GAME?OGENES!$ AME} RADMTION EFFECT 5 ON THE {QEPRGDUCTWE TESSUES QF OULEMA MELANOPé (L) Thesis for the Degree of M. S. MSCHIGAN STATE UNIVERSITY S. Kumararaj 1964 LIBRARY Michigan State University W GAMETOGENESIS.AND RADIATION EFFECTS ON THE BEPBGDUCTIVE TISSUES 0F OULEMA MEQAEOEA (L.) S. KumaraRaJ' The normal development of gonads, some histochemical aspects of the ovary, radiation reSponse, and histopathologi- cal effects of radiation on adult cereal leaf beetle, gnlgma melangpa (L.) were studied. Tetrahydrofuran-parlodion double embedding techniques were used for most of the histological preparations and Feulgen - Fast green stains were employed to trace the development of gonads. Dosage levels of 500r to 20,000r X-ray radiation were used to study the effects on reproductive tissues. It was found that the male beetles attained sexual ma- turity earlier than females of the same species. The ovary was in an underdeveloped state in most of the diapausing fe- males and developed after cessation of diapause. Observations indicated that the ovarioles of this beetle were of the acro- trOphic type and each beetle had in ovarioles, 7 on each side. Follicular epithelial cells also appeared to have a nutritive function, particularly in the latter stages of oocyte deve10p- ment. Radiation at levels of 5,000r and above caused over 90 Percent mortality within two weeks. Sperm motility was not S. KUMARARAJ affected by radiation up to 20,000r. Dosage of 2,500r and above affected oviposition drastically and 5,000r irradiation reduced viability of the eggs manly 100 percent. Radiation treatment of 7,500r and above resulted in considerable inhi- bition in the development of the ovary. The histopathological effects caused by radiation were degeneration of germarium, malformation, abnormal, and slow develoPment of the oocytes. The degenerating germarium of the irradiated beetles showed loss of DNA positive material in the nuclei of the cells of the germarium. GAMETOGENESIS AND RADIATION EFFECTS ON THE REPRODUCTIVE TISSUES 0F OUEMA MELQNOPA (L. ) By s . KUMARARAJ A THESIS . Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SC IENCE Department of Entomology 1961+ ACKNOWLEDGEI‘ENTS The author wishes to express his profound sense of gratitude to Dr. Roger A. H00pingarner for suggesting this thoughtful problem, counsel during the course of investigations, checking and helpful criticism in editing the manuscript, and for provid- ing all the laboratory facilities. He is grateful to Drs. Gordon Guyer and Robert Buppel for reading the manuscript and offering valuable suggestions. The author also has distinct pleasure in thanking Mr. Allen French for his kind help as and when needed, Mr. Thomas Castro for supplying the beetles and Dr. Mostosky Ulreh, Department of Surgery and Medicine, for irradiating the beetles with Kmrays. TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE IIA. . . . . . . . . . . . . . REVIEW OF LITERATURE IIB. . . . . . . . . . . . . . THE TEST INSECTS. . . . . . . . . . . o . . . . . . THE DEVELOPMENT OF GONADS IN THE ADULT CEREAL LEAF BEETLE. 0 O O 0 O O O O O O O O O O I O O O O O 0 material arld- MethOdS O O O O O O O O O O O O O O O S talningo 0 0 o 0 O 0 O O 0 O 0 O O O O O O O O O Bes‘llts O o 0 O O O O O I O O O O O O O O O O O O HISTOCHEMICAL STUDIES ON THE OVARY OF CEREAL LEAF BEE TE 9 O 0 O O 0 O O C O O O O O O C O O O O O 0 Material and Methods. . . . . . . . . . . . . . . ReSUltS 0 0 O O 0 0 0 O O O 0 O O O O O O O O O 0 EFFECTS OF RADIATION ON CEREAL LEAF BEETLES TREATED WITHX"BAYSooococoa-0000000... Material and Methods. . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . HISTOPATHOLOGY OF THE IRRADIATED GONADS . . . . . . Material and Methods. . . . . . . . . . . . . . . Results . . . . . . o . . . . . . . . . . . . . . DISCUSSION OF RESULTS . . . . . . . . . . . . . . . SUMMARY... LITERATURE CITED. . . . . . . . . . . . . . . . . . PAGE 12 12 13 13 37 37 38 42 42 44 53 53 53 63 72 74 LIST OF TABLES TABLE PAGE I Percent Mortality of Cereal Leaf Beetles Irradiated with X-Rays -- First Trial . . . . 44 II Observations on Oviposition and Eclosion of Cereal Leaf Beetles Irradiated with X'Bays '“" Fir-3t Trial 0 o o o o o o o o o o o “'5 III Percent Mortality of Cereal Leaf Beetles Irradiated with K-Rays -- Second Trial. . . . 47 IV Observations on Oviposition and Eclosion of the Cereal Leaf Beetles Irradiated with X—Rays -- Second Trial . . . . . . . . . 47 V Percent Eclosion from the Total Number of Eggs from each Radiation Treatment -- Seoond Trialo o o o o e o o O o o o o o o o o “’9 INTRODUCTION The cereal leaf beetle, ingmaflmelangpa (L.), an intro- duced pest in this country, has assumed serious proportions in some parts of Michigan and has caused impressive losses particularly on small grains. The purpose of this study was to consider the possibil- ity of controlling the cereal leaf beetle, Qulgmaflmglgngpa (L.) by releasing irradiated males with dominant lethal genes, which is usually referred as the sterile-male-release technique of insect control. Current interest in the sterile male method of controlling the cereal leaf beetle stems from the success- ful elimination of the screw worm fly, Egghligmyia,hgng;1gngg (Coqo) in Curacoa and Florida (Bushland & HOPkins, 1953, Baumhover et al., 1955), where this insect disappeared after repeated releases of sterile individuals. The present work reports a brief study of the testes and ovaries of the cereal leaf beetle, conducted to determine the sequence of events in normal spermatogenesis and oogenesis, in Order to establish a basis for evaluating the effects of radia- tion on the growth of the reproductive organs, oviposition, and eclosion. In addition, the present work deals with the doeages of Xerays required to induce sterility and also pro- vide information on the effects of radiation on the ovary. However, the present experiments were not designed for detail cytOIOgical study of sueh effects. IIA. REVIEW OF LITERATURE ON GAMETOGENESIS The first research on insect gonads was initiated in 1847 by Stein (according to Bonhag, 1958) with his histological study of the female reproductive organs of beetles. The next study that concerned reproduction in beetles was in 1900 when Bordas divided ColeOptera into two main groups, with regard to the types of testes, those with simple, tubular testes and those with complex testes. In the first group with simple tubular testes belong the Carabidae, Cicindellidae, Dytiscidae, etc., whose whole male genetal system exhibit a primitive con- dition. The beetles with complex testes Bordas further di- vided into those with fascicular testes and those with cluster or grape like testes. Saling (1907) in his investigation of the gonads of Tenepzio politor L. was primarily concerned with tracing the development ofthese organs and therefore presented only a cursory survey of the histological elements in the adult ovary. .And Gardiner (1934) used the ovaries of 2. 