncture was made about half vraiy between the mid- - 114 - dorsal lino axid tiie side of tlie animal. Care was taken not to piincturo the intestinal tract or to injure any otlier internal organs. Insects fed bacteria wore given broad soaked in the par- tlctilar cultm'a being used. Ho other food v/as allowed. New pieces of infected bread were supplied daily. All bacterial cultures were approximately 18 hours old when used for injections, inoculations, or feeding. Actual counts of bacterial concentrations in the cultures of Staphylo­ coccus aureus averap.ed 320,250,000 per c,c, of culture. Animals at S7°C. v/er© kept in a constant temperature oven. Small pieces of paper tov/eling v/ere placed on the bottom of the flasks to absorb the vmter which originated from the moist bread and banana. Insects at 5°C. were kept in a refrigerator whose temperature deviation did not exceed 0,5°C, To malce the hemolymph cell counts, the following technique was employed: Upon a clean glass slide was placed a large drop of mineral oil. Into this drop was put a small drop of the dilutins~3taining solution. This v/as made to settle to the top of the slide. A tiny drop of antennal hemolymph (obtained by clipping v/ith scissors) from the insect was quickly placed in­ to the colored solution, and immediately stirred with a dissect­ ing needle to prevent cell coagulation and to distribute the cells throu^out the droplet. Ho cover slip was used, but, to prevent evaporation, the diluted, stained hemolymph was covered 115 with, a film of the a-urromidins inlnoral oil, vrtrlch also served as imi'iiersion oil for the micr'oscope lens. Cells were counted randomly, using oil immersion of approximately 1,000 dianieters nxagniflcation. As the count progressed, the nuiiiber of cells in prophase, metaphase, anapliase, and telophase v/ere noted. Experience and tests showed tiiat a count of tv/o thousand cells was sufficient to ohtain true figures of the percentages of di­ viding cells, "both mitotic and ajnitotic, of multinucleated cells, and of other peculiar cellular elements present. Experimental animals were kept under observation until death occurred or were discarded when no significant chaioges any longer appeared in their records, or were eliminated when some other factor such as a bacterial infection was noted. Control animals were retained for various lengths of time, de­ pending on the duration of the experiment for wliich they served as comparisons. - 116 -

V. EXPERIMIAWS

Since the niiiabei'' of experimental, control, and other OID- served animals is ratlier large, direct reference to specific insects used will be omitted here in order to avoid "uimecessary duplication of infoairoation v/loich has to be included in "RESULTS". Primarily, the purpose of this investigation was to dis- cavev the nomal ratio of mltotically dividing cells occurring in the hemolymph of the insect, Blatta oi'ientalis, and to find how various agents and conditions affect the normal rate. Before actual experiiuentation began, it v/as necessary to deto3?mlne hov/ stable the mltotically dividing cell count (M.D.G.) was in nor­ mal animals. To establish this basic information, tMrty con­ trol animals v^ere used. Such variables as sex, age, proximity to ecdysis, oviposition, and room temperature fluctuations, were segregated and studied, in so far as they might influence division of hemolymph cells. Control counts were also made to determine if successive, daily, minute hemorrhages, such as are necessary in obtaining hemolyxaph samples, in themselves, affect­ ed the M.D.C. Methods of jDrocedure v/ere tested to find how many cells had to be counted in one preparation to obtain a fair and true figure of its dividiiig cell population. Originally it v/as intended to inject broth cultures of various bacteria to see if bacterial infections would inflvience 117 - tiie M.D.C. Naturally, sterile broth was also injected into some other animals as a control measure, and when it was fo\md that it, itself, produced a rise in the percentage of dividing cells, that method was abandoned. Instead, bacterial infec­ tions produced by needle inoculations were found to be entirely satisfactory. Bacteria washed free of their medium and sus­ pended in Hobson's solution were also used for injection. Fur­ ther work was carried out with the injection of broth, beef ex­ tract, peptone, and other protein containing substances or pro­ tein derivatives. Altogethei', ninety insects v/ere used as experimental sub­ jects for the follov/ing treatments: needle inoculations of Staphylococcus aureus; needle inoculations of two naturally oc­ curring bacteria found in this insectj feeding S. aureus; in­ jections of salt solution suspensions of washed Serratia mar- cescens. Bacterium coli« and Bacillus subtilis; injections of lO/i glucose, IQfc, albumin, rotenone, nutrient broth, 5/o beef extract, 10;,'^ dinitrophenol, various dilutions of insulin, lOyo hemoglobin, 1,0% lead chloride, certain concentrations of thyroxin, deflbrinatod rat blood, 10>^ glutathione, extracts of chick embryos, various concentrations of peptone, aspartic acid, 5^0 glutamic acid, 5^ glycocoll, ^fo alanine, 5/o tryptophane, and 5/j cystine; also for exposure to S7° and 5®C, Unless un­ avoidable leakaise occurred at the point of injection, l/20 c.c. of fluid was forced into the animal at one injection. Tliis - 118 amormt, in moat cases, approximated 10 per cent of tho aver- ago body weight of the species of insect used. In additional to the above experimental investigations, other observations v/ere made on specimens in certain pathologi­ cal and physiological conditionsc In the course of these and prevloixs x'esearches, the presence of bacteria in hemolyinph samples was sometimes noted. T^to distinct species (so far Tin- identified) of bacteria liave been seen; a coccus type and a short rod. Si:c animals suffering from infection of the former kind were studied; eight with rod Infection. Two nonaal in­ sects were inoculated irlth tlie rods and the subsequent effect on "tiie riiltotic rate v/as noted. Five animls possessing a peculiar form of paralysis were examined, and five v;hose hemo- lymph cells were full of very large vacuoles. Twenty-five animals v;ere used to find the effect ecdysls has on the H.D.C. In three cases, control animals molted, so that records are available for dividing cell rates dui?ing periods before and af­ ter molting. In the otlier cases, counts were begxxn while the animal v;as in the process of shedding its exoskeleton, or a few hours after the sMn had been cast off. Five ad\ilt female si>ecimens were studied during the time an egg-case vms forming, aM for a number of days after the ootheca had been deposited. He*ro all five cases vrlth control counts before and after the process ai'e ready for examination. - 119 -

More details concerning the experiments and observations v/ill be found in the next section of this paper. - 120 -

VI. RESULTS

Approxiioately 250 specimens of Blatta orientalis were used in the course of these investigations. Of tliese, 175 will be referred to directly by niimber in this paper; the remainder served as trial animals in developing tecliniques and in test­ ing methods of procedure, A total of 2,595 mitotically divid­ ing cell (M.D.C,) counts was made on these 175 animals; conse­ quently, the suminax'izing figures of percentages of prophases, metaphases, anaphases, telophases, amitotically dividing cells, and multinucleate cells are based on a total of 5,190,000 cells counted.

A, Results From Dotermining VJhat Number of Cells Con- stitu^e a. Fair SamiJle For MaElm M.D.G, Counts.

To ascertain vrhat number of cells had to be counted in each hemolympli sample in order to procure a M.D.G, count of some stability, a series of twenty animals was employed. Sam­ ples of hemolyniph sieve taken and prepared in the mamer already described. In each of the twenty samples, the percentage M,D.C. was noted after counts of 100, 500, 1,500, 2,000, 3,000, and 4,000 cells. These results are tabulated in Table I?. Exami­ nation shov/s that a count of 2,000 cells is the minimum number that can be used to determine a significant count-figure for this research. Below 2,000 cells the difference between the 121 - range end-points is in Inverse ratio to the ntiiiiber of cells counted, vKh-ile the range end-point differences above 2,000 cells are practically the same. Likewise, average counts in the region of and above 2,000 cells are more nearly of the same value than covints based on a thousand cells or less. - 122 -

TABLE IV

Comparison of M.D.C. Coxmta {%) Based on Different Kxuiibers of Cells Counted

Number Cells « « • • • Counted 100 : 600 : 1000 : 1500 ; 2000 3000 : 4000 M.D.C. Goxuit * » • * No. 1 0.0 : 0.00 ; 0.20 : 0.37 : 0.25 0.20 ; 0.20 •• 2 1.0 : 0.20 : 0.20 : 0.20 : 0.20 0.23 : 0.25 •* 3 0.0 : 0.20 : 0.30 ; 0.40 : 0.30 0.26 : 0.25 *• 4 0.0 : 0.00 : 0.20 : 0.27 : 0.20 0.20 : 0.20 •• 5 0.0 : 0.40 : 0.40 : 0.33 : 0.30 0.30 : 0.28 " 6 1.0 : 0.20 : 0.20 : 0.20 : 0.20 0.20 : 0.28 " 7 0.0 : 0.20 : 0.?^0 : 0.13 : 0.20 0.20 ; 0.25 " 8 0.0 : 0.20, ; 0.10 ; 0.20 : 0.25 0.33 ; 0.28 " 9 0.0 : 0.00 : 0.10 : 0.20 ; 0.25 0.30 : 0.28 " 10 0.0 ; 0.10 : 0.20 : 0.20 : 0.25 0.27 : 0,25 " 11 0.0 ; 0.60 ; 0.50 : 0.33 : 0.25 0.23 : 0.26 " 12 0.0 : 0.00 ; 0.20 ; 0.20 : 0.30 0.30 : 0.31 " 13 0.0 ; 0.00 : 0.10 : 0.13 : 0.20 0.20 : 0.20 " 14 0.0 ; 0.00 : 0.30 : 0.33 : 0.30 0.30 : 0.31 *• 15 1.0 : 0.20 : 0.20 : 0.20 : 0.20 0.27 : 0.28 •" 16 0,0 : 0.20 : 0.20 : 0.20 ; 0.20 0.26 : 0.25 " 17 1.0 ; 0.20 : 0.20 : 0.20 : 0.20 0.20 : 0.31 " 18 0.0 ; 0.00 : 0.20 : 0.33 : 0.30 0.26 : 0.30 " 19. 0.0 J 0.40 : 0.20 ; 0«33 : 0.30 0.26 : 0.25 20 0.0 : 0.40 : 0.50 ; 0.33 : 0.30 0,23 : 0.26 * Ranae 0.0 -l.(>.0.0-0.60-000-0.50; OJ3-a40:0£0-O.SQ•a20-0^a0.20-0J31 » • • • Difference be-- • • • • tween range • • • • end-points 1.0 ; 0.60 ; 0.40 : 0.27 : 0.10 0.10 0.11 Average of • • • • 20 counts 0.20: 0.18 : 0.23 : 0,25 : 0.25 0.25 0.26 - 123 -

B. Results Prom Tosts to Dirolicate ^5o^ta ^r6m" SeKe^

In checlclng the actual counting technique, eiglit hemo- lymph preparations v/ere made from the same animal and three counts of 2,000 cells each v/ere made on each sample, the sets of counts "being continuous in each case. These results are tabulated in Table V. The range of the entire twenty-four M.D.C, counts is 0.20^ to 0.35;^ and. the greatest difference between range end-points for any set of three counts from the same sample sample does not exceed 0,10/L

Table V

Duplicate Counts of Same Heraol:piph Sjamples

Preparation I II III IV V VI VII VIII M.D.C. Count No. 1 0.25 0.30 0.20 0.25 0.30 0.25 0.25 0.30 " 2 0.20 0.30 0.25 0.35 0.25 0.30 0.25 0.20 3 0.30 0.30 0.30 0.30 0.30 0.25 0.25 0.30

Althougli performed for another purpose (referred to later), the successive counts (Figure V) made on animal Ko.625a support the triistworthiness of results obtained by the counting tech- - 124 - nlque used here. Twenty-five successive counts from the same animal taken over a period of approximately twelve hours had a range of 0,20% to 0»4:0%,

0. Results from Control Animals

Thirty control animals v/ere used as checlcs foi' the various experiments during the course of these researches. SmKiax-ies of pertinent data are given in Table VI, along with other relat­ ed information. Important averages are uijderlined. Table VI contains facts that are particularly Important and v/hich, for coxwenience and clarity, need emplmsizing. It villi be noted that control animals v/ere kept different lengths of times, varying from ten days to 132 days. The latter period is considerably longer than taie longest period of observation of any experimental animal. The course of the per cent of M.D.C. for animal No.619, which was kept under observation the longest time of any specimen, is given In Figure I. The range of mi­ totic counts for this animal is from 0.0/^ to 0.50^0 and happens to be the widest range encountered in any control Insect; the average figure for 73 counts from It Is 0,202/6. Altogether, 568 counts of 2,000 cells each v;ere made on the thirty control insects, with an average of 0.186;^ and a range from 0,116/o (animal No,724) to 0.25/b (animal No,250). Table VI also shows that the 124 normal animals used in this re­ 1S5 search had an initial count range of 0,0% to 0,45/u, with an av­ erage of 0.196/0; and a range of 0,0% to 0.50/j vd-th an average of 0,181;^ for the tliree control counts made on all normal animsils during the first five days of observation. Table VI

Average Llitofcic Counts (Controls)

