The Rhodnius testis: hormonal effects on germ cell

The Rhodnius testis: hormonal effects on germ cell division J . B. DUMSER~ AND K. G . D A V E Y ~

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Institute of Parasitology, Macdonald College, McGill University, Quebec H9X3M1 Received May 25, 1975 DUMSER,J. B., and K. G. DAVEY.1975. The Rhodnius testis: hormonal effects on germ cell division. Can. J . Zool. 53: 1682-1689. The spermatogonialcells of theRhodniirs testis exhibit a basal level of division activity in the absence of morphopenetic hormones. Ecdysone, naturally induced by feeding o r injected in physiological cto~es. ~pproxirnstelydouble% the mitotic index as measured by colchicinemetaphose accumulation. Juvenile hormone production in the molting larvae or application of juvenile hormone m~rnicfamesyl methyl ether abolishes the ecdysone-stimulated increase in mito~icindex, but has no effect un the basal Icvel. Similar results are obtained in fed and unfed decapitates. and in surgicfilly manipulated insects. In contrast, natural induction of ecdysone secretion is shown to be ineffective in altering the duration of the meiotic prophase. DUMSER,J. B., et K. G. DAVEY.1975. The Rhodnius testis: hormonal effects on germ cell d~vision.Can. J . Zool. 53: 1682-1689. Chez les spermatogonies de Rhodnius, I'activite de division se fait selon un degre de base en I'absence d'hormones morphogenes. L'ecdysone, qu'elle soit stimulee normalement par I'alimentation ou injectie en doses physiologiques, double I? peu prks I'indice mitotique tel que m e s d par accumulation de cellules en mktaphase apres traitement B la colchicine. La production $hormone juvenile chez les lnrvesen ~nktamorphoseou I'adrninistration d'ether methylique farnlsylique irnitan! I'hormone juvtnile annule l'augmentation de I'ind~cemitotique stimulke par l'ecdysone. mais reste sans effet sur le tlegrk de base de I'activite. On obtient des resultats sernbtahlcs chez des spCcirnenrs d$capit&snourris et non nourris et chez des insectes manipules par chirurgie. Par contre, I'induction naturelle de secretion d'ecdy sone n'a pas d'effet sur la durke de la prophase de la miiose. [Traduit par le journal]

spermatogenesis in vitro (see review by Marks Introduction In an earlier publication (Dumser and Davey 1970). The notion of a germ cell division rate ac1974) we suggested a kinetic hypothesis of celerated by ecdysone is implied in the reports hormonal control of insect spermatogenesis which predicted an increase in the rate of cell of earlier authors using both in vivo (Nishiitsutdivision in the presence of ecdysone, and the suji-Uwo 1961; Takeuchi 1969) and in vitro absence of this ecdysone stimulation in the techniques (Schmidt and Williams 1953; Wilsimultaneous presence of juvenile hormone. liams and Kambysellis 1969; Yagi et ol. 1969; Juvenile hormone was further suggested to have Takeda 1972). An increased frequency of mitotic no effect on the basal, anhormonal rate of metaphase figures can be correlated with times division noted in unfed Rhodnius. Thus, a of known or suspected ecdysone presence in schema may be visualized which accounts Rhodnius (Dumser and Davey 1974) and in the for the changes produced by hormonal mani- mosquito Aedes aegypti (Jones 1967). This pulation during the larval stadia. and for the correlation is further supported in Rhodnius by continuation of spermatogenesis during the an analysis of changes in the size of the various endocrinologicalty distinct adult stadium (see differentiated germ cell compartments (Dumser review by Phillips 1970) and anhormonal and Davey 1974). These studies, however, provide evidence which is at best correlative: none of them has attempted to confront the 'Present address: Department of Entomology, Waite hypothesis with direct experimental methods. Agricultural Research Institute, University of Adelaide, The present paper, therefore, presents the results Glen Osmond, South Australia. 'Present address: Department of Biology, York of experiments which investigate the effect on University, Downsview, Ontario M3J 1P3. the division activity of the germ cells of Rhodnius

DUMSER AND DAVEY: RHODNIUS TESTIS


of various manipulations of the hormonal milieu in the insect.