391152; L., in addition to other tissues, in her study ofthe nucleolus but did not give an extensive account of the other structures in the ovariole. The general morphology of the female reproductive organs is given in Snodgrass (1935). He described a typical ovariole as consisting of three parts: a terminal filament, an egg tube, and a pedicel. In describing the various types of egg tubes, Snodgrass used the generally accepted classification of Berlese (1909), which divides them into panoistic, poly- trophic, and acrotrOphic or telotrOphic types. In the pan- oistic type there are no special nutritive cells differenti- ated from.the egg cells. This type occurs in the Apterygota, Ephemeroptera, Odonata, Orthoptera, and Siphonaptera. The polytrOphic type contains an alternating succession of oocytes and trOphocytes and is characteristic of the.An0plura, Neur0p- tera, ColeOptera;.Adephaga, LepidOptera, Hymenoptera, and Dip- tera. A few insects, the Hemiptera and the Coleoptera; Poly- phaga possess the acrotrophic type of egg tube. Here the oogonia give rise to nurse cells and oocytes. The former re- main in the upper part of the egg tube, while the oocytes separate from them as a series of egg cells, increase in size in the vitellarium. There are protoplasmic connnections be- tween the two types of cells and by means of these strands the oocytes in the egg tube continue to receive the yolk forming material from the nurse cells. There is some confusion as to which of the ColeOptera have telotrophic ovarioles. Stein (1847), Gross (1903), and Imms (1948) have stated that the Adephaga have polytr0phic ovarioles and the rest of the beetles contain telotrOphic ovarioles. Weber (1922) and Wigglesworth (1950), on the other hand, have characterized the Adephaga as having telotrOphic ovarioles and the Polyphaga as having the polytrophic type. Numerous other papers on the ColeOpteran ovary support the Original view of Stein. Bryan (1954) stated that the nutri- tive cords of telotrOphic beetles are not usually as conspicu- ous as those found in Hemiptera and may even be absent in 4 some. Bonhag (1958) points out that the criterian for distin- guishing between panoistic and telotroPhic ovarioles is the presence of trophocytes in the germarium. .According to him, in the absence of nutritive cords, a detailed study of oogenesis is often required to determine whether the apical cells of the germarium.are oogonia or trOphocytss. The relationship between nurse cells and oocytes has been investigated by many workers, i.e., the papers of Giardina, Korschelt, Nussbaummfiilarowicz (reviewed by Wislon, 1928), Shaffer (1920) and others. Such investigations suggested that nurse cells were abortive ova specialized for the formation of nutritive materials to supply the needs of the deveIOping egg. This problem was reinvestigated in the Hemiptera by Schrader and Leuchtenberger (1952) using cytochemical procedures. Their findings showed that the nurse cells did contribute material to the egg cytOplasm and furthermore that this nutritive substance was in part derived from the desoxyribose nucleic acid (DNA) of the nurse cell nuclei. Bryan (1954) made a cytological and cytochemical study of oogenesis in Popilus disjungtus Ill. (COleOptera; Polyphaga) and reported that the ovarioles of this beetle exhibit a modified telotrOphic condition. His cYtological and cytochemical study of the oogenesis of E. dis- Junctus also suggests large amounts of PAS positive substance in the apex of the germarium and also the trophic function of this region. His study did not show the presence of plasmatic strands connecting the apical region with the deve10ping oocytes. Krause (1946, 1947) studied the structure of the gonads and their deveIOpment in the wood eating beetle, ZBSSEIES cognutus Fabricius. He noted that in the long germarium the tightly packed cells appeared to be all alike when young, and the spiral like tip of the germarium had relatively few nuclei and great amoun s of brightly staining material (especially with Orange G and Eosin). Though he did not make a detailed analysis of this material he concluded that because of its apical position and characteristic staining it is likely that this region corresponds to the typical feeding chamber of the acrotrOphic type. However, he did not find definite and con- tinuous strands connecting these with the spiral tip of the germarium. He noted only that the smaller developing egg cells possess plasmic strands and large globules of bright staining material in the germarium prOper. This led him to conclude that the smaller oocytes receive nourishment from the apical feeding chamber via the plasmic strands until they acquire a follicle cell envelope of their own. In order to provide a detailed histologieal basis for further work on the postem- brycnic deveIOpment and histochemistry of the ovary of T. molitor L., Sehlcttman and Benhag (1956) made an extensive and detailed study on the ovary of this insect. They inter- preted the localizations of ribonucleic acid (RNA) and deso- xyribonucleic acid (DNA) in the ovary and also determined whether DNA was discharged from the apical trophocyte nuclei as had been reported in the telotrOphic ovariles of certain HeterOptera (Schrader & Leuchtenberger, 1952; Bonhag, 1955 a & b). In Tepebrio they noted the nutritive cords originate at the posterior boundry of the tr0phic tissue and extend to the developing oocytes in the vitellarium. So they concluded 6 that in contrast to the strongly develOped cords usually found in the telotrOphic ovarioles of hemdpterous insects, the nour- ishing strands in To molitor L. are of much more delicate na— ture and may be quite easily overlooked. IIB. REVIEW OF LITERATURE 0N RADIATION EFFECTS TO INSECTS Since a remarkable and extensive body of literature is available concerning the relation of X-rays and gamma rays to physiological and pathological processes on one hand, and with genetic change and its subsequent consequence on insects on the other hand, the topic under review demands restraint in the selection of published worko It was found necessary to bywpass certain papers simply because they did not bear a direct relationship to the subject selected here, or did not concern the order Coleopterao In his classic communications Muller (1927, 1928) men- tioned that untreated female grosophila flies mated to heavily treated males laid eggs that failed to hatcho He interpreted the mortality of eggs fertilized by irradiated sperm as re- sulting from chromosomal changes which he described as a “dom- inant lethal” effecto There are numerous other publications in the fields of genetics and cytology dealing with the effects Of radiationo The various papers have been reviewed and inter- Preted in comprehensive publications by Muller (1940, 19#1), Lea (1947) and Catcheside (1913»8)o There is some early work with radiation effects on the Coleopterau For example, Runner (1916) studied the effects of roentgen rays on the tobacco or cigarette beetle, Laslgdgzma seprigorne (F0), and reported that infertile eggs were depos- ited after exposureo He also reported that the treatment had the effect of stepping the activity and deve10pment of larvae and causing death of the treated larva before reaching pupal stagec Davey (1928) showed that the life of Tripolinm Egg: EEEHE Jacquelin duVal may be prolonged by the use of X-rays given in a series of small daily doses and the prolongation of life is greater than that of larger doses given all at onceo He emphasized that by merely varying the quantity of the dose, a purely physical agent, X-ray, may be made to pro- duce at will a stimulation, a destructive effect which occurs only after a latent interval, and an instantly destructive effecto The successful application of the sterile-male technique in the control and eradication of screw-worm flies, (Bushland & Hopkins 1951, 1953; Lindquest, 1955; Baumhover, gt ‘a;., 1955) has stimulated interest in the possibility of using this method to control other insect specieso Their laboratry observations showed that it was most efficient to irradiate pupae