Animal Ooimts Days Cells Counted Rarsge :Average Humber Description Made Observed (slO®) (/=) : (^) 6S1 ^ Nymph 25 48 50 0.00 - 0.40 : 0.166 623 29 71 58 0.00 - 0,45 : 0.162 12n 25 29 50 0.15 - 0.35 ; 0.250 728 17 22 34 0.05 - 0.45 ; 0.229 621,623,727,728: 96 170 192 0.00 - 0o45 ; 0.203 614 ^ Ad^t 19 38 38 0.10 - 0.30 : 0.205 625 28 72 56 0.05 - 0.40 : 0.194 725 •• 20 33 40 0.05 - 0.30 : 0.160 614,625,725 • « 67 143 134 0.05 - 0.40 : 0.186 619 Q llT^jnph 73 132 146 0.00 - 0.50 ; 0.202 620 T •' 31 63 62 0.05 - 0.45 : 0.225 624 17 42 34 0.05 - 0.40 ; 0.211 723 24 26 48 0.05 - 0.35 ; 0.137 6K,620,624,723 145 262 290 0.00 - 0.50 : 622 QT Adult 26 48 52 0.05 - 0.40 : 0.238 721 17 26 34 0.05 - 0.35 : 0.188 722 25 32 50 0.05 - 0.40 : 0.222 622,721,722 68 106 136 0.05 - 0.40 : 0.216 • Sex and age m 751 not checked 10 11 20 0.05 - 0.30 : 0.140 750 19 23 38 0.00 - 0.30 : 0.176 747 11 13 22 0.10 - 0.35 : 0.200 745 13 16 26 0.00 - 0.40 : 0.238 744 14 20 28 0.05 - 0.40 ; 0.203 742 16 19 32 0.05 - 0.40 : 0.193 741 15 19 30 0.00 - 0.40 ; 0.216 737 9 11 18 0.05 - 0.30 : 0.155 733 17 27 34 0.05 - 0.35 : 0.200 Sable VI (continued)

Animal Cotints Days Cells Coimted Range Average NiEiiber Description Made Obsex'ved (xlO®) Ci) i%) Sees and a^e 731 not checked 12 13 24 0.05 - 0.25 0.loo 730 14 19 28 0.05 - 0.35 0.153 729 10 10 20 0.00 - 0.30 0.155 726 11 12 22 0.05 - 0.20 0.127 724 9 14 18 0.05 - 0.20 0.116 675 9 12 18 0.00 - 0.30 0.133 613 3 12 6 0,10 - 0.20 0.150 All controls of urLknown 192 251 384 0.00 - 0.40 0.172 sex and age All controls 568 932 1136 0.00 - 0.50 n-lflfi All (124) Initial normals count 124 0 248 0.00 - 0.45 0.196 Control All (124) counts:3 372 5 744 0.00 - 0,50 0.181 nornials •v/ithin 5 days Figure I

M.D.C, Counts from Control Animal Wo.619 0.5 0 0.4 0

O ZO

O. I o Discarded

O.O O IOC lOl toz i03 lois- 103 105. lOT ICa 102 S lO H! 0.4-0

o.oo 73 74 7!r TG SO 53 as ea, ar as as e>o si! *33 s-3- e>5 ex:- •S't •36

O. ZO o. \ o o.oo 3 5. 3" 36 39 40 41 4Z 43 44- 45 4G 47 46 4e> 50 5! 52

O.ZO O. lO O.OO lO 14. l3 lei 20 Z4. ss Z7 za t?! DAYS - 130 -

D. Results Prom Anlmala SeKrep^ated B'or Sex and Af^e

Differences in sex and age were considered among those variables v/hich rai^t influence the per cent M.D,G, of this in­ sect. Resxilts of counts on animals sesi-egated for these fac­ tors are inclToded in Table VI. In all cases the range of counts came v/ithln normal control limits and average counts v/ero 0.203/^, 0.186;^, 0,194/b, and 0.216;o, respectively, for male nymphs, male adults, female nyrapha, and female adults. The average for all nymphs is 0,198^^; for all adults, O.SOlJoj for all males, 0.195^3; for all females 0.203^.

E. Results From Animals GheckQd For Possible Effects ^roSi Change a"Tn Tat'orai^ory (Sa^oratur^

Since counts were made anytime between 7:30 A.M. and 10:00 P.M., and since the temperature v/as noted at each count a fairly good check on room temperature fluctuation was obtained. These sttidies were done during a period of more than a year; conse­ quently, the range of temperatures is raiiier wide, namely: 17 to 32®C,, with an average of 23.3°C. for the 2,695 counts. To check the possibility that laboratory temperature changes mi^t be a variable v/hose effects affected the anitotic rate, three controls. No,727, Ho.783, l!Io,750, have been selected for Figure II, (A), (B), and (C), Two of these controls. No.727 and No. 750, were selected because their counts happened to be made at - 131 ~ temperatures which range from 19 to 32° and 20 to 32°, re­ spectively j while Wo.723 v/as picked, "because its counts were made within a comparatively nari'ow range of 19 to 26°, Ex­ amination of Figure II shov/s no correlation between variations in per cent M.D.C. and fluctuations in room temperatiore. Figure II

Con^arison of M.D.G. Ctjrves smd Laboratory Temperature Curves for Control Animals Ro.7S3, (A); No.727,(B); and No.750, (C). 0.35 0.30 - o Temp. • M.D.C

Disccnrded 0.15 -

O. lOO O Q05 o.oo 33 0) M.D.C. Disccrded :o.3o

H-O.EO* UJO.15 H^QIO 0.050-

O.OO 0.3D oTemp. M.D. 0.30

o.is Discarded O.IO-

QOO

DAYS - 134 -

P. Results B'rom Ovipositing Pemalea

The possibility that egg formation and egg deposition might he reflected in the M.D.C. count was checked with five animals. No.663, No,670, No.677, No.746, No,753. Results are indicated in parts (A) and (B) of Figure III. (A) represents a composite curve hasad upon the counts from the five animals. The points through vfliich the composite ctirve is drawn are the average values for the counts made on each particular day. The point "E" on tlie axis of abscissae indicates the day v/hen the ootheca was deposited; figures to the left of "E" mark days be­ fore deposition; those to the ri^t, after deposition. The range of the composite curve extends from 0,10/j to 0,26/o. (B) is a curve based on the daily variations of M.D.C. counts from adult female IIo.677, The counts range from 0.05% to 0,25%. Complete data from the five test animals is given in Table VII. It will be seen that both before and after deposition of tJie egg-case the M.D.C, counts remain within the normal range limits. Figure III

Composite Ctu?ve (A) and Ifypical Ctii'va /TB), from Animal No ,67^ for Poniales Durixig Egg Formation and Deposition. - 136 -

0.40 O. 3 0 , 0.2 O

o.oa

Discarded O.OO E I 2 3 4 5 C 7 3 iO 11 DAY5 Table VII

Stmimary of Data from Ovipositing Females

Cells :Mitotic Range:Mitotic Range Expeidment Animal Condition Counts Days Counted:(/S) During Egg:{^) after Egg Terminated Nianber Made Observed (xlO®): Formation i Deposition by

663 Ovipositing 8 10 16 :0.10 - 0.25 io.io - 0.25 Discarding 670 •* 11 17 22 :0.10 (1 day) :0.10 - 0.30 677 •• 14 20 28 ;0.10 (2 days):0.05 ~ 0.25 •« 746 *• 6 7 12 :0.15 - 0.30 :0.10 - 0.30 ••

•• 753 10 16 20 jo.10 - 0.25 :0.10 - 0.40 «« - 138

Gr. Results Prom Molting Animals

Soon after test coimts v/ere begun on molting aaiiiuals it became evident tlmt the process of ecclysis was definitely re­ flected in decided deviations from tho normal M.D.G. range. In all, thirty animals were obseirved* Resiilts are plotted in Figure IV (A) along with a composite curve based on the counts from tlae various animals. The points of this curve v/ere deter­ mined in the same way as for Figure III, The point "m" on the axis of abscissae mrks the day during wMch the exoskeleton v/as shed; figures to the loft of "U" are days previotis to ecdysis; to the right, after moltizig. Quite plainly evident is a tendency for the M.D.C. coimt to fall preceding and during molting, and as plainly marked io the tendency fox* a rise in M.D.C. counts after shedding. The maximum point of the rise occurs usually on the third day after ecdysis. Within five days after the process is completed, the counts I'eturn to their normal limits, (B) of Figure IV shows a typical curve from a single molting insect. The characteristic features in -{Jie com­ posite curve are again demonstrated here. A summary of data from molting animals is given in Table VIII. The highest count after molting was found to occur in animals No,646 and No»757 with 1,0/L All other animals have counts above, or just at the maximum of, the normal range. Figure IV

Composite Cixrve (A) and Typical Curve /TB), from Animal No.619a7 from Molting Specimens. oo

O. 8 5

0.75

0.70

^ O G5

O. 35

O. 3 C Discarded O. 2 5 0.20'

O. I O

0.03 O.CO

PAYS Table VIII

StffiBnary of Data from Moltliig Animals

: : : : Cells :Mitotic Range:MiT;otic Range:Experiment Animal: Condition :Gounts: Days ;Go;mted: {%) Before : {,%) After :Terminated number; - : Made :Observed: (,30.0^ : Molt : Molt : by

* « • • • • • 616 ;Kewly molted: 5 : 6 : 10 :0.30 {one day):0,10 - 0.55 :Death (Inoom- : : : : : : : plete molt) 683 : ** " : 17 : 22 : 34 :0.10 - 0.20 ;0.00 - 0.65 ;Discarding 758 : " [] : 10 : 12 ; 20 :0,00 - 0.20 lO.OO - 0.70 : Discarding 634 t [[ : 7 : 9 : 14 ; —- :0.05 - 0.45 : Discarding 635 : : 21 : 57 : 42 : - t0.05 - 0.55 : Disc^ding 637 : II H t 18 : 3§ : §6 i ——- :0,05 - 0.60 : Discarding i 638 : ^ : 11 : 14 : 22 : —— :0,00 - 0.85 : Discarding 639 : " " ; 4 : 4 : 8 : :0.10 - 0.80 :Death (Incom- ^ ! : : : : : : jxLete molt 640 : II II : 13 : 25 : 26 : :0,20 - 0.70 :Discarding ' 641 : ** " : 3 : 3 : 6 : —— :0.00 - 0,50 rDeath (Ihcoia- ; ; I i J I : plete molt 644 : " H : 19 : 34 : 38 : :0.00 - 0.70 :Discarding 646 : " ! 6 : 6 : 12 : • :0.00 - 1.00 :Death (Incom- : : : : : : : jxletemolt 648 : : 11 : 17 : 22 : :0.00 - 0.65 :Discarding 650 : II II : 15 : 27 : 30 : :0.00 - 0.70 :Discarding 651 : " : 15 : 25 : 30 : ;0.05 - 0.55 :Discardinf^ 652 : : So i 3? I :0.,00 - 0.80 :Discarding 653 : " " : 13 : 16 : 26 : :0.05 - 0.75 :Discarding 654 : II II : 14 : 24 : 28 : :0.05 - 0,75 :Discarding 655 : : 13 : 23 : 26 : —— :0.00 - 0,60 :Discarding 657 : " " ; 11 : 17 : 22 ; ;0.00 -- 0.50 :Discarding 661 : I 2i : 28 : 32 ; :0.00 - 0.85 ;Death 738 : ** " : 8 : 13 : 16 : :0..00 - 0.80 :Discarding 739 : " " : 9 : 12 : 18 : :0.05 - 0.70 ;Disca3?ding 749 : ** " : 10 : 10 : 20 : :0.00 - 0.80 ;Discarding 755 : " : 11 : 12 : 22 : :0.05 - 1.00 ;Discarding: 142 -

H. Reaulfcs From. Annals Sub leGted to Suooesalve wyiaiin" Sljor^ ervaXa

Altiio\igh no experimental animal was subjected to such drastic treatment, several attempts were made with different animals to ascertain if successive sampling of the hemol^^nph over a ten or twelve hour period would produce any change in the M.D.C, count during that period, or if any change would manifest Itself later, provided the animal lived. An example of the results is shovm in Figure V based on counts from ani­ mal No.625a. Counts v;ere made at the times noted on the ab~ scissa axis. Twenty-five counts v/ere made between 8:40 A.M. and 6:10 P.M. when hemolymph samples could no longer be ob­ tained. Ho deviation from the normal range is noticeable tlirou^out this series of counts. During the experiment the animal Imd access to food and v/ater. At 9:20 P.M. of the same day another count v/as made with a M.D.C. pep cent of 0.50. This was higher than any obtained so far from the animal, but still within the normal range. However, twelve hovirs later

the count had risen to 0.95;J and was still 0.95;;j at twenty hours. The animal was dead the following day. Figure V

Curve of Successive Counts from Animal No.625a also Counts Made on Following Day after many ' Small Samples V/ere Taken. 0.9 5

0.50.