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elsewhere (Dumser 1974). These later experiments also employ the more rigorous counting procedures described below. It was previously determined (Dumser and Davey 1974) that the Rhodnius testis, at the age and instar Materials and Methods selected for experimentation, contains only spermatoRhodnius has been reported Rearing and breeding gonial cells surrounded by a sheath of various mesodermal elsewhere (Pratt and D a v e ~l972). Surgical procedures cells. Before incubation in sodium &rate solution, the have also been described ( ~ u m s e r1974). Additional sheath was removed, thus reducing the mesodermal techniques include decapitation of fourth-instar insects, cell presence to those cells lining each tubule, cyst-wall This procedure is carried Out between 23 and 25 cells, and a few plug cells and vas efferens cells. The following the blood meal to insure a reasonably con- proportion of mesodermal cells was thus very small in sistent exposure of the individual samples to endogenous relation to the germ cell population, and no effort was ecdysOne lo days was made to separate cell types when counting cells or A recovery time provided following removal of the head. All structures metaphase plates. anterior to, and including, the suboesophageal ganglion The germ cell cysts increase in cell number in an were and the sealed with Tacki- exponential fashion; hence, the great proportion of cells wax (Cenco Ltd.). p-ecdysOne (ROhto will be those of the most advanced cysts. In Rhodnius, Pharmaceuticals, Osaka. Japan) were prepared in distilled the three largest categories of germ cell cysts would be water and stored frozen. Injections Were administered expected to represent 87.5% of the total germ cell populathrough the last thoracic leg and the wound sealed with tion. This permits the experimenter to ignore the pasTackiwax to prevwt leakage- A 5-p1 sibility of a progressive and regular change in cell cycle with 33-gauge need'e CO.* duration during the course of germ cell differentiation. was e m ~ l o ~ e dSimilar , techniques used for In- However, the statistical validity of the mitotic index jetion of colchictne, also prepared in distilled water, within any given microscopic field in the squash may be and for injmions of I3Hithymidine (5.Q P C ~ / ~reduced ~ ; by the synchronous development of these large *ppIications of Samesy' cysts. In fact, the resulting preparations are not at all Amersharn %Ie* Hoffmann-La Itmhe homogeneous as to frequency of division figures. Forether (FME)* a gift IBasel, the were made tunately, the testis at the time selected is of such a size that abdomen. Solutions prepared in iso-octafie and the ,,tire cellular complement is displayed in a single stored under nitrogen at - 20 "C. slide. Twenty-five to 50 fields, comprising between 2000 and 4000 cells, were sampled on each testis. Since these Radioautography preparations were not particularly homogeneous as to Testes were removed from the bug under 0.8% sodium cell density, all cells within the ocular grid were counted citrate, desheathed, and placed in fresh citrate for for each sampling as well as metaphase figures, and the 15 min. The relevant portion of the tubule was squashed numbers summed to provide an average mitotic index for under a coverslip by thumb pressure, and the coverslip the entire testis. frozen with Freon 22 to permit its easy removal. PrepAlthough all preparations except the first were carried arations were dried overnight at room temperature, out by the improved aceto-orcein technique, insufficient fixed for I h in acetic acid -ethanol (1 :3). washed 1 h pressure would still occasionally result in ambiguous in absolute ethanol. and permitted to dry before being chromosome spreads, and such slides were discarded. dipped in Kodak NTB 2 emuls~on. R s d i o a u t o ~ a p h ~All the slides were blinded with opaque white tape, waq carried out by the method of Kopriwa and Leblond shuffled, and numbered randomly before counting. These (1962). After development of the emution, the prepamtechniques are time-consuming; so much so that the tions were stained in a 1 % solution of toluidine blue in sample size of any given experiment is limited by the 41% borax, dehydrated in ethanol, and mounted in Per- to 5-day usable life of the squash preparation. Nonethemount (Fisher Scientific). less, the highly reliable and unbiased estimation of mitotic index so produced outweighs the disadvantage of small Mitotic Index sample size. Colchicine injections were carried out as previously Temporay preparations for this purpose were initially described. It should be noted the volume and concentramade by squashing the testis in acetotmine by thumb tion varied somewhat from experiment to experiment, pressure applied to the coverslip and w m i n g the prepM best results finally being achieved at 2 p1 of aration with a gas flame. This prwdure in Rhodnilrv solution, with an exposure time of 24 h. Concentrations yields a highly condensed chromosomal appearance, and durations used in earlier experiments should not be and it is very dimcult indeed to direrentiate between typical "ball metaphases" and potentially pycnotic considered invalid, but rather non-optimal. Failure of nuclei. Furthermore, these slides were not blinded before the colchicine to block mitosis completely resulted in the appearance of anaphase and telophase figures, and such countjng the metaphase figrrres. Only the first experiment mported here used this procedure. Subsequent preparations were discarded. cxperirnenls employcd a cold aceto-orcein "tap" squash The mitotic index was calculated simply as the ratio rnethcd with pretreatment in 0.8% sodium citrate; of metaphase figures to total cells within the testis x 100. recommended by Ms. G. Fontana of the Department d Means, standard deviations, and tests of significance Entomology, Macdonald College, and described in detail (t-test) were carried out as described in Dunn (1967).