within ‘wo days of emergence and a dosage of 2,500r sterilized the males and twice that dosage sterilized the femaleso In addi- tion their laboratory studies also indicated that sterilized males competed about equally well with normal males for mates. Bushland and Hopkins (1953) also made a comparative study of the effects of Keray and gamma rays and investigated the effects of gamma radiation on longevity, fertility and fecundity of adult screw-worm flies. Their comparative study on the effects of X-rays and gamma rays indicated no difference between the two kinds of radiation. Knipling (1955, 1959) and LaChanoe and Knipling (1962) formalized the approach which involves sustained release of sterile males in numbers exceeding those in a normal pOpulation and also discussed insect papulation control on an area wide basis. They stressed the need for investigations to determine the feasibility of applying the sterile male technique for the control of other insect pests. In a paper on radiation control of insects, Grosch (1956) reported that irradiated Braconid wasps, with large single doses of erays, under starvation diet, induced leth- argy in the animals and at a certain level increased the life span. Jaynes & Godwin (1957) made a preliminary study of sterilization of the white pine weevil, Pigsodes strobi (Peck) with gamma radiation. Their study indicated that an exposure of 5.000r to 10,000r to be the best for this weevil because longevity of the weevils was not reduced and egg sterility was at an acceptable level. However, they pointed out that diminished feeding and oviposition rates suggested the possié bility that other activities such as frequency of flight may also be altered or reduced. Cork (1957) from his study on gamma radiation and longevity of the flour beetles reported that life span of a given number of flour beetles may be ex- tended by several peroent by radiation with gamma rays and beetles that survive a single large dose of gamma rays appear to have a survival rate superior to those that receive no radiation. According to him, this may be a single dose of 3,000r or chronic daily doses of 100r. He also pointed out that 20 percent of the animals receiving 100r daily dosage lived more than #50 days and in that time they received 45,000r. This was more than twice the amount that would have produced complete annihilation in a single irradiation. However, his conclusion was based on a single experiment, though done under ideal conditions over a period of two years. Park, gL_ gl. (1958) studied the relation of X-radiation to the fecundity (number of eggs laid) and fertility (egg hatched) of two species of flour beetles, W W Duval and Tribolium castanegm Herbst. They tried four dosages of erays (2,000r to 5,000r) in their experiment, and advanced conclusions that there were differences between the two species with respect to fecundity and fertility. They noticed fertility was more affected by practically all components of treatment than was fecundity. They also concluded that the relation of increase in dosage to decrease in reproduction was essentially linear rather than curvilinear and irradiation at 2,000r to 5,000r levels did not reduce adult longevity. Rogers and Hichey (1960) studied the post irradiation feeding activity of g. castaneum and arrived at the conclusion that the nutritional state of beetles after radiation affects their life expectancy. Dennis (1961) reported that an expo- sure of 100,800r gamma rays killed all of the confused flour beetle adults and larvae, saw toothed grain beetle adults, lesser grain borer adults, granary weevil adults, and rice weevil adults. He pointed out that confused flour beetles 10 rposed to 8,#00r did not reproduce during a two month period after erposure. Eletchly (1962) studied the effects of a single X irradiation at #9000r on the larvae of powder post beetles, chtus brunneus (Steph.) and suggested that irradia- tion of larvae at a dosage too low to prevent completion of development would be of little value as a practical method of control, because of recovery in the pepulation level of sub- sequent generations of the treated strain. Hoover9 Floyd & ichardson (i963) subjected all develOpmental stages of rice weevil, figipohilus orygae (L.) to radiation by Xurays and found resistance to the effects from radiation exhibited by this insect increased as develOpment increased. In addition, they reported that the LD5O and the point of complete sterility for the adult stage was between 7,500r and 10,000r. THE TEST INSECTS The cereal leaf beetle has an adult diapause during winteru Preliminary histological study of the pre-diapause and diapausing insect indicated that most of the male beetles had fully matured spermo But in case of female beetles, it appeared that the insect was not sexually mature at this period as indicated by ovarian deve10pmento In order to find out how sexual deve10pment takes place in the adult beetlesy a brief study of gametogenesis was undertaken as it is one of the important prerequisites for controlling the insect by sterilemmale release. Most of the insects used in this study were summer adults, collected in the field by the Department of Ento- mologyfl Michigan State University° DEVELOPMENT OF GONADS IN THE ADULT CEREAL IEAF BEETLE Materials and Methods. The beetles used for studying the development of gonads had the following treatments: They were collected from the field on July 25, 1962, and maintained in a bioclimatic chamber on living plants until July 31, 1963. They were then placed in one pint paper cartons at a constant temperature of 43° F on.August 8th until September 16th. From September 16th to September 17th they were maintained at 25° F for 15 hours. In the bioclimatic chamber they were given 15% hour daylight. The temperature at day was 82° F and at night 77° F. The cold treatment apparently was necessary to break diapause. Three hundred and fifty beetles were taken out from the cold storage on September 17th, and put into cages (50 each) made of a.glass chimney and having oat seedlings. Samples were taken each day, except the first sample which was taken after 18 hours, and fixed in KAAD (Peterson, 1948) immediately. (KAAD = Kerosine, Acetic acid, 95% Alcohol and Dioxane in ratio of 1:2:7:1.) Samples were taken up to 14 days. 'When the beetles started feeding voraciously, they were changed to new cages containing fresh oat seedlings. Each day about 20m25 insects were sampled. Before fixing in KAAD, the heads of the beetles were out off with a sharp razor blade and the legs and wings were carefully removed. This enabled a better penetration of the fixative. The vials containing the 13 beetles in the fixative were kept under vacuum to remove air bubbles. The beetles were left in the fixative for a couple of days and then dehydrated in tetrahydrofuran for two hours and double embedded as per method suggested by Salthouse (1958), in tetrahydrcfuran containing 1% and 2% parlodion for 24 hours and 36 hours respectively. Some were embedded with tetrahydrofuran containing 3% parlcdion. In that case the beetle was double embedded in tetrahydrofuran containing 1% and 2% parlodion for 24 hours each and in tetrahydrofuran containing 3% parlodion for 36 hours. After this treatment, the beetles were given a quick rinse in tetrahydrofuran and embedded in tissuemat (610 C) for 3 to 4 hours, making 2 changes. The blocks were then sectioned on a microtcme at 10 microns thickenss. In add- ition, a few laboratory reared larvae were allowed to pupate and