UJ OAS I ^ 0.4Q

I

O. I O 0.05

^ ^pco-^rOcvj'^OwincJ-^OOininuiininminoOinOO — OOOOO lAinOC OCOOO 06 — — — CO— — CO cjco'v • " ~--:i^d , 'u^ DAYS A.M. 5th. DAY Grn DAY - 145 -

I. ResTolts Prom Animals Subjected to Hi^ or Iiow Teg^eraturea

Results of counts from animals subjected to 37°G., and others kept at 5°C,, are summarized in Table IX. Also, Figure VI shows graphically the results from animals No.610 and No. 626, both of v/hich were placed at the hi^er temperature; and Figure VII (A), (B), and (C) gives the counts from insects No.767, No,768, No,671 kept at the lov/er temperature. Subjec­ tion to 37°C. results in a slow increase in the value of the M.D.C, counts until a maximum is reached a few days before death. In three trials, peak counts of l,50/o, 0,85/^, ond 1.00/^ were reached. In each case the count then began to decrease until death occurred. In general, this species of roach does not well withstand low temperature. Usually, after about five days at 5®G., ani­ mals become so v/eak that another day or so would probably re­ sult in death. However, several were placed at 5°G. and, when signs of v/ealmess became evident, were returned to room temper­ ature, The results for No,768 and No.671 are shown in Figure VII, (B) and (C), respectively. These curves, along vdth that of No,767 figure VII, (AJ[7, would seem to indicate a trend to decreased M.D.C, coxints from animals subjected to low tempera­ ture. Also a return to normal room temperature seems to be as­ sociated with a sudden but improloiiged rise in the M,D,C, count. Figure VI

Curves of M.D.C. Counts from Animals No.610 and Ho•626 Subjected to 37°C. Ci O.0O O^QZG

5 0.70

U O.QO u Q50 R / \ \j nsad-i' « '/\^0-0/ \j O'^ o. /v\/

9 10 II 12 13 14 15 IG 17 18 I"? ZO 21 22 23 24 Z5 ZG 27 DAYS Figure VII

Curves from Animal No.767 (A), 110.768 (B), and No,671 (C) Subjected to 5°C. and Room Teioperatur©. - 149 -

OA a 0.30

O-ZO

O. IQ Dead O.OO EOOM T 5°C.

O. GO. . 0.5 O 2 0.40 h- o.3o; U o, 20. u O. lO Dead Q. O.OO 5°C. ROOM T 5°C.

0.50 0.40 0.30 O.ZO O. lO' Deod O.OO

EOOM T" 5°C. ROOM T° DAYS Table IX

StErama3?y of Data from Animals Subjected to 37®C. and 5°C.

Cells Mitotic Range Mtotic Range Experiment Animal Temperature Counts Days Counted (JO During 5 {%) During Terminated Number Made Observed (xios) Day Control Treatment by 610 370 c. 15 23 SO 0.00 - 0.05 0»15 - 1,30 Death 618 370 c. 8 12 16 0.10 - 0,25 0,20 - 0,85 Death 626 37°0. S3 32 46 0.10 - 0,20 0.10 - 1.00 Death 671 6°C. 9 12 18 0.10 - 0,35 0.00 - 0.10 671 Room Temp. 4 4 8 0,10 - 0.35 0.30 - 0.55 671 5®C. 2 2 4 0.10 - 0.35 0.00 - 0.05 Death 767 50c. 15 17 30 0.15 - 0,35 0.00 - 0.10 Death 768 5®C. 10 12 20 0.15 - 0.30 0.00 - 0,20 763 Room Temp. 3 4 6 0.15 - 0.30 0.20 - 0,65 768 5®C. 4 4 8 0.15 - 0,30 0.00 - 0.15 Death - 151 -

J. Resxilts Prom Various Experiments Uainp; p^ferent^aeteri^

Table X includes data from insects used in experiments conducted in an effort to find if the presence of various bac­ teria in the body hemolymph stiimilates or retards the multipli­ cation of hemolymph cells. In only one case each, using Bac. coli and B« subtilis, v/ere Inoculations successful. No,692, vri.th coli, reached a maximum count of l«70/a; No,691, witli subtilis, showed no re­ action, the highest count of 0,40^ being well within the normal limits. Negative results v/ere also obtained in five animals v/ith Serratia marcescens; counts remained witdbdn noiroal ranges. Insects into which suspensions of Staphylococcus aureus v/ere injected died before any decided changes in hemolymph counts occurred. This happened in many cases, but only four. Wo,600, No.601, Ho,602, No.607, are referred to in Table X, However, when needle inoculations were adopted, with the result that the bacterial increase in the hemolymph v/as slov/, maximum counts of l,45^a, 1.45/^, 0,55%, 2,80%, 2,05%, l.SS^-S, and 1.25^0 re­ sulted. Tliree records, from No,631, No,632, and No.720, are plotted in Figure VIII. These particular curves were selected to show the difference in the types of curves obtained, and to point out that death of the animal, in itself, is not associat­ ed either with hl|^i mitotic counts or with low mitotic counts. These features will be discussed more fully in the next section - 152 - of this paper. Animals fed S. aureus reached maximum counts of 1,10/s and 1,50/d in lTo.609 and No,627, respectively. No bacteria were noted in the hemoljrmph of ?Jo,609 throughout the entire time of observation; v/hereas bacteria were seen in the body-fluid samples from Ho»627 on the tv/enty-fifth day after feeding be­ gan, and continued to be present in increasing numbers ixntil death on the fiftieth day, when the bacterial population was exceedingly higli. Natural bacterial ijofections of two kinds were uncovered duriiig this work. Six specimens iirPected x^ith a micrococcus liad peak counts of 2,35/b, l,55i^, l.SOJij, 1,15/i, and 1.00/i. In most cases tlie period of observation was short due to the fact that the anilmais condition was not discovered tin- til tlie Infection was quite far advanced and was not placed under observation until tlie infection was seen in the cotirse of selecting nomal animals for experimental puiTposes, One nor­ mal animal. No,752, inocxilated by needle with bacteria from one of the infected animls reached a M.D.O. count of 1,70/2 within nine days after inoculation. In Figure IX are plotted tv/o curves: that from No,633 whose infection v/as present at the beginning of observation; and that from No,752 whose infection was the result of a needle inoculation. The two curves exhibit similar characteristics. 153 -

The other naturally occurring bacterial Infections were produced by a short rod bacterium, distinctly different from the micrococcus and easily distin(;;ulahed from it. Eiglit in­ sects afflicted v;ith this form v/ere oliminated from the stock aniimls and kept under observation. Maximum M.D.C. counts were as follows: 1.25%, Z.10%, 1.20?^, 1.45fo, 1,40%, and l,35/o. Two normal animals, No,660 and Ko,748, inoculated v/ith th3.s bacterium had liigh counts of 2*10fo and 1.05^0, respec­ tively. Figure X illiistrates graphically the results from No. 656 with a iiatural rod infection, and from No.660 with an in­ fection by way of a needle puncturo. Table X

Suinnia3?y of Data from Experiments v/ith. Bacteria

Cells Mitotic Rai3ge Mitotic Range Experiment Animal Condition ; Counts Days Co\mted {%) Control (^) Aftetr Terminated Ifumber : lSB.de Observed (xlOS) Period ^Treatment by 692 1/20 c.c. 11 16 S2 0.05 - 0.35 0.10 - 1.70 Death Bact, co35 suapensict t 691 1/20 c.c. 10 15 SO 0.05 - 0,10 0,00 - 0.40 Death B, 3Ubtf11 3 suspension 693 1/20 c.c, 3.1 16 22 0.00 - 0.15 0.00 - 0,35 Death S, mar- ^escens suspension 769 «• 12 15 24 0,10 - 0.15 0.10 - 0.40 Death 770 *• 10 14 20 0,05 - 0.15 0.05 - 0.45 Death 771 •• 13 15 26 0.15 - 0,30 0,00 - 0,35 Death 772 •« 11 16 22 0.15 - 0.35 0.05 - 0.40 Death

600 1/20 c.c. 4 10 8 0.15 - 0.35 0.45 - 0.45 Death S. aureus suspension

•• 601 4 10 8 0.20 - 0.35 0.25 - 0.25 Death

• • 602 4 10 8 0.15 - 0.40 0.55 - 0.55 Death 607 •• 6 9 12 0.20 - 0,30 0.45 - 0.55 Death Table X (continued)

Cells Mitotic Range Mitotic Range Experiment Animal Condition Goimts Days Counted i'/s) Control (%) After Terminated Htnaber Made Observed (xlO®) Period Treatment hy 608 S. au3?eus 11 16 22 0.25 - 0.30 0.40 - 1.45 Deatli keedle in­ oculation 615 32 51 64 0.25 - 0.35 0.10 - 1.45 Death 617 8 15 16 0,15 - 0,30 0.05 - 0,55 Death 630 42 50 84 0.10 - 0.15 0.10 - 2.80 Death 631 22 50 44 0,15 - 0.25 0,15 " 2»05 Death 632 20 27 40 0.05 - 0.15 0,00 - 1.85 Death 720 12 19 24 0,05 - 0.25 0.10 - 1.25 Death 609 Fed S. aur- 22 28 44 0.05 - 0.30 0.10 - 1.10 Death eus 627 38 50 76 0,05 - 0.05 0.05 - 1.50 Death

633 Natural 16 23 32 "• "" 0,40 - 2.35 Death coccus in­ fection 636 •« 10 12 20 0.20 - 1.55 Death 643 •• 2 2 4 0,60 - 0.80 Death 743 «• 10 11 20 0,40 - 1.50 Death Table X (conbimied)

Cells Mitotic Ravage Mitotic Range Experiment Animal Condition Counts Days CoTinted (Ja) Control {%) After Terminated Nisnber Made Observed (slO®) Period Treatment 754 Natural 12 13 24 0.45 - 1.15 Death ooccus in­ fection 756 #• 10 10 20 0,25 - 1.00 Death 752 "Coccus" 14 15 28 0.10 - 0.30 0.20 - 1.70 Death needle in­ oculation 645 Natural rod 16 16 32 0.15 - 1,25 Death infection 656 15 15 30 0.20 - 3.10 Doath 662 • • 10 10 20 0.30 - 1.20 Death 666 • » 19 25 38 0.15 - 2.45 Death 667 «• 14 18 28 0.45 - 1,35 Death 683a •• 5 3 6 0.o5 — 1.45 Death 734 •* 3 3 6 0.60 - 1.40 Death 736 •* 8 8 16 0.75 - 1.35 Death 660 "Rod" needle 28 38 56 0.10 - 0.15 0.20 - 2.10 Death inoculaticai 748 •• 15 18 30 0,05 - 0,20 0.20 - 1.05 Death Figure VIII

Curves from Animal Ho.631, No.632, and No,720 Inoculated vdth Staphylococcus aureus. E.IO ZOO

Dead ^0.50 O.QO

O.IO O.&O

0.50 0.40 0.30

O.ZO O. \o .^Irioqulqfe4 ' Dead lO I I IZ 13 14 !5 16 17 IS 19 ZO 2! 22 23 24 25 2G 27 2S 2? 3031 DAY5 Figure IX

Curve from Animal No,633 Infected with Coccus Bacteria When Taken from Stock Gage; and from Wo,75S, a Hoimal Animal Inoculated \T)ith This Same Bacterium. - 160 -

2.40

30

2. lO Z.OC

1. 00 1.80 I.70 l.GO O 1.50

i.30 Z I. SO y I.oo a 0.30 0.80 0.70

O.SQ Dead 0.40 / \ O.ZO • o.lo O.OO 1 z 3 A- 5 G 7 6 3 10 ;i IS 13 14- 15 ic. 17 16 15 SO 21 DAYS Figure X

Curve from Animal lTo,656 Infected v;lth Rod Bacteria Vftien Talc en from Stock Gage; and from Ko.660, a Horraal Animal Inoctilatod with Tills Same Bacterium. - 162 -

-—•

-—o^GGO

P\ I'l I \ I \

A\ r Dead7 I V \

I J \

x/ Inoculation, Dead

I I I I I I I L t 3 4 5 G 7 5 0 \0 11 It 13 14- 15 16 17 18 19 ZO 21 24 DAY5 - 163 -

K. Results Prom Animals in Certain ^HoloHc^rcoSHB:^

In addition to naturally occurrin^j bacterial infections, two' other pathological conditions were encountered among these insects. In one affliction, external symptoms were recognia- able; in the other, no discemable external changes manifested themselves until the very last stages of the disease. At various times when animals were being taken from stock cages, certain "arched" individuals, with the body convexly curved, were noted. That the specimens were not normal was easily seen. Counts v/ere made on the five of these partially paralyzed insects which were encoimtered. In all cases, mitotic ranges exliibited by them were hij^er than that from normal ani­ mals; the highest cotint v/as found in No«727, namely: 1.85;«, In other respects, as seen through the microscope, the hemo- lymph and its contained cells appeared normal. No bacteria were obsei*vedj the condition, however, was plainly seen to be as­ sociated with some derangement of the nervous system. Results from these specimens are recorded in Table XI and data from tv/o of them, No,717 and No,735, are plotted in Figure XI, (A). No nonaal .mitotic range for the different individuals is available since the symptoms were already present when the counts were begun. Because no outv/ard signs were present tmtil the later stages of the condition, certain roaches with abnormally vacuo- - 164 -

lated cells (See Plate I) could not be Isolated until counts vrero begun. Five indlvidmls with this peculiar type of body- fluid cell were found v/hile these investigations were being conducted. Results of the counts are given in Table XI and shoxv M.D.C. count per cent ranges of 0.95 - 1,60, 0.60 - 0,85, 0,55 - 2.05, 0,85 - 1,60, and 0,35 - 0,85. Complete data from two specimens. No.715 and Ho,716, are plotted in Figure XI, (B), P03?lods of obsearvation with insects afflicted with partial

paralysis or v/ith vacuolated hemolymph cells are ratlier short; in every case the exaininations were begun as soon as the con­ dition was discovered and terminated by death of the specimen. Table XI

Summary of Data from Aiilmals in Certain Pathological Conditions

Cells Mitotic Range Experiment Arjimal Condition Counts Days Counted (^) VVhile Terminated Uujjiber Made Observed (xlO®) Observed by 642 Paralysis 12 14 24 0.20 - 0.85 Death 717 6 8 12 0 0 2o ** 1« 85 718 3 3 6 0.50 - 1.20 735 8 9 16 0,40 - 1.35 757 4 4 8 0.20 - 1.10