the freshly emerged adult beetles were fixed in KAAD, with- in 3 days after emergence. These were also sectioned, stained and studied. §ggjggggg. Most of the slides were stained with.Feulgen Stain, made as reported in Stain Technology (1951), and counter stained with fast green. The length of acid hydrolysis varied between 6w? minutes at 60° C. The slides were treated with Feulgen Stain for 45 minutes and with fast green for about 1% t0 2 minutes. Results. The sections from beetles fixed after 18 hours to 3 days after removal from the cooler show a suspensory lig- ement9 6m? OVariole: cn.each side of the ovary and the stalk bearing them. The ovary on each side is composed entirely of 14 the germarium and the slender stalk or pedicel bearing it. The germarium is composed of densely packed, extermely Feulgen positive9 more or less spherical cells (Fig. l). The cells in the germarium.all look alike at this time. (In many of the sections 6 ovarioles and in some 7 ovarioles on each side of the ovary were observed. However, this depends on the plane of sectioning. but the maximum number of ovarioles observed on each side of the ovary was 7.) The stalk and the lateral ovi- ducte are slender during the early period. No young oocytes are visible at this time9and the ovary appears to be immature. After 3 days or so, the vitellarium, where the egg cells grow, deve10ps gradually. The cells in the immature, densely packed. germarium contain large nuclei and are almost devoid of any cytoplasm. But when oocyte deve10pment and enlargement begin the cells in the germarium appear to clearly have cyto- plasm, as is evidenced by the presence of the counter stain, fast green9 around the large nuclei. After ooeyte development starts9 the posterior end of the germarium shows a diminution of Feulgen positive material, and the cells appear granular at this time. The young oocytes, which are i—b rregulariy located, are intermixed with slightly elongate pre folliculer cells (Fig. 2). Very rarely, disinte~ Eration or pycnosis cf the brightly staining, Feulgen positive cells in the germarium is noted. The young oocytes in the posterior portion of germarium appear to have filamentous chromosomes. As the deve10pment progressesS the young oocytes appear tobe situated more or less Side by side among the preefollioular cells. When the oocytes 15 grow larger, they change from a spherical to an oval shape and become oriented in a line. Various sizes of oocytes are seen in a developing ovariole. The first stage small oocyte and the second stage oocyte appear to have columnar fOllicular epithe- lium (F180 Bag 3b)o The second stage oocyte is larger in size than the first and in both the nuclei have Feulgen positive chromatin strandso The follicles are quite characteristic in the vitellarium in that they have a different kind of follicular epitheliumo The third stage oocyte has a cuboidal follicular epithelium and the individual cuboidal follicular cells appear to be horizontally elongate (Fig° “)0 The follicular cells at the third stage have a well defined cytOplasmo The nucleus of the occyte at this otage appears faint and clear Feulgen posi- tive chromatin material is not visibleo However, it is evident that the nucleus undergoes a gradual transformation and deve10ps into the germinal vesicleo The transformation that takes place in the cccyte nucleus during the deve10pment of the germinal vesicle is too complicated and is not handled here in detailo The fourth stage oocyte is quite large and also has a cuboidal follicular epithelial cells? but these appear slightly elongate verticallyo The cubeidal follicular epithelial cells at this stage also have a well defined cytoplasm and a brightly stain- ing Feulgen positive nucleuso The cuboidal follicular cell nucleus appears to be little larger than in the previous stageo Yolk synthesis seems to proceed hereo The fifth stage is quite characteristic in that the follicular epithelial cells have attained a squamous condition (F180 5). A great amount of yolk Synthesis has taken place by this time (Figo 6)o The chorion 16 is not laid down until yolk formation has been completed. Since the formation of follicles is a continuous process, the exact number present at any specific time is subject to variation. However, it appears that 3 to 5 follicles, frequently 3, appear in the vitellarium before the largest, most posterior oocyte is released into the lateral oviduct. When oocytes are found in the vitellarium, the cells in the germarium appear to be less densely packed and each cell appears to have a well defined cytOplasm (Fig. 7). The interfollicular tissue (the mass of cells in between two successive oocytes) seems to be composed of small follicular cells which have accompanied the oocytes during their growth and have consequently become wedged in between the successive follicles of the vitellarium. Beyond the last egg chamber a mass of follicular cells form the plug, which closes the proximal portion of the egg tube. At the time of ovulation the epithelial plug appears to break down, and the preceeding follicle then takes the terminal position and the accompanying interfollicular tissue becomes the functional plugo The pedicel (Fig. 8) or stalk, is a duct that connects the vitellarium.with the lateral oviduct. The wall of the Pedicel is composed of simple columnar cells covered exterior- ly by a continuation of the outer epithelial sheath of the ovariole. The cells of the stalk are also brightly Feulgen Positive. Before the development of the oocytes, the stalk 0f the oviducts are comparatively slender, and the cells in this region appear to be densely packed. Also the Feulgen 17 positive material is prominent because of the compactness of the columnar cells. When oogenesis commences the stalk and the oviduct becomes distended. In the beetles which have started oviposition, the ovi— ducts are characterized by'mrapresence of remains of the de- generating egg follicles at the base of the vitellarium. In beetles which are actively forming eggs, these bodies are dif- ficult to distinguish. It is important to note that the nutritive cord or plas- matic strands connecting the trOphocytes in the germarium.with the develOping oocytes were not observed in this beetle during the entire course of this work. Not even a slightest trace of this cord was found. (c.f. Schlottman and Bonhag, 1958) The testes of the adult male cereal leaf beetle consists of 2 pair of lobuler testes9 one pair on each side of the body in the abdominal region. They appear pale white with a yellow- ish tinge. They are moderately big for this small beetle. Each lobe of the testes is more or less bean shaped and in be- matured spermatoza are seen. Around the testes, there is an investing coat composed of a single sheet of cells. sections from the few laboratory raised adult male beetles showed at least a few fully matured spermatoza within 3 days after emergence from the pupal stage. Mostly, sex cells in different stages of deve10pment and a few mature sperma- toza in bundles were seen at this period (Fig. 9a). In the case of females, the development of the oocytes were never noticed at this period. This indicates that the males become sexually mature earlier than females. 18 Near the outer periphery of the lobular testis sperms» tocytes in different stages of meiosis are visible (Fig. 9b & 10). Towards the inner side9 spermatids and transformation of spermatids into flagellated spermateznican be observed (Fig. 11). The mature spermatozoa appear in bundles towards the central duct. The spermatids are at first compressed and round and then they become elongate and tappering on both the sides giving an appearance of a spindle (Fig. 12a, 12b). Later they are dissolved and released. It appeared that when the beetles entered diapause, the males had fully matured spermatozoa and also sex cells in dif- ferent stages of develogment. Many sections from diapausing males showed large amounts of matured spermatozoa, and also 'the males that emerged from diapause showed large amounts of matured spermatozoa. 19 Emmi LONGITUDINAL SECTION OF THE OVARIOLE OF OUIEMA MELANOPA (L.) Fixed in KAAD and Stained in Feulgen and Fast green (265x). Showing suspensory ligament, ovariole sheath, bright staining germarial cells, prefollicular oocytes and prefollicular cells. EIQEE§_Z HORIZONTAL SECTION OF THE OVARIOLES OF OUIEMA MEIANOEA (L.) Magnification ShOx. Showing the germarium and three successive develOping oocytes in the vitellarium. Elfi!§§.3a HORIZONTAL SECTION OF THE OVARIOLE OF OULEMA MELéflOEA (L.) A germarial stalk oocyte and the second stage oocyte with Colanmnr follicular epithelial cells. 1100x UREb CROSS SECTION OF TEE OVARIOLES OF OULEMA MELANOPA (L.) First stage oocyte of an ovariole and cuboidal follicular epithe- lium of a third stage ooeyte of a neighbouring ovariole. 540x EIQQE§_& HORIZONTAL SECTION OF THE CUBOIDAL FOLLICULAR EPITHELIAL CELLS OF THIRD STAGE OOCYTE OF OULEMA MELANOEA (L.) Mangification 2,700x. 25 mass: HORIZONTAL ssc TION OF THE SQUAMOUS FOLLICULAR EPITHELIAL CELLS OF ms FIFTH STAGE OOCY‘I'E OF OULEMA MELANOPA (L.) Magnification 1080x. EIGQQ 6 HORIZONTAL SECTION OF THE FIFTH STAGE OOCYTE OF OULEMA W Showing the squamous follicular epithelial cells and abundance of yolk.5#0x FIGURE 2 HORIZONTAL SECTION OF THE GERMARIUM OF A DEVELOPING OVABIOLE OF 931% MELANOPA (L.) Cells in the germarium are less densely packed and cytOplasm is well defined. 540x FIGURE 8 HORIZONTAL SECTION OF THE. FIFTH STAGE OOCY’IE OF OUQQMA MELANOFA (L.) ON THE PEDICEL Portion below the oocyte is the plug. 265x Frgggg 2a LONGITUDINAL SECTION OF THE TESTIS OF A YOUNG ADULT a d spermaton Showin mostl dividing cells, a few spermatids an zoa. .Errow ifidicates a single bundle of spermatozoa. 265x I. Illr‘ws- aCH... FIGURE 93 31 FIGURE 2b SPERMATOCYTES, SPERMATIDS AND THE TRANSFORMATION OF SPERM INTO SPERMATOZOA IN THE TESTIS OF OULEMA MELANOPA (L.) THREEDS DAYS AFTER EMERGENCE FROM PUPA. 1080: GURE (EEOSS SECTION OF ONE OF THE LOBULAR TESTES OF OULEMA MEMOPA Showing different stages of spermatogenesis near the periphe and abundant spermatozoa in bundles towards the inner side. §Z0x FIGURE 11 TESTIS OF OULEMA MELANOPA (L.)SHDWING EARLY SPERMATIDS AND FLAGELLATED SPERMATOZOA IN BUNDLES 1080x GURE 12a SPERMATIDS IN THE TESTIS OF OULEMA MELANOPA (L.) IN THE PROCESS OF TRANSFORMATION INTO SPERMATOZQA Magnification 2,700x EIGURE 12b CROSS SECTION OF TESTIS OF OULEMA MELANOPA (L.) SHOWING SPERM- .ATIDS IN GROUPS BEING TRANSFORMED INTO SPERMWTOZOA IN BUNDLES 1080x FIGURE 10 FIGURE 12b. HISTOCHEMICAL STUDIES ON THE OVARY OF CEREAL LEAF BEETLE The germarium of the cereal leaf beetle showed bright staining cells when stained with Feulgen Stain, or Pyronin I and methyl green. Both Feulgen and methyl green are specific for nuclear DNA, and Pyronin I for RNA. To confirm.whether the germarium is rich in DNA and RNA, some of the histological preparations obtained from the female cereal leaf beetles were treated with DNAase and some with BNAase (Pearse, i961) and then subject to the usual staining procedures. Matezial and Methods. The histological preparations made by the tetrahydrofuran and parlodion double embedding process were treated with DNAase after the usual dehydration process in graded alcohol series and rinsing finally in glass distilled water. Then the slides were treated for 30 hours at room temperature with 3,000 Dornase units of DNAase per mill- iliter in 0.025 M. vernol buffer at pH 7.5 containing 0.003 M. MgSOh. The enzyme solution was renewed after 15 hours. At the end of 30 hours the slides were rinsed in water and the standard Feulgen reaction was performed (The DNAase used in this experi~ ment was supplied by Nutritional Biochemical Corp. containing 30,000 Dornase units per mg.). The controls were not treated in the enzyme preparation. 38 The Ribonucleic acid extraction. was done as follows: the slides were dehydrated in graded alcohol series in the usual manner and rinsed in glass distilled water and incubated for 1-1% hours at 37° F in a solution of RNAase (1mg/ml.) in glass distilled water. (RNA'se was supplied by Nutritional Bio- chemical Corp.) After this treatment, the histological pre- parations were washed in distilled water and stained with Pyronin I and methyl green. The controls were also stained with Pyronin I and methyl green without treating them in the enzyme preparation. Resulpgg The histological sections treated with.DNA'se were Feulgen negative (Fig. 13) and the controls Feulgen posi~ tive (Fig. 14). This indicated that the germarium.was very rich in DNA. The sections treated with RNA'se and stained with Pyronin Y and methyl green showed absence of RNA in the germarium. It appears that the germarium of the cereal leaf beetle is rich in both.DNA and RNA. This result is suggestive of the trOphic function of the germarium in the cereal leaf beetles although no nutritive cord was observed to connect the develOpIng oocyte with the germarium. The nucleic acid contents of the follicular epithelial cells was also removed by the DNAase and RNAase treatments, particularly in the later stages of deve10pment. 39 FIGURE 1:} GERMARIUN.OF OULENA MELANOPA (L.) TREATED WITR.DNAase AND STAINED WITH.FEULGEN AND FAST GREEN Showing loss of DNA positive material. SAOX FIGURE 14 GERMARI‘UM OF OULEMA MELANOPA (L.) STAINED WITH FEULGEN AND FAST GREEN Showing abundance of DNA positive material in the nuclei. 265x EFFECTS OF RADIATION ON BEETLES TREATED WITH X-RAYS Nategials and Methods. Two trials were conducted at dif» ferent periods. The beetles used in.thefirst trial were col- lected from field on July 26, 1963 by the Department of Ento- mology. The beetles had the following treatments before they were used in this experiment: The beetles were placed in a bioclimatic chamber on July 31, 1963, until they were moved to cold storage at 43° F on August 6, 1963. Five hundred and sixty beetles were removed from the cooler on October 14, 1963, and divided into lots of 80 beetles each. Except the control, the lots of 80 beetles were irradiated with the following do- sages: 198r/min., no filter, ISMA, FSD5O CM. Dosage Time Reguized 1,000 Roentgens 5.5 min. 2,500r 12.6 min. 5,000r 25.2 min. 7,500r 37.8 min. 10,000r 50.4 min. 20,000r 100.8 min. The beetles were not sexed. Characters for sexing the living beetles were not available. Many attempts were made to sex them but met with failure. Ten beetles from the control, and from each treated sam» P18, were taken for study of their sperm motility immediately 43 after treatment and the rest were left in cages containing oat seedlings. The beetles were changed to new cages containing fresh oat seedlings when they started actively feeding. This was done more or less every day. At the start of oviposition the beetles were transferred to new cages containing fresh seedlings every day in order to take an egg count each day and to keep the eggs laid in a day for viability counts. The sperm motility was Observed in the following manner: The insects were dissected carefully and the