651a Vacuolated Cells 2 2 4 0.95 - 1.60 Death 714 3 3 6 0,60 - 0.85 715 8 8 16 0.55 - 2.05 716 7 8 14 0.85 - 1,60 752 9 10 18 0.35 - 0.85 Figure XI

(A) Shows M.D.C. Counts from Two Animals, No.735 and Ho,717, Afflicted wltli Paralysis; (B) Shows H.D.C. Counts from TITO Specimens, Ho,715 and Ho,716, with Abnomally Vacuolat­ ed Homolymph Cells. - 167 -

Z.oo

I.80 !.70 .GO 1.50 1.40 !. 30 I.20 . lO GO X) 0.90

OA' O 30 715 aeo 735 7IG o. lO o.oo ^ 3 4 5 G 7 S ) ^ 3 4- 5 G DAYS 168 -

L. Results From Inflections of Sterile Nutrient Brotii Medium, or various Oohcentratibns of Peptone, And ofn^T^ Percent Beer

Viilaen attempts were first made to inject bacteria into the hemocoele of these roaches> the bacteria were suspended in the nutrient medium in v/Mch they had been grown. Therefore, con­ trol infections with sterile broth were also used on other aia- imals. Vifhen these latter insects exhibited i'ises in M.D.C. counts on the injection of this fluid, injections of solutions of peptone and beef extract, constituents of the broth medivm, were tried. The sturmiarized results are tabulated in Table XII Althoiagh all insects used were approximately the same age wel^it, and sljse, and althou^i amounts injected were approxi­ mately the same, the results vrf.th nutrient broth are not con­ sistently of the same magnitude. While there is no doubt that the injection results in a rise of the number of dividing cells, that rise luay vary from 0.55Ji, as in Wo,611, to 2,15J6, vri-th No.604; in all cases the maximum of the mitotic range af­ ter injection exceeds the maximum normal control limit. A typical curve, based on counts from No,606 is illustrated in Figure XII. Of the insects vdilch were Injected with five per cent beef extjract, the highest count (2.105'^) v/as reached by No.729; while the other two had maximum counts of 0.65/a and 1.40^, Results following peptone injections proved interesting 169 - in that concentrations from 0,10/o to 20,0/6 Imd the ability to produce rises in M.D.O, coxmts. The Biaximum effects seemed to be ISae result of Injections of 10,0^; the peak count of all trials being 1,60^ f3?om No,679 injected with this concentra­ tion. A typical cu37ve of the variations in M.D.O. counts af­ ter peptone injections is plotted in Figure XIII. An examina­ tion of results in Table XII seems to indicate, too, a graded effect, in direct ratio, to the strength of the injected pep­ tone, but this will need furtlier confirmation with additional examples before definite conclusions can be dravm about that feature. Control injections of sterile salt solutions produced no changes in the M.D.C. rate. Table XII

Suroraary of Results froni Injections of Sterile Ifutrient Broth Meditun, Peptone, and Beef Extract

Cells Mitotic Range Mitotic Range Experiment Animal Injection OoTints Days Ooiinted (JiJ) Dtiring (Ja) After Teisiinated Nxanber Made Observed (xlOS) Control Infection by 604 1/20 c.c. SO 37 60 0.20 - 0.40 0.35 - 2.15 Death nutilent b3X>tib. «s 605 6 11 12 0.20 - 0,50 0.45 - 0.70 Death 606 •• 27 54 54 0.15 - 0.35 0.30 " 1,95 Death 611 IS 15 26 0.20 - 0,35 0.10 - 0.55 Death 612 •• 8 15 16 0.15 - 0.35 0.20 - 0,80 Death

680 1/20 c.c. 8 IS 16 0.10 - 0.30 0.20 - 0.65 Death (Diir 5% beef ing molt) extract 685 11 14 22 0.00 - 0.25 0.20 - 1.40 Death •V 709 16 19 32 0.10 - 0,40 0.00 - 2.10 Death

628 1/20 c.c. 37 47 74 0,30 - 0.40 0.05 - 0.65 Discarding 0,1^ pep­ tone

629 1/20 c.c. 47 60 94 0.10 - 0.25 0.05 - 0.70 Discarding 0^5% pep­ tone Table XII (continued)

Cells Mitotic Range Mitotic Range Experiment Animal Injection Counts Days Counted i/o) During i%) After Terminated Husiber Made Observed (xlO®) Control In.lection by 649 1/20 c.c, 25 34 50 0.25 — 0.30 0.15 - 0.95 Death 2.0/0 pep­ tone 659 1/20 c#c# 44 52 88 0.05 - 0.20 0.05 - 1.15 Discarding 5,0?j pep­ tone

681 1/20 c.c. 17 22 S4 0.00 - 0,40 0 * 10 ** 1.10 Discarding 10^ pep­ tone 664 •• 27 32 54 0.15 - 0.45 0.00 - 1.35 Death 679 »« 7 11 14 0.15 - 0.30 0.10 - 1.60 Death 629a »• 6 8 12 0.10 - 0.25 0.15 - 1.20 Death

672 1/20 c.c. 15 19 SO 0.15 - 0.25 0.15 - 1.00 Death 1J5% pep­ tone

665 1/20 c.c. 15 18 26 0.15 - 0.15 0.00 - 1.10 Death 20/1 pep­ tone Plgiore XII

Ctirve from Animal Ho.606 Injected with Ster­ ile Hutrient Broth. .CO

1.60 I.50 1.4 O U 1 30

M

OGO 0.3 0 0.4 0 0.30 O. ZO

njecfed o.oo lO I 1 DAYS Figure XIII

Guj?v

02 0.50 U Cl 0.40 0.3 0 Dead O.ZO - O. lO O.OO I 2 3 4 5 G ( S 2) lO I I IE 13 14- 15 IG 17 IS O SO 2( 23 24 25" 27 28 29 30 31 DAYS - IVCJ -

M. ResiJlts Prom Aniinals Inlected V/ltii Ghlclt Embryo l^t:ra!c€7^1 th^^Tbrlmred Conr Hemoglobin Solution, or VW&IER!R Albumin

Table XIII contains the rosxilts obtained after injection of tJie above substances. Five insects were injected vdth extsracts of macerated chich embryo. The maximum coxmt appeared in No.676 with an M.D.C. percentage of 2.70 and a range of 0.30/o - 2.70/o; other ranges, in per cent, follow: 0,0 - 0.55, 0.05 - 1.70, 0.0 - 0,55, and 0,0 - 0.80. Two specimens injected v/ith small amounts of defibrinated rat blood reached maximum H.D.C, counts of 0,90^ in No.673, and 1,20% in No.740. Injections of a ten per cent solution of hemoglobin crystals \vere followed by high counts of 1,15/a in No,684, and 3.8^^ in Ho.708. Use of egg albijmin was complicated by the fact that the usual ateriliaatlon procedure could not be carried out without coagulation of the solution. Preparations had to be made with precautions against contamination, but v/ithovit actual sterili­ zation, In spite of great carefulness and many trials, only one animal was injected v/ith albumin v/ithout subsequent appear­ ance of bacteria in the hemol^nph. The results frcwa this in­ sect, Ko,668, are included in Table XIII, The mitotic range before injection was from 0,10^ to 0,40^; after injection, from - 177 -

0.35^b to 0.70/0. The dtiration of the exxjeriment v/as short - only nine days. Control Injections of salt solutions, such as Hobson'a, v/ero followed by no changes in the normal division rate. Table XIII

Summary of Data from Aniaiala Injected vd.th Embryo Extract, Blood, Hemoglobin, and Egg Albumin

Cells"' liitotic Range iaitotic Range Experiment AniTnal Injection :Counts Days Counted {%) Diaring (%) After Terminated Nmaber ; Made Observed (3J.0®) Control In.i ection by >

674 l/lSO c ft c • : 18 24 36 0.05 - 0.35 0,00 - 0.55 Death cMck extinct 676 «» 9 14 18 0.15 - 0.40 0.30 2.70 Death

*• 678 34 41 68 0.05 - 0.10 0.05 - 1.70 Death

»• 689 11 15 22 0,10 - 0,25 0.00 - 0.55 Death 699 *• 20 26 40 0.05 - 0.15 0.00 •w 0,80 Death

673 1/20 c,c. 8 14 16 0.00 - 0.10 0,00 — 0,90 Death deflbidnated rat blood

•• 740 IS 18 26 0.10 - 0.30 0.10 - 1.20 Death

684 1/20 cjc. 10% 27 35 54 0.10 - 0,35 0.05 - 1.15 Death heiQOglobin

«• 70S 6 9 12 0.20 - 0.30 0,30 - 3.80 Death 668 i/SO c.c. 10/4 6 9 12 0.10 - 0.40 0.35 0.70 Death egg al­

bumin • - 179 -

N. Results Prom the Use of Some Amino Acids, ParticuisS'ly Cysteln^^ glutathibrie^""oF'insulin

Five per cent solutions of aspartic acid, glutamic acid, glycocoll, alanine, tryptophane, and cysteine were used. Re­ sults are listed in Table XIV. Complete data are given only in the case of cysteine; tabulated data from the other insects into which amino acids were injected are limited to selected

examples showing some of the Mghest M.D.C. counts for the particular acid used. Significant Increases in M.D.C. counts resulted only in those specimens into wliich cysteine was in­ jected, It is true that the maximum range end-point seems to be shifted higher with the use of the other acids (see Table XIV), but with the exception of lIo.766 and its higja count of 0,55/j, the ranges after injection are all within the normal range limits of 0.0 - 0.60^$, This high count fiom No,766 is of doubtful, if any, significance, Nine insects v/ere used to test the effects of cysteine.

High M.D.C, counts, in per cent, are: 1,20, 1,30, 1,10, 0,60, 0,90, 2,50, 0.70, 1,15, and 1.0, Counts from No.682, Ho,700, and No,707 are plotted in Figure XIV. Maximum counts appear from four to seven days after the injection. All experiments v/ith amino acids were terminated by death. Figure XV allows the count record of animal No,696 inject­ ed v/ith five per cent glutathione. The maximum count of 1.10/a - 180 -

\ms repeated three times, namely, on the tliirteenth, foiar- teenth, and twentieth days after injection. Tahle XIV shows

ranges of 0.10 - 1,10/w for No.696, and 0,20 - 0,60/j smd 0.0 - 1,10/0 for No,697 and No,698, respectively. Of all the animals used, tiie highest M.D.C. count v;as ob­ tained from animal No,706 injected with undiluted 40 U. insu­ lin. This high count was 5,^0%, Results are tabulated in Table XIV and plotted in Figure XVI. Anotlier animal. No.687, injected v/ith diluted insulin had a maximum count of 1.40/L Table XIV

Stcmnary of Data from Injections of Various Amino Acids, of Glutathione, and of Ins'olin

Cells Mitotic Range Mitotic Range Esperinent Animal Injection Counts Days Counted i/t) Before (/O After Tei^ttinated Utanber Made Observed (xios) In.lection Injection by 759 1/20 ae. 5% 10 20 20 0,10 - 0,30 0.00 - 0,45 Death aspartic acid 760 1/20 c.c. 5% 12 22 24 0.15 - 0.25 0.20 - 0.50 Death glutamic acid 761 1/20 c.c. 5^0 19 27 S3 0.15 - 0.20 0.10 - 0«45 Death glycocoll 762 »• 13 19 26 0.10 - 0.10 0.05 - 0,40 Death 765 1/20 c.c. 5^j 11 16 22 0.10 - 0,15 0.10 - 0,40 Death alanine

•• 766 15 20 • 30 0.15 - 0.25 0.10 - 0,55 Death

763 1/20 C.C. 5/0 10 15 20 0.15 - 0.20 0.10 - 0,40 Death tryptoriiane' 764 •» 17 22 34 C.IO - 0.40 0.10 - 0.50 Death

682 1/20 C.C. 5/i IS 17 26 0.10 - 0.30 0.00 - 1.20 Death cysteine Table XIV (oontinued)

Cells Mitotic Range Ilxtqtic Range Experiment Animal Injection Counts Days Counted (;i) Before (/i) After Terminated Iluraber Made Observed (xlO®) Infection Infection by

636 l/SO c.c. 5?i 27 35 54 0.15 - 0.25 0.00 - 1,30 Death cysteine

•* 700 18 25 36 0.10 - 0.40 0.00 - 1.10 Death

«» 701 16 21 32 0.15 ~ 0,25 0.10 0,60 Death

«• 70S SO 27 40 0.15 - 0.30 0.00 - 0.90 Death

4* 707 9 12 18 0.15 - 0.25 0.20 - 2.50 Death

•• 710 12 15 24 0.15 - 0.20 0.10 - 0.70 Death

•• 711 11 14 22 0.10 - 0.15 0.10 - 1.15 Death

«• 712 15 18 26 0.10 - 0.10 0.00 - 1.00 Death

696 1/20 C.C. 10;?i 19 27 38 0.10 - 0,25 0.10 - 1.10 Death glutathicgie

•• 697 7 12 14 0.15 - 0.25 0.20 - 0,60 Death

• * 698 25 32 50 0,C5 - 0.20 0.00 - 1.10 Death

687 1/20 c.c.40U^ 26 32 52 o.co - 0.40 O.CO 1,40 Discarding insulin di­ luted 40x

706 5 1/20 C.C. 40IL 17 21 34 0.15 - O.SO 0.30 5.30 Death • insulin Figure XIV

Ciirves from Three Aniioals, No.682, No.700, and No,707, Injected with 10/S Cysteine. - 184 -