testes were re- moved and placed on a slide containing two drops of Yeager's Saline (Yeager 1939) or housefly saline (Brebbia and Ludwig, 1962) and squashed with a glass rod having a bulbous tip. Then the squashed preparation was taken on a cover glass and ob- served by the hanging drOp method under a phase microscOpe. Visual observation on the feeding habits of the normal beetles and the irradiated beetles were also made. The insects used in the second trial of irradiation were Summer adults, collected on June 9, 1963. These beetles were Put in bioclimatic chambers on July 15, 1963, and became in- active on July 21, 1963. They were transferred to the cooler (43° F) on July 23, 1963. The beetles were taken out of the cooler oanebruary 7, 1964, and divided into lots consisting of 100 each. Then they were treated at different dosage levels ofiadiation using Xe rays. The following dosages were used in the second trial: 1) 500 Roentgens, 2) 1,000r, 3) 3.000r, 4) 5,000r, 5) 0. After irradiation, they were put into chimney cages containing oat seedlings, for feeding and oviposition. Approximately 50 #4 beetles were left in each cage. Some of them appeared to be injured or damaged while handling and soon died (See Table III fOr detail). When the beetles started feeding actively, they were transferred to new cages containing oat seedlings every two days. Mortality records and egg counts were made at this time. The eggs laid in two days were kept for studying the effect of X-rays on eclosion. 3222122. mam; Percent Mortality of Cereal Leaf Beetles Irradiated with.X~Rays -- First Trial. No. of Beetles No. of Beetles % Mortality Treatment left in cage alive after in 15 days 15 days Control 61 40 3u.u 1,000r 7O 43 “7.1 2,500r 7O 34 51.4 5,000r 70 2 97.0 79500r 7O 3 90.0 10,000r 7o 6 91.4 20,000r 70 20 71.4 W Observation on Oviposition and Eclosion of Cereal Leaf Beetles Irradiated with Xwfiays -- First Trial. 45 No. of % Beetles Eggs laid in No. of eggs Eclo- Treatment left 1 day hatched sion for egg laying Control 2#—25th Oct. #3 #9 28 57.1 25-26th Oct. #3 99 63 63.6 27-28th Oct. #1 157 50 31.8 28-29th Oct. #0 1#9 #9 E238 Total #5# Total 190 Ave. 1.8 1,000r 2#-25th Oct. #3 72 1# 19.# 25-26th Oct. #3 6# 18 28.1 27-28th Oct. 36 81 none hatched O 28~29th Oct. 33 63 none hatched 0 Total 280 Total 32 Ave. 11.# 2,500r* 28-29th Oct. 34 29 2 hatched 6.9 29-30th Oct. 3a 12 none hatched o 2- 3rd Nov. 20 _8 none hatched 0 Total #9 Total 2 Ave. E01 5:000r 10 #0 in 3 days nil 0 5,000r (29—30) 2 __1 in 1 day nil o #7 Total *OViposition was considerably delayed in beetles treated with 2,500r and 5,000r #6 Table II (Continued) 7,500r did not lay any eggs - 10,000r did not lay any eggs m 20,000r did not lay any eggs - There was motility of the spermatoza in all treatments immediately after irradiation and thus they were not immediately affected by the radiation. Some of the histological prepara- tions from the irradiated female beetles also showed spermatozoa in the spermatheca up to 20,000r radiation. This indicates the sperm motility is not affected by X-ray radiation, particularly the first few days after irradiation Egeding habits of the irradiated beetles. The beetles irradiated with 10.000r and 20,000r,did not feed as normally as the control. The 10,000r irradiated beetles during the first week after radiation were feeding on the leaves to a lesser extent than the control, but during the second week they stOpped feeding to a considerable extent. The 20,000r irradiated beetles did not feed actively either during the first week or the second week after irradiation. In other treatments, the difference in feeding habits were not pronounced. #7 TABLE III Percent Mortality of Cereal Leaf Beetles Irradiated with X-Rays a» Second Trial. No. Alive No. Alive % Mortality Treatment Initial No. After After in 10 days 7 days 10 days Control 91 60 55 39.7 SOOr 96 50 #7 51.0 1,000r 96 #3 28 70.8 3,000r 100 #2 11 89.0 5,000r 100 #0 8 92.0 (In the second trial, first eggs were noticed on February 11th.) TABLE IV Observations on Oviposition and Eclosion of the Cereal Leaf Beetles Irradiated with X-Rays m» Second Trial. Treatment No. of Eggs Laid Eggs Hatched % Eclosion Insects 11th-13th, Feb. Control 71 2#0 126 52.5 500r 65 144 ' 17 11.8 1,000r 7# 2 nil o 3,000r 85 nil - o 5.000r 82 - - O 13th‘15th’ Feb. Control 61 508 180 35.# TABLE 500r 1,000r 3,000r S’OOOI 15th-17th, Feb. V (Continued) Control 500r 1,000r 3.000r 5,000r 17th"19th, Feb. Control 500r 1,000r 3.000r 5,000r 19th"218t, Feb. Control 500r 1.000r 3 9000]? 5.000r Many had died by ‘ this time and rest were used for histom logical work the 56 56 75 69 60 50 #0 33 32 55 #7 28 ## #0 9 nil nil 500 344 35 nil nil #63 294 23 nil 2# nil nil 185 25 nil 119 #8 8.3 38.8 26.# 8.# 14.2 39.9 8.5 8.7 to #5.2 14.1 8.3 Can’t-nag. “was“ 49 TABLE V Percent Eclosion from the Total Number of Eggs from Each Treatment of Radiation -- Second Trial. Treatment No. of Eggs Laid No. Hatched % Eclosion Control 1.97# 7#2 37.6 500r V 1,253 121 9.6 1,0001: ' 9o 15 16.6* 3,000r 5 2 #0 5,000r nil --- 0 * The total number of eggs laid in this treatment was far less than the control and 500r, but the percent eclosion was higher in the small number of eggs. Mortality of the beetles was very high in beetles re- ceiving X—rays over 2,500r in the first trial (Table I). Some Of the beetles receiving 1,000r lived up to one month. 0bser~ vation was not made beyond this period. Some beetles receiving 20,000r were observed alive at the end of 15 days (Table I). However, they died subsequently. The percent mortality in control was 3#.# in the first trial, whereas the percent more tality of beetles irradiated with 5,000r, 7,500r, and 10,000r was over 90 percent; this was in 15 days. In the case of 20,000r irradiated beetles the percent mortality in 15 days was 71.# percent (Table I) and the 20,000r treated ones did not feed actively. 50 From the results obtained in the first trial, it appears that radiation affects egg production. There was considerable reduction in the number of eggs laid by the irradiated beetles (Table II). Beetles receiving over 7,500r did not lay any eggs at all during the period they were under observation. In the case of beetles receiving 2,500r and 5,000r radiation, the egg production was extremely low when compared with the control. Also oviposition was delayed by 3—4 days in these two treat~ ments as compared to the control and the 1,000r irradiated beetles (Table II). There was also considerable difference between the eclo— sion 0f the control and the irradhflufl.beetles. Percent eclo- sion in the first trial, in the control was 41.8; 11.