, a o . 7 0

1.4o I. 30 -Dead 1. 20

0,30 o. 30 0.1 o O. GO O 5 O 0.4 0

\ -

0.00 -J I <. ^ 1 I 1 I ! I 12 13 14 15 IG IT IS leiZOZI 22 23 DAYS Figure XV

Curve from Aiiimal No»G96 Injected with 10^ Solution of Glutathione. O.0 5

O.SZ

rS0.70

QGQ [- ZQ55. ^jQ50UJ C^q4^ Dead uJ Cl0.40 0.35 a 30 a E5 O.ZO O. 15

O. 1 O

0.05 In iecTed O.OO 12 13 14 15 17 16 19 EOEI 22 £3 24 Z5 ZG Z7 2S S-? DAYS Pisure XVI

Curve from Animl No*706, Injected with Undiluted 40U. In3\ilin. 188 -

4.4 O

4.00

Q 3.20

^ 2.80 H Z.GO u 2 40 2.2 O

1.40

In iected 3 4 5 G 7 8 ^ lO I I 12 13 14 15 16 17 18 19 20 DAY5 - 189 -

0. Results From the Injection of Dlnltrqplienol, IThyroxin^ Glucpse^ Lead cElorlde, or Rotenone

Data relating to the above experiments are collected to­ gether In TalDle 3CV,. Two metabolic stimulants, dlnltrophonol and thyroxin, v/ere tried. Three animals. No,688, Ho,702, No,,719, Injected with the former substance had peak counts of 1,50/i, 1»20%, and 0,95^0, respectively; fcvro. No.,705 and No,713, injected with thyroxin, had high counts of 1,90/d and 0,90^^, respectively. Several trials with injections of glucose v/ere attempted, but no rise in mitotic counts resulted,. A typical example, No,699, is recorded in Table XV,, Most of the animals treated with g3.ucose had to be discarded because of the subsequent ap­ pearance of bacteria in the hemolymph. Lead chloride lias been found by other workers to be in­ hibitory or retardltive toward karyoklnesis,. Injection into the roach, however,, seems to have no effect on the M.D.G,. rate. The per cent of indirectly dividing cells remained within the normal raiige after injection., until death from poisoning oc- ctirred, Tliree examples from No,,690, No,,694, aid No,,694a are recorded in Table 3CV. Injections of rotenone were vd.thout effect on the M.C.C. count,. In only one trial did the count rise slightly above the normal rangej this was with animal No.695,, recorded in Table XV, whose M.D.C, count after injection ranged from 0,0^ - - 190 -

0,55/0. Tlala slight rise is also of doubtful significance. Cable XV

Summary of Data from Animals Injected vrf.th Dinitrophenol, Thyroxin, Glucose, Lead G^oride, or Rotenone

• Cells Mitotic Range Mitotic Range Experiment Animal Injection :Counts Dficrs Counted ifo) Before {^) After Teiaiinated Huniber : Made Observed (xlO®) Injection Injection by • 688 1/20 c.c, 10/a: 24 S2 48 0.15 - 0.20 0.15 - 1.50 Death dinitrc^iiancS. 702 •• 17 21 34 0.10 - 0.25 0.05 - 1.20 Death 719 *• 11 13 22 0.25 - 0.25 0.20 - 0.95 Death

705 1/20 c.c. ti^ 16 20 32 0.25 - 0.25 0.05 - 1.90 Death 3?axin {Igc^a in 10 c.c.) 71S •• 15 20 30 0.10 ~ 0.15 0.10 - 0.90 Death

669 1/20 c.c. io;;i 5 8 10 0.05 - 0.20 0.05 - 0.20 Discarding glucose (Bacteria)

690 1/20 c.c. 1% 9 12 18 0.15 - 0.40 0.05 - 0.40 Death PBCLA

694 •• 13 17 26 0.00 - 0,25 0.15 - 0.40 Death 694a •• 5 7 10 0.15 - 0.35 0.10 - 0.35 Death

695 1/20 c.c. Ifo 14 20 28 0.25 - 0.35 0.00 - 0.55 Death rotenone ~ 102 -

P. ,?ot;al Hmber aM Per Cents of Dividing Cells

Of the 5,190,000 cells actually counted in the propara- tS-ona from tlie 175 insects Included in this thesis, 1,203,000

Y/ere from control animals or experimental ani:nala dtiring their control period of observation. Tho remainiiTg cells, 3,982,000, were from experimental and pathological specimens, and from a fev/ in certain types of physiological conditions. (See Plate I). In Table XVI are tabiilated the nviabers of cells found in the different phases of mitoses. Tolojihases wei'e encountered most often (8,053); prophases next (6,7SV); followed by ana­ phases (4,611) and metaphases (1,309). The same order holds v/hetlier seen in homolymph samples from control or experimental animals. (See Plate I). There were 1,353 multinucleate cells seen. By far the vast majority were bi-nucleate; 1,313; tiiirty-one Imd three nuclei; six had four nuclei; one had five nuclei; and tv?o had six nuclei. The per cent occurrences of the above are, respec­ tively, 0.0253, 0.0006, 0,0001, 0.00002, and 0.00004. Tliere seems to be no significant distribution of the multinucleate cells between those foiind in control samples and those in samples from experimental specimens. (See Plate II). Ajnitotlcally dividing cells v/ere rare; only 345, or 0.0066 - 193 - v;©re found among the entire 5,190,000 cells counted. Altliougli only about ono-tlrL3?d as many control cells as exporlmsntal cells Y/ere counted, almost Imlf of the total number of amitoti~ cally dividing cells recorded v/ere from the former group, (See Plate I). Table XVI

Total Ntariber and Per Cents of Dividitig and Multinucleate Cells Observed

Cells ;Propl mses:Met^)ihase3:Anapl oases :Teloi jhasea Ami^botic:Multii lucleate cf Animals Counted JNO. % :No. % ;No. :Ho. JO No. % :No. : • • All 5,190,000:6,727 0.150tl,309 0.026:4,611 0.089:8,033 0.154 345 0.00661,353 0.026 • • • • ContPcGls 1,208,000: 803 0.0^: 159 0.013: 519 0.043: 937 0.07S 112 0.0927 322 0.027 • • • • • « Experi- • • aentals 3,982,000:5,924 0.149:1,150 0.029:4,092 0J03:7,095 0.178 233 00059:1,031 0,026 • • • • • •m ~ 195 -

VII, DISCUSSION

Hov; iiaportant is the comatiiig of sufficient cells from each homolyriph sample, in studies of the kind, described, may well be pointed out in Table XV. Ilad the M.D.C. figures been based on counts of 100 colls, for example, as is often done vrith differential blood cell counts from vertebrate animals, the results v/ould have been entirely misleading - both frora the standpoint of hov; often no dividing cells Vifould be found, as v/ell as from the number of times counts of 1,0>^ or more would occur. As a matter of fact, using coiints based on samples of 2,000 cells, counts of 0,0% {or less than one mitotically dividing cell in 2,000) v/ere listed only 63 titties in the 2,595 counts made; of these, 20 v/ere from control ani­ mals, and 43 from experimental or abnormal insects. Likewise, counts of 1,0% or greater are relatively high in the co-unt range when based on 2,000 cell samples, being two or more times greater than the maxinjum of the normal range. But, counts of l.O^o or greater v;ould sometimes actually be the max­ imum for the normal M.D.C. range if based on coujits of 100 cells. To a lesser degree, but, nevertheless, of nearly equal Importance, the same argmnents hold for not basing dividing cell frequencies on counts of 500, 1,000, or 1,500 cells. Naturally, as the number of cells used is increased, the chance ~ 196 - of determining a misleading datijm ia decreased. As shown in Table IV, however, a count of 2,0U0 cells seems to "be a suf­ ficient nuDiiber upon v;hich to base data of soiue moaning; to count more, for the most part, only increases the accuracy in the fourth or fifth decimal place. Table V shows that the technique of counting employed in these researches is dependable and gives results which ceoi be duplicated within reasonable limrlts. At first glance, a dif­ ference of 0,10/6 betv/een range end-points for a set of tliree counts seems largo, but it must be remembered that the divid­ ing cells in a normal animal are comparatively rare and a chance observation of one or tv/o more mitotic cells may make considerable difference in the end results. This difference in range end-points of duplicate, counts from the same hemo- lymph saaiple or from different samples from the same insect may also be an indication tlmt the dividing cells are not even­ ly distributed throughout the body hemol^/mph, or if equally distributed there, become unequally placed on the slide in the process of preparing the mount. The fomer alternative is probably the more likely since there seems hardly a v/ay in v/hich the dividing cells mij^t become isolated or segregated during the talcing of the hemolytnph sample, during the mixing of the hemolyipph with the diluting-staining fluid, or while the cells are settling to tlie top of the slide. Considering the large amount of data collected from nor­ - 197 - mal control miimals and from the control periods of obsea^va- tion from insects destined for experimental work, it seems quite evident that the nomal specimen of Blatta orientalis. be it male or female, adult or large nymph, has a mitotically dividing cell rate somewhere v/ithin the liBiits of 0,0 to O.SOJi). Counts of 0,50^ are rare - not more than three occurring in the 604 control counts; and, never, with all 124 normal ani­ mals used, was a value of 0,50% found on the initial count. The extent of the range of average counts, from 0,116/3 to 0,250ya, might lead one to suspect some factor or factas of influencing various Individual specimens. However, high and lov/ averages were found, iiidiscriminately, among males, fe­ males, adults, and large nytaphs, with no correlation to the sex or age factors mentioned. Thus it seems that a liigh or low average M.D,G. count is a specimen clmracteristic allied with individual variation. A significant feature to notice in Table V is connected with these figures; the average M.D.O. value of 0,186"! from all counts from the entire group of control specimens; the average of 0,196/^ from the Initial count of all 124 normal in­ sects; and the average of 0,181^o from the three control counts made within the first five days during which all normal ani­ mals were kept before subjected to experimentation. The close agreement of these figures provides a three-way check on the average M.D.O. value, which can be said to lie, approximately. 198 - within the limits of 0.180 to 0,200%. Most of the control specimens were retained only as long as needed for comparison with restilts from experimental Insects kept at the same time, but a few were continued over quite long periods - much longer than any experimental speoiaiens were maintained. One male nymph was kept 71 days; another, 48 days; a male adult, 72 days; two finale nymphs for 132 and 63 days, respectively; and a female adult for 48 days. These were all in good condition as long as the counts were continued, and so indicate that the methods of handling, feeding, and cag­ ing were satisfactory; and, more important, that the slight necessary hemorrhages concomitant v/ith taking of hemolymph samples, in themselves, did not produce any superficially evi­ dent damage to the insect, or any harm or effect v/hlch might be reflected in the liemolpnph picture. So far as indicated by the studies made in this research, M.D.C. values seem unrelated to sex and age, the latter v/hen restricted to the age of large nymphs and active adults as used in these experiments. No investigation was made of the

M.D.C. counts at different instars, but in view of the changes taking place at ecdysis (these will be discussed later) when the animal undergoes rapid growth, it may be that the younger, more rapidly grov/ing instara have average counts v;hich differ from those of older roaches. Likewise, no linlcage between

M.D.C. figures and fluctuations in daily laboratory tempera­ - 199 - ture v/as uncovered. (See Figure II). TMs factor, by chance of location and time, was rather thoroughly tested "because the counts were made over a period including winter cold and sunrmer heat with variations in room temperature from 17 to 32®C. Since Tauber and Yeager (545, 546, 547) and Yeager and Tauber (606) previously pointed out that high total hemolymph cell counts seemed to be associated with egg formation and de­ position in various species of insects, a close check was made on the possibility that the some processes might be connected with changes in dividing cell numbers. No deviations from the nonnal rate v;ere found to take place, however, eitlier before or after the ootheca was deposited. Some females were put un­ der observation as long as five days before deposition and con­ tinued for as long as eleven days after, with no change fi-om the normal M.D.C, rate curve. (See Figure III). On the other hand, records from molting animals proved to be extremely interesting because of the effects that phenomenon has on the normal percentages of dividing cells, /^ee Pig\xre IV, (A) and (Bjj. one accustomed to observing the usual ap­ pearance and habits of specimens of Blatta orientalis. those approaching ecdysis can often be recognized by their sliglitly bloated appearance as well -as by the softness of the body - the exoskeleton seemingly not being as rigid or as firmly at­ tached to the enclosed tissues as normally, A fev/ of these were recognized in the stock cages so that there is one spec- - 200 -

Imen wMch was put under observation five days before ecdysis, but most of those on which there are data before molting were not isolated until a day or two before jdiedding the exoskele- ton. Twenty-five of the thirty molting specimens used her© were placed under observation either while undergoing ecdysis or within a few hours after the procedure had been completed. Figure IV (A) shows the general trend followed by the M.D.G. rates from molting animals. The act itself is preceded by and accompanied by a dlstxnct decrease in the number of di­

viding cells. Of the twenty-five specimens included at "M" on the graph, eleven had counts of O.O^j; ten, of 0,05/i; two, of O.lO/oj and two, of 0,15^. All of these values are within the lower third of the normal M.D.G. range, while the majority are in the lov/er tenth of the range. After ecdysis the coiant tends to remain low for about a day, then begins to increase on the second day, and usually reaches an optimum count on the third day after molting. Two specimens reached counts of l.O^^j; four of 0.80^; one, of 0,15%', six of 0.70^; and the remainder, counts of 0.65/u' (the average of the thirty animals for the tliird day after molting) or leas, none falling below 0,25% which is the mid-point of the normal range. The values on tSie fourth day are definitely decreased, although six of the thirty had counts above or equal to the laaximum of tiie normal liisiits. On the fifth and following days, all counts have come down within the limits of the normal M.D.C. - 201 - range. No explanation for these fluctuations can bo offered at the present time. It has been noted that this species ceases to eat and becomes, in the main, more inactive Just before ecdysis. The decrease in M.D.C. at the same time is probably associated v/ith this general let-up of activities. There is, liovrevex', no clue as to what initiates tliis slowing of activity. In considering tilie rise in M.D.C, rate after molting, there is a possible explanation other than that this post-molting per­ iod is allied with a general body gz>o\7th. Including an increase in the number of hemolyraph cells - a fact tliat was guessed at by Tillyard (550), Landois and Landois (315), and Graber (183); and established by actual counts by Yeager and Tauber (606) and Tauber and Yeager (545, 546, 547), This other explanation is linked v/ith the possibility that the cytolytic prodvicts from the breakdown of connective tissue before and during molting contain some protein derivative v/hich is stimulatory to cell division, just as cysteine injections (See Figure XIV) result in pronounced rises of M,D.G. values, Reiche (465) has shown that tissue autolysates favor mitosis. Of course, it is not inferred that tlais product of cytolysis is cysteine or that It is selectively stimulatory to the hemolymph cells alone; other potentially multiplying cells might probably react similarly. Counts f3?om control animals have proved that the slight losses of hemolymph., which the talcing of hemolymph samples en­ - 208 - tails, are of no effect on the M.D.C. values. However, Plgu??© V shows that 3lov/ "but extensive hemorrhage may result in sig­ nificant increases in the percentages of Icaryokinetic colls.

V/hat sets the mitotic mechanism to v;orlcing cannot be explained at present. Since it seems unlikely that this insect has def­ inite compact hemopoietic tissues, tlie mechanism probably v/ould not be comparable to that which stimulates the bone marrows of vertebrates after henaorrhage. Hemopoietic activity is more probably diffused throu^out the circulating cells of the in­ sect. The resialts from specimens subjected to successive counts accompanied by large loss of hemolymph are particularly valuable in deraonstrating that hemorrhage must be quite exten­ sive before any variation in tiie LI.D.C. figure reveals itself. Gold has long been knovm to be retardative to coll divi­ sion, just as Indicated in Figure VII; but more interesting in the present examples is the sudden idse of M.D.G. values when the animal is returned to room temperature, subsequent to treat­ ment with cold, A plausible explanation may be that those cells ready to divide are hold in check and accumulate in the hemolyo^h during the cold period; then, when brou^t into v;armth, rapidly complete their division. This is borne out by the records which show that the rise is not prolonged and that the count rapidly comes back to the normal limits. The failure of tMs particular Insect to withstand cold is not unusual because those roaches vdiich liave become accustomed 203 - to living indoors are, for tlie most part, war-mth loving, l'ii6 increased covmt from irdivlduals at 37°C, v/as more or less expected since the application of heat, up to the optimum temperature, has been often sliown to speed cell division. In some cases this has been found to be due to the inci'eased con­ centration of toxic products reatilting from the increased rate of metabolism at the higher temperatures. This may possibly be the explanation for tlie results obtained with this insect. An obsei''vation v/liich may be significant in tliis connection v/as noted in the abnormal vacuolation wliich became evident in the hemolymph cells of ardmals at the high temperatures. Gytolo- gists often interpret increased vacuolation as a sign of de­ generation, particularly of boginning cytolysis brouglit about by toxemias of various kinds. Gu6not (108, 109), in another coniiection, has also noted an increase in hemolymph cell vacuoles in degenerating cells from the inaect, Dombyx trifolil. Prom the difficulty met \iritli in trials to inoculate specimens of filatta orientalis with Bacterium coli. Bacillus subtilia» or Serratia marcescens. it seems reasonable to con­ clude that tMs insect has quite an efficient mechanism to com­ bat some bacteria which may enter the hemocoele. Such large amounts of culture had to be injected that the animals died be­ fore data of any value cotild be obtained, except in tv/o cases, one each of coli and sub tills. but none vd.th marcescens. On the contrai»y, susceptibility to inoculations of Staphy- 204 - lococcus aureus is so pronouncad that the introduction into tho hemocoelo of a naodle wet vd,th the culture is sufficient to in­ fect th.e contained body fluids, Accoinpanying the Increase in the nuniber of bacteria is a rise in the M.D.C. values. At least two explanations may be put forv/ard for this result. First, since somo of the liemolytnph cells are phagocytic, the

presence of the bacteria may stimulate the fo3?iaation of phago­ cytes, just as in vertebrates; or, second, that the bacteria throw out a toxin which, like most toxins in dilute concentra­ tions, stimulate the hemopoietic activity of the circulating cells. It is Impossible to say wMch is the real exx)lanation; the increased karyoldnetic figures may be due to a comblimtion of the tv/o causes mentioned, or may be due to an entirely dif­ ferent factor. As the Infection v/ith aureus becomes more pronounced, var­ ious symptoms begin to appear. Food is taken in small quanti­ ties, if at all, v/ith the result tlmt a general body weakness follows. Movements are slow and feeble. If placed on its dor­ sal surface, an afflicted specimen is often so sluggish and v/ealc it cannot right itself. Later there Is evidence of a real neurotoxin: a progressive, anteriorly moving paralysis sets in; the meta-thoracic legs are affected first and when tlae ani­ mal is stimulated to move, this pair of legs is useless and dragged along behind; the anterior part of the body starts to Jerk spasmodically; the mesa- and pro-thoracic legs become tise- ~ 205 - less and tiaey, v/ifch the last pair, are folded close under tlie body, aasxanlng tiao same positions as v/hen the animal dies; the body begiJis to arch, v/ith the dorsal sijrface convexly cta?ved; tlie head folds dovmward under the pronotum and its append-ages move slov/ly and ierlcily. During the latter part of tliis series of symptoms tlie animal aeema unable to eat or drink and vrlll not talie water ovon if a droplet is placed on its mouthparta. Internally, other chsjagea occur in tlTS hemolyniph in ad­ dition to the Increase in the number of bacteria. Tlia cells be­ come filleiJ'. v/ith vaciioles, stain more easily and deeply, and bocome "ragged". Cellular disintegration with its debris be­ comes evident. The hemolymph becomes thicli: and millcy-v/hite v/ith bacteria, and Just before deatii sometimes turns a yellov/lsh- or orange-brown. This insect apparently cannot overcome even sli^t infec­ tions vri-th Staphylococcus aureus. In some cases, the infec­ tion is held in check for a considerable time, but eventually the bacteria become dominant. The course of tlie M.D.O. counts from infected aixlmals seems to follow no pai'ticular scheme. Pigvire VIII shov/s three dif­ ferent types of curves. lTo,631 has a record that is very er­ ratic, v/ith a slow rise in counts until the twelfth day after inoculation and a subsequent series of rises and falls for six­ teen more days. Ho.652 also possesses a curve with a slov/ in­ crease until the eighth day whan a sudden rise took place. For - S06 -

eleven days tMs level, approxlioately 1.75/i, vias maintained at a plateau, followed by a sharp decrease until death. Wo,720 shows a series of counts which remained vAthin the normal limits for seven days after inoculation, then rose suddenly. Just what malces different animals react dissimilarly is not Imown. All received approximately the same initial infection, and all \7ere approximately of the same age and weight. Indi­ vidual susceptibility, just as in other animals, may be the ex­ planation. The wide fluctuations of values in some animal can hardly be explained in any other wa:ss except that the variations, for some reason or other, ^ occur in the animal; or that the distribution of dividing cells is not equal. As stated in the previous section of this paper, the three graphs of Figure VIII were selected also to show that death of the aniicals was not corx»elated with any special count-level or trend. In one case death followed a decline in the cotmt, but still at a point imich above the normal range; in another, death follov;ed a sudden decline but at a count that was well with nor­ mal limits; and in the third example, death came when the ani­ mal's M.D.C. cotmt was close to its maximum. An examination of other curves in the various figures and of counts in the different tables will shov; similar non-correlation betv/een death and dividiiig cell levels. An observation of some importance relating to deatli and bacteria, however, was made. It lias already been mentioned - 207 - that in aOEie cases tMs insect seems to have an effective means of oombatln(s bacteria which enter its body. Another fact tliis connection \7as often seen in insects which had died, but previous to death had had no visible bacteria in the hemolymph. In many cases, although the animals could not have been dead more than a few hours, or at the most, ten hours, the hemolymph would be tliick with bacteria. No inoculations had been made, but apparently a latent or controlled infection of some kind had been present all the time, and broke out as soon as the checking mechanism had lost the ability to function. The rapidity with which tlie hemolymph would become filled with bac~ teria was astotxnding. A clue as to how natural bactei»ial infections may be spread throu^^out a group of insects v/as furnished by the records from animals No.609 and No.627. (See Table X). These tv;o v;ere fed bread wMch had been soalced in twelve hour cul­ tures of Staphylococcus aureus. Ali2aough no bacteria were ever seen in the hemolymph of No.609, bacteria did appear in No,627 after twenty-five days of feeding. Cultui'lng of bactei'ia iso­ lated from the hemolymph of this insect proved them to be Staphylococcus aureus. Thus it is clear, when one remeiubers the cannibalistic habits of the roach, how natural infections can be spread from one animal to another. It is Hkely also that bacteria may enter through breaks in the exoskeleton, es­ pecially at the time of molting, Kraus (305), working vrfLth a - 208 ~ locust lias also shovm that this species can be infected by feeding bacteria; he used Coccobacillus acildioruin, an ento- raophyte found principally in grasshoppers. In addition to the above concltision regarding the spread of infections, the record from No»609, which also had a M.D.C. coxmt considerably above the normal level, althougji no bacteria v;ere present in the hemolympla, indicates tliat bacterial toxins absorbed from the alimentary tract are capable of affecting the dividing cell rate. The body-flxiid cells had the same vacuolated, partly disintegrated appearmace that had cells from hemolytnph containing bacteria. Symptoms in those animals infected with eitlier of the two natiirally occ\irring bacteria are much like those already described for Staphylococcus aureus. The same loss of appe­ tite, sluggislmess, paralysis, and cytolysis of hemolymph cells are present. One notable difference was seen, particularly vdth those infected vAth the rod bacteria, which seems tiie more viinilent of the two. In the last stages of this disease, tlie conjunctival folds at the various joints of the body break and the thick, white hemolymph oozes out. This was noticed partic­ ularly at the joints of the meta-thoracic legs and in the folds between the dorsal abdominal plates. Both the coccus and the rod infections could be easily transferred to a normal animal. The increase in the number of bacteria when inoculated into a new animal was very rapid and - 209 - v/ltMn a fev/ daya would be followed by an increase in tho di­ viding cell rate. Figures IX and X shov/s the results obtained from four series of these counts. Althou^ those animals sviffering from paralysis, without the presence of bacteria in the heraolyinph, had syoiptoras that resembled in many v/ays the extei^nal symptoms in animals infect­ ed with bacteria, the differences, internally, were quite dis­ cernible. No abnormality of any kind (except a high M.D.C. count) could be found in tho hemolym,ph; no cells with many vacuoles, no evidence of coll disintegration, no heavily stain- ii-sg cells v/ere seen. Tlie only possible explanation available in view of the information on hand is that a heavy infection of some kind was present in the aliiaentai^y tract and killed the animal before being able to break throu^ tlae gut wall to make its presence known. Toxins, liov/ever, v;ere absorbed, just as in the case of animl Ho.609 wMch was fed StaphYlococcus aiar- eus. Viflmtever the cause, the result could plainly be seen to be lirjked in some way with a deranged function of the nervous system, similar to that found in animals with a very evident bacterial infection of the hemolymph. Even more baffling is an explanation of the results from animals having abnormally vacuolated cells, v/ith no other syii^tom, eitJrier external or internal, except a feebleness in the last stages of the affliction. Wo signs of paralysis v;ere observed so it is not likely that the trouble was bacterial in 210 - origin, auch aa described above. ITo explanation can be offer­ ed at tills writing. Dustln's (129, 130, 131, 132, 133, 134) experimQnta with the injection of peptone, albumins, sera, and other protein or protein derivative containing substances into mice in order to study the resulting increase in the number of dividing cells in various tissues were amoog the first and moat important of this nature. Ho satisfactory theory as to the reason for the phenomenon has ever been brought forward. Dustin' s rather vague 3?eferonces to a "regulatory mechanism of defense" are of no value in trying to find an explanation of the phenomenon. However, a lead may be fo\xnd in the recent investigations Ilammett (218, 219, 220, 221, 822, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235) has undertaken in connec­ tion vd.th the part played by sulfhydryl (-SH) compounds in stimulating cell division. He believes that sulfur in this form is the essential stiimilating factor governing cell divi­ sion and has acc\an.ulated a mass of reproducible evidence to support his theory. Assuimaing that Hoimnett's contention is correct, it seems plausible that such substances as peptone and beef extract may be stimulatory to the Icaryoldnetic pi'ocess be­ cause of the presence in them of glutatMone, a di-peptid con­ taining glutamic acid and cysteine, the latter having as a part of its malceup the sulfhydryl radical necessary for the de­ velopment of the suggestion which is to follow. - 211 -