# percent in 1,000r; and 4.1 percent in 2,500r treated beetles. The eggs of 5,000r irradiated beetles did not hatch at all (Table II). The beetles treated with 2,500r and 5.000r laid only #9 and #7 eggs respectively during the period they were under observation. Out of this only 2 eggs of 2.500r irradiated beetles hatched (Table II). Usually the eggs of normal beetles take 5 to 6 days for hatching. However. in the present study the eggs were ob~ served for eclosion till the 12th day of their oviposition. Mortality in the control was 39.7 percent in 10 days in the second trial (Table III) whereas it was 34.4 percent in the first trial in 15 days (Table I). Mortality of 1,000r irradi~ ated beetles was #7.1 percent in 15 days in the first trial and 70.8 percent in the second trial. The 5,000r treated beetles had 51 a mortality of 97 percent in 15 days in the first trial and 92 percent in 10 days in the second trial (Table I and III). In general, mortality in the second trial was higher than in the first trial (Table I and III). This may be due to the fact that the insects used in the second trial were older than the insects used in the first trial. The insects used in the second trial were collected from the field on June 9, 1963, and remained in the cooler from July 23, 1963 to February 7, 1964, whereas the insects used in the first trial were col- lected from the field on July 26, 1963 and given cold treat» ment from August 6, 1963 to October 14, 1963. This difference in the pretreatment may also account for the disparity and higher mortality in the second trial. The total number of eggs laid by control and 500r treated beetles were 1,97H and 1,253 respectively from February 11 to February 21. However, only 37.6 percent of eggs from control and 9.6 percent of eggs from 500r treated beetles hatched (Table V). It appears from the data obtained in this experiw ment that as low a dosage of radiation as 500r affected ovim Position to some extent, and eclosion considerably (Table V). 1,000r irradiated beetles in the second trial also oviposited fewer eggs than the control. Out of the 90 eggs oviposited by 1,000r treated beetles, only 15 hatched in the second trial, giving percent eclosion of 16.6. Though the total percent ecloe sion in 1,000r treated beetles in the second trial appeared to be higher than 500r treated ones the total number of eggs laid by 500r irradiated beetles was much higher than the 1,000r irradiated beetles (Table V). 52 Beetles irradiated with 3,000r laid only 5 eggs during the period they were under Observation. Out of this 5, only 2 hatched, giving a percent eclosion of 40, though the total number of eggs oviposited was far less than the control and 500r treated beetles. 5,000r irradiated beetles in the second trial did not lay any eggs during the period they were under study (Table V). However, in the first trial a total number of 47 eggs was obtained in this treatment, out of which none hatched (Table II). The data obtained in the present study suggests that X-ray radiation over 7,500r brings total steril— ity as far as the female beetles are concerned. Beetles treated with 7,500r to 20,000r did not oviposit at all during the period they were under vigilant observation (Table II). The data obtained in the present study also indicates that 5,000r irradiation of the adult beetles caused total egg sterility in the few eggs oviposited (Table II). HISTOPATHOLOGY OF THE IRRADIATED GONADS Since there was considerable diminution in the fertility, of the irradiated beetles, it was thought worthwhile to in- vestigate briefly the effect of X-ray radiation on the deve10p- ment of gonads. Material and Methods. Beetles used in the experiment on oviposition effects of X-rays were sampled for this experi- ment. The beetles were removed from the cage at certain per- iods and fixed in KAAD and histological sections of 10 Microns thickness were made in the same manner as mentioned earlier. Feulgen and fast green were used for staining the histological Preparations. Results; It was observed that in beetles receiving higher dosage of radiation, e.g., 10,000r and 20,000r the de- velopment of ovary was at a standstill. The ovary was in the same stage as it was found in the diapausing beetle. Different stages of oocyte deve10pment were not at all noticed in the small samples (about 6 to 12 beetles per sample). Beetles re- ceiving 5,000r also showed no devakgment or a slow deve10p- ment of the ovary (Fig. 15). In the case of male beetles, the testes did not show immaturity or absence of spermatozoa. It 18 important to note that the males had fully matured sperma— tozoa at the time of irradiation. In case of beetles sampled from the second irradiation trail, beetles receiving 500r showed oocytes in different 54 stages of deve10pment and also fully matured eggs (Fig. 16a). But frequently malformations and distortions in the oocytes were observed (Fig. 16b, 160). These kinds of abnormalities were observed in beetles sampled 15 days after irradiation. In addition degeneration of the germarium.was also noticed (Fig. 17). In some cases oocytes in different stages of development were noticed Just below the degenerating germarium. Many cells in the degenerating germarium did not indicate any Feulgen positive material (Fig. 18). Instead9 they took the counters stain, fast green. Some cells in the germarium showed com~ plate absence of the nucleus and in some cases only traces of Feulgen positive material was observed. Cells which showed traces of Feulgen positive material seemed to be in the pro~ cess of degeneration. In some instances, the degeneration and disappearance of the nuclei were observed in patches in the germarium (Fig. 18). A larger number of partially deve10ped oocytes were observed on the pedicel than in the control (Fig. 16a). In these cases the germarium above them was in varying degrees of degeneration. However9 in some cases the deve10pu ment of the ovary was normal and no germarial degeneration or abnormal development of the oocyte was observed. In beetles receiving 1,000r, 3,000r and 5,000r degeneration of the germm arium was also observed in some beetles along with a normal germarium.in other beetles. Beetles receiving 3,000r and 5:000r X-rays showed frequently only a few stages in deve10pm ment and many did not show advanced stages of oocytes9 para ticularly the 5,000r irradiated beetles. The deve10pment of the oocytes in these beetles appeared to be rather slow when compared with the control. 55 Male beetles9 some which received higher dOsages, e.g., 3,000r and 5,000r, showed some reduction in the amount of spermatozoa after 10 days of irradiation. It seems likely that after they exhaust the mature spermatozoa the production of new is affected. Some of the beetles which died after 3,000r and 5,000r radiation were also studied within few hours of their death. These histological preparations also indicated a reduction in the amount of mature sperm. However, it is not known whether it is due to radiation or exhaustion of the sperm supply. This study on histOpathological effects of radiation is rather indicative more than conclusive because of the small number of insects sampled. 56 W ARRESTED DEVELOPMENT, MALFOBMATION AND DEGENERATION OF THE OVARIOLES OF OULEMA MELANOPA (L.) TREATED WITH 5,000r X-RAYS. 265: W Two PARTIALLY DEVELOPED OOCYTES AND ONE FULLY DEVELOPED OOCYTE ON THE PEDICEL OF THE OVARIOLES OF OULEMA MELANOPA (L.) TREATED WITH 500r X-BAYS. 265x EIQUQE 1§b MALFOBMATION AND DISINTEGRATION OF A FOURTH STAGE OOCYTE OF QELEEQ,M§LANOPA (L.) TREATED WITH 500r XmRAYS. 