So far as the results from these investigations hold, the siiggestion that the -SH radical is stimulatory to mitosis is a reasonable one to make. That it is the essential stimulus, as Haimnett says, is not entirely proved, and seems somewhat doubt­ ful since sulfur compounds not containing this element in the sulfhydi'yl form, in some cases, also stimulate mitosis. It is lonovm that all proteins, except protaanilnes, contain sulfurj in many cases, hoiirever, the sulfur linlcage has not yet been v/ork- ed out. Be that as it may, however, Table XIV and Figure XIV show how injections of cysteine are followed by rises in M.D.C. values, as much as to 2,5^ which is five times the maxiimm of the normal range. Likewise, Figure XV and Table XIV shov; that injections of glutathione produce a rise in karyolcinotic cell percentages. Glutathione, as stated previously, is a di-pep- tid consisting of cysteine and glutamic acid. Table XIV also shows that injections of glutamic acid, and other non-sulfur containing amino acids, have no effect on the M.D.C. values. Cysteine, then, must be the responsible agent, so far as tlae sulfhydiyl radical tlioory is concerned. To continue further: , glutathloiie is a constituent of mxiscle and blood punter and Eagle (S72J7'« Tables XII and XIII and Figures XII and XIII show that injections of rat blood, beef extract, peptone, and nutrient biHDth all produce increas­ ed per cents of dividing cells. Could these results be due to the presence of glutathione in these substances? - 212 -

Murray (387) has dontonstratod tliat the glutathioiie con­ tent of cMck embryos is invei-sely propertioiial to age, and Yaoi (602) has shown the aarao to be tnis for rat. Table XIII shows that injections of niacerated yotui^ chid: embryos into Blatta orientalis causes the mitotic rate to go up, aa liigh as 2.7/^0 wh3,ch is more than five times the maximum of the normal M.D.C, range. Could tM.s rise also be due to the glutathione content of the embryos? The presence of sulfm-- in instilin lias been ioiovai for some time, but there still seems to bo saaio question as to the fOMi of this constituent. Jensen and Evans (282) state that in tiie 88;^ of the insuJAn molecule so far identified are found cystine, tyrosine, glutamic acid, leucine, arginine, histi- dine, and lysin. This v70vi3.d make it appear that all the sul~ fu.r is present as the disulfide and not the sulfhydryl linkage, but Unminett refers to Vign-eaud's (567) results which indicate tilmt part of the sulfur may be in some other form, perhaps the 3ulfhyd3?yl linlcage. Vihatever its foi^a, insulin seams to pos­ sess some property which has the ability of increasing the per cents of dividing cells in the ioemol^imph when injected into the Oriental roach. The highest M.D.C. count in the entire grotip of 2,595 counts considered in this papei^ was S.SO/o from a specimen injected with undiluted instilin. This count is over ten times the maximum of 0,50/J at the upper lianit of the normal ran^e. The data farom this animal can be fotuTd in Table XIV and - 213 -

Pig-ure XVI. Although there is also considerable uncertainty as to the forra in which sulfur occurs in albumins, it is Imovm that this class of simple proteins is high in this olemont. Table XIII aiiOT/s tlmt injections of albumin solutions restilt in an in­ creased M.D.C. count, so it may bo that some of the sulfur is in a form that is ntiraulatory to mitosis. Another class of aimple proteins vAiich contains sulfur la the his tones, an example of v/hich is found in the glob in part of vertebrate blood pigment. Here, again, the exact sulfur combination is not Icno^m, but it, too, may be in such a form to be stimulatory to the karyokinetic process. In reviev;ing, briefly, this subject of suDjCur st3.mulation it miGht be pointed ou.t that the investigations described above show that a nunil^er of sulfur containing substances seeai to be able to produce a rise in the mitotic index of heraolyc^oh cells from the particular insect used. Incltided among these aulfixr possessing materials are beef extract, peptone, nutrient broth, glu.tathione, insulin, vertebrate blood, hranoglobin, cMck em­ bryo extracts, and cysteine. In some of these, as in gluta­ thione and cysteine and in others in vjliich either of these oc­ cur, the sulfur is Icnov/n to be present in tlie form of the sulf- hyd3?yl (-SH) radical; in otherD there is uncertainty as to the exact nature in which the stilfur is arranged in the various molecules. - 214 -

Ho suggeation is offered at the present time in explana­ tion of the rise of M.P.C, counts after injections of dinitro- phenol or thyro^cin. Both are drugs which cause an increase in the metabolic activities of an-inal organisms. More is Iniovm about the action of thyroxin tlian about dinitrophenol because the use of the latter to increase the B.M.R. of humim beings is comparatively recent; bixt in neither case has sinytMng been offered v;Mch raicht explain its abilitj- to cause increased mitotic activity in the insect's hemolymph cells. The only clue at present may be tlmt the toxic products of Increased metabo­ lism in some vra.y stimulate the mitotic mechanism. The appearance of bacteria laa tlie majority of animals in­ jected with glucose leads to interesting conjectures. Were the bacteria introduced with the injected solution, or were they present inside the body and did the presence of the glucose make favorable conditions for their multiplication? The for­ mer inference seems "unlikely since the glucose solutions and instruments v;ere sterilized and r^o stich difficulty appeared after similar injections of other materials. Seemingly the iia- sects possessed, somev/here in the body, a latent infection, held in check by some meclianism, but unleashed by the presence of the glucose, which hapx^ens to be an excellent food-stuff for bacteria. The results lead to a possible surmise that many of these insects contain bacteria in the hemolyrnph (or in closely associated tissues), even though not evident in nomal animals. - 215 -

TMs s-U£;gostlon is svipported "by the rapid development of bac- toria in the body fluids of many specimens after dentb.. • Hov; did those bacteria get there? V/ere they present all the time, but gained the upper hand only at the death of the organism?

IlGgative results vTith lead ch3.oride are unusual in viev; of

the many times lead salts have been reported to be rotardative

to nitosis. Since these particular injections were all of a

solution v;ith the sarao concentration of lead, and more or less

of a proliv-ina3:>y natxire, it is suspected that the concentra­

tion v;as leliial and 3d.llod the insects before any effect on

the division I'ate could be revealed. Rotenone is an insecticide v/Mch is becoming more populai* as its properties and usages are discovered and developed. It v/as used in this series of researches mainly to find if its letlaal properties v/ere connected in any way -v^th effects on the hemolymph picture. Ho disturbances were noted. A consideration of the data in Table X^/I leads to a num­ ber of interesting observations. First it mtxst bo assumed that it is possible to ascertain relative duration of tlie phases of mitosis by noting the actual freqi.iency with wMch the var­ ious stages are found in examined preparations. With that as- sianption^ it is possible to conclude that tlie diu'ation of the different phases is identically the same ks tlie relation be­ tween the freqiaency of their occurrence. In the 2,595 counts made, 8,033 telophases, 6,7S7 propliases, 4,611 anaphases, and - 216 -

1,309 metapliasos were encountered. This means that the dura­ tion of these particular stages of division are in the same or­ der as given above; that is, the time talcen by a cell in going throu^ the telophase stage is longer than that necessaxy to ccanplete any other phase. This is not in agreement with seme observations made on other types of cells in which the prophase stage has usually been found to be the longest phase. However, so far as is knovm, this Is the fix'st observation of cell-divi­ sion phase frequency made on insect hemolymph cells and it may be that the phase times in them is not idontical v/ith those in dividing echinodeim eggs, or snail eggs, or tissue culture cells. It can also be seen that the metaphase is apparently the shortest phase, while the anaphase is next in length of time necessary for completion. These tv/o relationships agree v/ith observations made on other kinds of cells. According to the frequencies observed here, the duration of a telophase is approximately six times tliat of a metaphasej about 1.7 times that of an anaphase (which Is 3.5 times that of a metaphase); and about 1.2 times that of a prophase (which is five times that of a metaphase). A metapliase pe3?iod is about 0,16 times that of a telophase; 0,2 times that of a prophase, and about 0,3 times that of an anaphase. Some workers on insect hemolymph cells have made a point of distinguishing types of cells by their mode of division. - 217 -

Ho distinction could be aeeii in tliis species; cells of all sizes and types divided by mitosis, but usually the very large cells divided directly. Amitotic divisions were seen so rare­ ly that this method of multiplication could be of little use in maintaining hemolj/mph cell populations. Only 545, or

0.0066iiS, directly dividing cells were seen, (See Plate I). Multinucleate cells were usually quite large - the size increasing \vith the nuniber of nuclei. (See Plate II). Several cases of nuclear division, vrlthout any Indication of a cyto­ plasmic fission to follovr, were observed, (See Plate I). These vrere probably examples of the formation of polynucleate cells. In most of these large cellular bodies, however, tlie nuclei seamed fully formed and in the resting stage. No ex­ ample TiTas seen in which a nuclear fragment of a bi-nucleate cell was in the process of dividing to form a tri-nucleate cell, although some of tlie latter were encountered, Plate I also shows other cells which were observed, Uo attempt will be made here to classify these bodies. - 218 -

VIII. CONCLUSIOKS

1. Sffiiples of 2,000 cells are entirely satisfactory for detemining the per cent of mitotically dlvidins cells found in the hemolymph cells of the insect, Biatta orientalis. Counts based on less than this number are lilcely to be erroneous; counts founded on more than 2,000 cells usually increase their accuracy only in the fourth or fifth decimal place. 2, Mitotically dividing cell counts detexroined by the method described in this paper can be duplicated on the same sample of hemolymph, or on different samples of hemolymph from the same an3.mal, v/ithin reasonable limits. S, Nonual specimens of B. orientalis have a M.D.C, count between Q,Q% and 0.50>i. Counts of 0,0/a occ\ir about tliree times in 100 comts vihen based on sasiples of 2,000 cells; coxints of 0.50/j are rare in noiroal animals, occurring about three times in six hundred counts. 4. Average coxmts based on control aniirals kept for var­ ious lengths of time range from 0.116/^ to 0,250^5 with an aver­ age of approximately 0.190/1. 5. Control animals cared for and handled as described in this paper can be kept for over four montl'is vdthout showing any effects of the treatment. This is much longer than it was fovind necessary to retain any experimental animal. 6. Sex and age, the latter restricted to the age of large - 219 - nymplis and active ad\ilts, have no effect on the M.D.C. rates of these insects. 7. Slight hemorrhages such as those accompanying the tak­ ing of hemolymph samples are not reflected in any way in chajiges of the dividing cell rate. 8. Extensive hemorrhage over a short period, however, causes an increase in the mitotic cell count. 9. Daily room temperature fluctuations are not correlat­ ed with fluctuations of the M.D.C. rate. 10. Egg formation within the body of the female and deposi­ tion of the ootheca are not associated with any deviation from the normal karyokinetic cell count-time curve. 11. Records from molting animals are characterized by de­ creased M.D.C. counts for a day or two preceding, and during, ecdysis; by increased counts above the normal range on about the third day after molting; and a return to vdtliln the noxinal limits on approximately the fifth day after shedding the exo- skeleton. The rise in the co\int is associated with the general body grov/th after molting and may be due to a stimulation of the mitotic apparatus by some product of cytolysis from the breakdown of tissues during molting. 12. Subjection to cold (5°C.) results in a decrease in M.D.C, rates. 13. Subjection to heat (3V®C.) tends to produce a gradual rise in dividing cells. Hemolymph cells from insects kept at 220 this temperature become increasingly vacuolated; this may "be a sign of degeneration, as interpreted by many cytologists. 14. Judging from the difficulty associated v/ith attempts to make Bacterium coli. Bacillus aubtilis. and Serratia marcos- cens gi>ow in the hemolymph of B. orientalis, it seems tliat this insect lias a strong defense mechanism against the multiplica­ tion of sOEie bacteria in its body fluid. 15. B, oilentalis is hi^ily susceptible to infection with Staphylococcus aureus. Accompanying the increase in the num­ ber of bacteria is a rise in the M.D.C. values. 16. Infection v/ith Staph, aureus is liiilced v/ith a definite set of symptoms: loss of appetite, sluggishness, an anterior­ ly progressing paralysis beginning v/ith the metathoracic legs and terminating v/ith the whole body convexly arched. Inter­ nally, other signs appear: the hemolymph cells become filled with vacuoles, stain more easily and deeply, and shows signs of general disintegration. The hemolymph becomes milky v/hite with bacteria and may become darker just before death. 17. Death, in itself, from any cause - natural or other­ wise ~ is not correlated with any chaiige in M.D.C. values.