540x GU “ c DISINTEGRATION OF YOLK IN A FOURTH STAGE OOCYTE 0F OQLEMA MELANOPA (L.) TREATED WITH 500r X-BAYS. 540x w DEGENERATING GERMARIUM AND A MALFORMED POST GEBMARIUM OF A NEIGHBOURING OVARIOLE OF A 500r IBRADIATED OULEMA MELANOPA (L.) 5 0X0 ELGQEE l8 DEGENEBATION OF THE GERMARIUM OF A 5,000r IRRADIATED OULEMA MELANOPA (L.) SHOWING PROGRESSIVE LOSS OF NUCLEAR DNA. 1080x a ed 958E v-‘TfiT-r A "\ DISCUSSION It appears from this investigation that the females of the cereal leaf beetles attain sexual maturity later than the males. Histological preparattxm made Of female beetles withm in B-h days of emergence9 prewdiapause beetles and diapause beetles showed arrested deve10pment or immaturity of the ovary. Whereas many of the histological preparations made of males 3- # days after emergences preediapause and diapause beetles showed spermatooytes, spermatids and some mature spermatoza. The female beetles seem to require at least a short period for the full development of the ovary immediately after diapause. Most of the female beetles sectioned after they came out of diapause showed gradual deve10pment of the ovary after 2~3 days. Beetles sectioned in the prewdiapause, diapause, and post—diapause state showed bright staining.DNA and RNA positive cells in the germarium with very little cytOplasm during the underdeveloped period of the ovary. Although nutritive cords, connecting the bright staining cells in the germarium with the develOping oocytes, were never observed during this investiga» tion, it seems quite likely that the bright staining cells in the germarium do perform some sort of nutritive function. The findings of Krause (19E6) on Easgalps, and Sohlottman and Bonhag on Tenebrio molito; L. also support this View. Krause (1946) noted bright staining material at the tip of the ovariole 0f Passalus and the smaller deveIOping egg cells possess plas~ matic strands. However, he could not demonstrate direct and 64 definite protOplasmic connections between oocytes and the distal tip of the ovariole,but presented evidence that some sort of protoplasmic connection does exist. Schlottman and Bonhag (1956) noted definite protOplasmic strands unite the folliculate oocytes with the nutritive cells in the germarium and suggested that in contrast to the strongly develOped cords usually found in the telotroPhic ovarioles of hemipterous insects, the nourishing strands in ngebrio mglipgg L. are of a much more delicate nature and may be quite easily overlooked. Wilde, gt a1. (1959) working on Colorado potato beetle also reported that they could not find cytOplasmic connections from the in- conspicuous nutritive cells with the oocytes after being re- leased into the vitellarium. In the light of these previous reports on other beetles and the present observation of apical and dense bright staining nature Of the germarium, it is sug~ gested that the ovarioles of cereal leaf beetle, Qulema melanOpa (L.) are of acrotrophic type. Although the oocytes grow and enlarge in the vitellarium. the presence of yolk is not clear until the oocytes reach the stage when the surrounding follicular cells become cuboidal in Shape. This finding is quite in agreement with that of Schlottu man and Bonhag (1956) on Tenebrio molitor L. The presence of bright staining Feulgen positive (DNA) and RNA positive substances in the follicular cells. particum larly in the later stages, suggests that the follicular epithem lium in the cereal leaf beetle may also play at least some part in the nutritive function of the deveIOping oocyte along with the DNA and RNA rich germarium. It may be probable that the 65 increasing nutritive demands of the develOping oocytes require additional supply of this kind of material which is not solely met by the germarium.during this period. This view is also supported by the fact that bright staining, large and cuboidal follicular cells rich in nucleic acids are present in the third and fourth stages of the oocytes. However, investigators in this field, particularly Schlottman and Bonhag (1956), attri- bute the alteration in the size and shape of follicular cells to changes in volume of the growing oocyte. According to them, as the oocyte within the follicle grows larger, it ex- erts an ever increasing pressure upon the cells of the folli- cular epithelium. This pressure is alleviated in the incipient follicles chiefly by cellular multiplication and growth of the daughter cells. Since they noted that the mitotic ability is apparently lost by the epithelial cerkgof the more advanced follicles, they concluded that accomodamon to changes in volume of the oocyte in Tenebpio molitor L. occurs through an alteration in the size and shape of the follicular cell. The oocyte growth and continuous change in shape of the surrounding follicular epithelial cells by the gradual transition of the columnar epithelium into a cuboidal condition and finally into a squamous type was also observed in the present study on the ovary of cereal leaf beetles. However, Schlottman and Bonhag (1955) agree that there is evidence that at least in some telotrOphic ovarioles nutritive substances are passed from bOth the follicular epithelium and the apical trophocytes to the develOping oocytes. Also their cytological observations indicated that nucleic acids (DNA and RNA) or their derivatives 66 may be contributed to the enlarging oocytes by the follicular epithelium, The observatnniof bright staining DNA and RNA positive follicular epithelium, particularly in the later stages of the oocytes of the cereal leaf beetle also supports the view of Wilde, 23, al. (1959) and Schlottman and Bonhag (1956) that follicular epithelium also makes a nutritive contribution to the deve10ping oocytes in some telotrophic ovarioles. The present study on the effects of radiation on the re- productive aspects of Qulema melan09a (L.) was designed to form the basis of experiments more directly related to the practi- cal problem of controlling this insect by the sterile-male technique. Radiation, even as low as 500r, appeared to affect the normal deve10pment of the ovary of a fair number of sus- ceptible adult female beetles sed in this experiment. How- ever, radiation up to 20,000r did not seem to affect much the mOtility of the spermatozoa particularly in the first few days after radiation. This conclusion is supported by the fact that in some of the 20,000r treated beetles, the females sec» tioned after about two weeks showed spermatozoa in the seminal receptacle. .At this point, it is important to point out that dominant lethality does not mean the cessation of the motility Of the sperm. "Dominant lethafixy'is the change caused by radiation in genetic constitution of a sperm which may not prew vent fertilization of an egg, but may make the zygote incapable 0f deve10ping to maturity, usually causing death in the embry- onic stage" (Bushland and Hopkins, 1953). 67 The histological preparations made from the irradiated females showed lack of deve10pment of the ovary above a radia~ tion dosage of 5,000r. Many of the 3,000r and 5,000r treated females also showed a kind of inhibition of the deve10pment of the ovary. Oviposition was drastically reduced in beetles treated with X-rays 2,500r and over, and mortality was greatly increased in beetles receiving over 2,500r radiation. In gen- eral, the cereal leaf beetle appeared to be radio sensitive above the dosage of 2,500r. The sterilewmale method may not be feasible with certain species of insect because of the damaging effects of radiation in the vigor of the insect. As LaChance and Knipling (1962) pointed out, one of the most important requirements that must be met in applying the sterilewmale technique successfully is that the sterilized males must not be seriously affected by the sterilizathniprocedures so that they will be reasonably competitive with normal males. The present study showed that in general radiation increased mortality, particularly the doe sages over 2,500r. This finding is contrary to one of the im~ POrtant requirements of sterilemmale technique of controlling insects. Secondly. the dosage of 2,500r and 3,000r appeared to allow some eggs to deve10p in these treatments and also seemed to affect the normal deve10pment of the ovary in many. Higher dosages of radiation also appeared to affect the feed» ins habits, particularly l0,000r«—20,000r. Since at 5,000r treatment, the females