18. Multiplication of bacteria in the insect's hemolymph is exceedingly rapid after death. Even though no signs of in­ fection may liave been noticed during life, bacteria often ap­ pear soon after the aniinal dies. This seems to indicate that most of these insects are infected \7ith some bacteria whose 221 -

Qvovrth. is held in check during tiie life of the individual. 19. Bacteria, such as Staph, aureus, may enter the hemo- lyraph hy way of infected food coining in contact with breaks in the exoakeleton or possibly by passing through the wall of the alimentary canal. This latter method v/ould confirm iCfaua's (305) v/ork v;ith the feeding of Coccobacllliis acridiorum to grasshop­ pers. Because of the cannibalistic habits of the roach this may es^jlain hov; bacterial infections are spread from one animal to another. 20. Bacterial toxin, such as that from Staph, aureus, ab­ sorbed from the gut produces a rise in dividing cell values. 21. Two naturally occurring bacteria, a coccvis type and a short rod, v/ere found in the hemolymph of this insect* Their presence v/as alv/ays combined with a high M.D.C. count. Symptoms of the infection were practically the same as those seen v/ith Staph, aureus, except that in the last stages of tiie diseases the conjunctival folds of the metathoracic legs, or betv/een the abdominal plates, would break and allow the thick white hesnolymph to escape. 22. Both the coccus and rod infections could be transfer­ red to normal uninfected animals, with a subsequent rise in the M.D.C. coimts as the bacteria become more numerous. 23. The rod type was more easily transferred to now ani­ mals and perhaps is the more virulent of the two naturally oc­ curring forms. - 222 -

24. A peculiar paralytic condition, not accompanied by bacterial infection, was associated with lii^ M.D.C. counts in some animals. It is suggested that the condition is due to the absorption of to:d.ns from a heavy bacterial population in the gut. 25. Specimens afflicted with a strange abnormal vacuola- tion of the hemolyraph cells may also have high M.D.C. counts. 26. Injections of nutrient broth, of solutions of peptone, beef extract, glutathione, insulin, cysteine, and hemoglobin, of vertebrate blood, and of chick embryo extract cause definite, significant rises in dividing cell rates. It is suggested tliat this is due to the presence of some fom of sulfur in these substances ^amuiett's (-SH) theox^j^. 27. Injection of sulf\irles3 atnino acids such as glutaciic acid, t37yptophane, etc, do not produ,ce an Increased M.D.C. count. Injectj[.ons of sterile salt solutions, such as Hobson's, do not affect the rate of dividing cells. 28. Injections of dinitrophenol or thyroxin are followed by higli M.D.C. counts. 29. The appearance of bacteria in many normal aniinals in­ jected vrfLth sterile solutions of glucose also leads to the s-urmise that most of these ijisects contain latent bacterial infections which may be unleashed when conditions are favorable for bacterial multiplication. 30. Injections of lead chloride or rotenone do not produce - 223 - any cliange in tlie h.emolymph piclTuire 'bofore doatli •bakes place. 31. Baaed on frequencies of observation, it seems that tho telophase stage of mitosis talcas longer tlian any other stage to pass tlirough its phase. Its time is approximately six times tlaat of the shortest phase, the raetaphase. In order of decreasing duration time, tho phases are: telophase, pro­ phase, anaphase, and metaphase. 32. Mitosis is not confined to any particular type of cell in this insect, although the smaller fonns seera to be the ones that usually uiadergo irjdirect division. S3, Arjxtosis is rare and must take very little part in maintaining the normal heraolsiuph cell population. It is con­ fined to the large cells, usually, 34. Multinucleate cells, some having as many as six nuclear fragments, are also rare, but raoi'e numerous than ami- totically dividing cells. Some evidence v/as seen that they are formed by fragnientation of tho nucleus without subsequent cyto­ plasmic division. - 224 -

IX. SmiARY

1, Literature surveyed for tMs thesis includes some of the more "basic early papers and books dealing \7ith tlie Cell Theory, tiie Pa^otoplasralc Doctrine, and the Organismal Theory, with particular reference to cell division. Prom the stand­ point of this dissertation it is interesting to note that in­ sect hemolymph cells v/ere among the first colls seen; Swammer- dam made the first observations, records, and Illustrations of the ameboid-like cells from the body fluid of the dog~louse. Another section of the survey is devoted to Insect hemolymph cells and contains a table which compares the nomenclature used by the leading v/orkers on the morphology of these bodies. A third part comprises a selection of more iK5>ortant publica­ tions about bacteria foiand in insect hemolymph. Another sec­ tion reviews the more accessible papers on factors influencing mitosis; especial attention being given to the contributions of Alexander Gurwitsch and his mitogenetic rays, and to Fred­ erick S. Hammett and his sulfhydryl theory. The last part in­ cludes an analysis of those few papers dealing with tlie stimu­ lation of mitosis of insect hemolymph cells. Altogether 617 literature citations are given. 2. Teolmiques employed in this research are the same as described previously by Yeager and Tauber (606) aaxL Tauber and Yeager (545, 546, 547), with the exception that a definite num­ - 225 - ber of cells, 2,000, was co-unted In each preparation used, 3. A total of 175 large n;ympha and adults of the insect, Blatta orlentalia, was used for these investlgationa; 124 were normal, so far as could be discer-ned; the remainder were in some pathological or physiological state, 4. A total of 2,595 temporary mounts of heuiolymph cells were made for the mltotically dividing cell (M.D.C.) counts; 5,190,000 cells were actually observed and counted, 5. normal individuals of this species have M.D.G. counts which range botvieen 0,0^ and 0,50^J. The minimum count is found about- three times in a hmidred preparations; the maximum about three times in six hundred samples. Nonnal an3.raals under ob­ servation for more than four months maintained counta within the above limits. 6. Average counts for control animals Icept various lengths of time varied from 0,116/J to 0«250;&, with a mean of approxi~ mately 0»190/i. 7. M.D.G. counts are not affected by such variables as sex, age (restricted to large nymplis and active adults), fluc­ tuations of laboratory temperature, egg fomuation and oviposl- tion, or slight hemorrhages concomitant with the obtaining of the necessary hemolymph samples. 8. Ejitensive hemorrhage within a sliort period ca\ises a rise in M.D.G, values. 9. Counts f3X)m molting anlimls decrease before and during ~ 226 - ecdysis; Increaao (as high as 1,0/0 about three days after moltirjgj and retu3?n to normal approxlnjately five days after shedding the exoslceleton. It is suggested that this rise is due to some stimulatory factor contained in the cytolytic pro­ ducts from the brealcdov/n of tissues during ecdysis, 10. Cold {5°C.) causes a decrease in M»D,G, counts; heat (37°C.) produces a gradual rise (as Mgh as 1.30>a). 11. Inoculations with Bacterium coli. Bacillus subtil is, and Serratia marceacens v/ore generally unsuccessful, indicating an active defense mechanism against these bacteria. A heavy emulsion of dead B. coli injected into a specimen, however, did cause an increase (1,70^0 in dividing cell values. 12. Susceptibility to Staphylococcus aiareus is high. In­ fections produce counts as high as 2,80^. 13. Infections of Staph, axireus can be induced by feeding infected food, and counts reached a maximum of l,dO%, Since this insect is habitually cannibalistic, this may be one method by wloich bacterial infections are spread throu(^ a colony. 14. Toxin of Staph, aureus absorbed from the gut increase M.D.C. counts as much as l,10/&.

15. TVJO kinds of nattirally occurring bacterial infections v;ere uncovered: a micrococcus producing peak counts of 2,35^; and a short rod infection with counts as hi^ as 5,10%, ®iese bacteria can be easily inoculated into nonaal animals with a subsequent rise in M.D.C. counts. - 227 -

16. Bacterial infections are accompanied by sytnptoma of a neurotoxic nature as well as a vacuolar degeneration of henio- lyinpii cells. 17. Paralyzed animals v/ith no bacteria in tiie hemolyraph laad counts as high as 1,85/i. It is suggested that these ani­ mals have heavy bacterial infections in the alimentary canal (perliaps from eating parts of a specimen which had been Icilled by a bacterial infection), and absorbed toxins are responsible for the Increased counts. Death results before the bacteria break through the gut v/all. 18. Animals v/ith abnormally vacuplated cells had counts as high as 1,60/L 19. Injections of nutident broth, peptone, beef extract, ch5,ck embryo extract, rat blood, hemoglobin, alljumin, cysteine, glutathione, and insulin resulted in high counts, respectively, of 2,15;;^, 2.10;^, 1,60%, 2,70%, 1,20%, 3.&%, 0.70%, 2,50%, 1,10%, ami S.SOJIi. This last count la the highest obtained in the 2,595 samples exatnined. It is suggested this stimulation to iihe mitotic meclianism may be associated with the presence of sane form of siilfur - perhaps, in some cases, as explained by Hammett's sulfhydryl theory. 20. Sulfur less ataino acids are not stimulatory to the mitotically dividing cell mechanism. 21. Injections of dinitrophenol or tliyroxin are followed by liigli counts - a maximum of 1,50% and 1,90;^, respectively. - 228 - being recorded. 22. Lead chloride and rotenone injections proved fatal but noil-stimulatory to the mitotic process before death occur­ red. 23. Injections of glucose were \vithoxit effect on the mitotic index, but v;ere often follov;ed by the growth of many bacteria in tlie heraolymph. 24. Death, in itself, is not associated v/ith any particu­ lar level or change in the M.D.C. count range. Hov/ever, after death, even in ani;rials in wiiich no bacteria vjere previously soon, a sudden and heavy gro\Tbh of bacteria often occurs. This, and the results follov/ing glucose injections, seem to indicate the presence in most of these animals of a latent in­ fection which is held in check until the defense mechanism is upset or stopped. 25. In an increasing order of frequency and duration of tiri?.e necessary to complete the phase, the stages of mitosis were found to be metapliase, anapliase, prophase, and, te3.ophase. The telophase takes approximately six times as long as the metaphase; 1,7 times as long as the anaphase; and about 1«2 times as long as a prophase. 26. Multinucleate colla of two, tliree, four, five, and six nuclear fragments were seen v/ith the following respective per cents of frequ.ency; 0.0253, 0.0006, 0.0001, 0.0002, and 0.0004. - 229 -

27, Aniltotically dividing cells were rare. Only 345 or

Q,0066J!> were found among the entire 5,190,000 cells. 23. i.iitosis is not confined to any one kind of cell in this species, althougli the smaller forius seem to undergo In­ direct division, and the large, rare cells, direct division. - 230 -

X. LITERATURE CITED

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13. Arnold, Julius. (1908) Haben die liaberzellen Meanbranen und Blnnennetze? Anat, Anzeig, 32;257-260. 14* (1908) Supravitale Parbung Mitochondrien ^iniicher Graniila In den Knorpelzellen nebst Bonier- kungen iiber die Morphologi© des Knorpelglykogens. Anat, Anzeig, 32;S61-366. 15. (1908) Plasmosomon, Granula, Mitochon­ drien, Chondriomiten und Hetzfiguren. Anat. Anzeig, 640-648. 16. Auerbach, L, (1874) Organologische Studien. 320 pp., Broslau.

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XI» ACKIIOWLEDGMENTS

The xvriter vrishes to express his appreciation to the Iowa State College administrators of the Rockefeller Fluid Research Fund and the Department of Zoology ai-jd Entomology of Iowa state College for making available part of the funds vdiich made possible this research; to Dr* J* FraiUclin Yeager of lov/a State College for his suggestions and critical read­ ings of the manuscript; to Mlas Ruth Gonfare for her steno­ graphic assistance; and to Miss Ann Hager of Union University for her help in arranging the rather long list of literature citations. Thanks are also due to Mr. J. Roger Porter, formerly in the Department of Bacteriology of Iowa state College and now at Yale University, for preparing the pure cultures of bac­ teria used in certain of the experiments. - 282 -

XII. VITA

Full name of father: Emil Tauber, Maiden name of mother: Anna stephan. Date and place of birth: May 23, 1908; Decatur, Illinois. Elementary school: Grade schools of Decatur, Illinois. Secondary schools: Central Junior Hi{^ School and De­ catur Senior nigji school of Decatur, Illinois. Undergraduate colleges: James Millilcin University of Decattar, Illinois; and University of Illinois at Champaign- Urbana, Illinois. B.A, from Millikin \7ith a Major in Biology and a combined minor of English and History, Major work taken under Dr. Frederick C, Hottes of the University of Minnesota. Graduate work: Iowa state College with M.S. in 1952. Major v/ork in Insect Physiology under Dr. J. Pi-anklin Yeager of new York University; minor work in Physiological Chemistry under Prof, V, E, Helson. Major work for Ph.D. was also un­ der the direction of Dr. Yeager in Experimental Zoology; studies for a first minor in Human Physiology mider Dr. Erma A, Smith; and for a second minor in Biochemistry under Dr. El­ lis I. Fulmer and Dr. R. M. Hixon. - 283 -

XIII. ILLUSTRATIONS Plate I.

Sketches of Hemolymph Cells from Blatta orientalls as Seen in Hobson-Acetic Acid-Gentian Violet Diluting Fluid. (Approximately 1,000X) *> A A,B; large cells v/itia large nuclei; £, D, E: large spin- dle-ahaped cells; F, G: small cells v/ith large nuclei; H, I, J: small cells %vith small nuclei; K, L, M: very small celXs vrf-th little cytoplasm; N: very large cell vAth tiny nucleus (rare); large cell v/ith extremely large nucleus (rare); distorted spindle-shaped cell; Q: extremely large spindle shaped cell (rare); R: ^^small spindTo-shaped cell (rare); S^i group of non-nucleat'ed "plastids" of various sizes. eai'ly prophases of large and small cells; S_, Q metaphase of^lai^e and small cells; 7, 3^; early anaphases; _8, 12: late anaphases; 3^: various telophase stages "of HTvidiris cells; 14, 15; stages of nuclear division without cytoplasmic fission; 16, r?, 3^: examples of amitotically di­ viding cells; 18: late telophase of a small cell (rare); 20, 21: cells showliag "plastid" formation; 22, 23: cells witE" abnormally vacuolated cytoplasm; 24: an abnormally vacuolated cell in early telopliase stage.

Plate II.

Sketches of Multinucleate Ileraol^mpii Cells from Blatta orien- talla as seen in nobson-Acetlc Acid-Gentian "Violet Diluting Fluid. (Approximately 1,000X)

SL> Z- binucleate cells (No,4 contains a small vacuole); 8, 9_, jW, 11, IS# 13: trinucleate cells; 14, 15, 16, 17: "cells with four nuclear fragments; 18: cell \^tliTive nu^eij 19, 20: cells with six nuclei. - 287 -

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