Nevada)

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In all cases, methods used included Bessel's correction for small sample size.

TABLE 1 The effect of ecdysone injection and FME application on

the mitotic index in the testis of fed fourth-instar Rhodnius Results lacking their neurosecretory cells and corpora allata Colchicine Mitotic Index A preliminary experiment in this series was No. Treatment Mitotic index carried out to investigate the effect of exogenous Group of insects morphogenetic hormones on the insect rendered 8.8+1.8* A 5 Inject water, free of such hormones by surgical removal of apply iso-octane B 3 Inject water, 8.3k1.8 the appropriate endocrine organs. The counting apply FME techniques used did not guarantee an absence C 3 Inject ecdysone, 19.8 + 4.2 of bias, and technical difficulties in some cases apply iso-octane resulted in a very small sample size. Neverthe8.9k0.8 D 2 Inject ecdysone, apply FME less, the results show at least a statistical significance and are in good agreement with later N O T E : A V S . B , D ; B V S . D>; P0 . 4 . C v s . A , B , D ; p c 0.05. *Standard error. trials using different preparations and methods. Sixty-six insects of similar size and history were isolated 20 days following the molt from effect on the basal mitotic index in the absence third to fourth instar. All were subjected to of ecdysterone, as predicted by the kinetic removal of the corpus allatum, corpus hypothesis. A more physiological, albeit less direct, cardiacum, and neurosecretory cells of the pars intercerebralis. During the postoperative re- demonstration of these effects was provided by covery period, 30% of the sample died. Following the following experiment. Siblings, approxithis 10-day period, the remaining bugs were mately 1 month after the molt to the fourth permitted to feed, 80% (56% of the original instar, were subjected to removal of the corpus allatum alone. Particular care was taken to operated group) gorging to repletion. The gorged insects were held for 24 h in the avoid damage through overheating when sealing incubator at 28 "C to permit diuresis, and the wound with Tackiwax in order to favor the randomly segregated into four groups of nine normal feeding response, brain hormone reinsects each. Inability to discriminate the sex of lease, and prothoracic gland activation. Morthe insects at this stage resulted in a further tality was 25% during the 10-day postoperative reduction in ultimate sample size. The combined period. Sham-operated controls, in which a losses from postoperative mortality, refusal to similar incision was made and one salivary feed, and inability to sex the fourth instar re- gland reservoir removed, suffered a 40% sulted in a yield of usable insects of about 20%. mortality. The insects were fed to repletion 10 days Two groups were injected with 1.0 p1 of 2.5 x M colchicine solution in distilled following surgery, and returned to the inwater containing 1.0 pg of P-ecdysone, and two cubator. Twenty-four hours after the blood groups were injected with the colchicine alone. meal, 30 allatectomized individuals received an One hour following this treatment, one group application of 0.7 nl FME in 0.5 p1 iso-octane, receiving P-ecdysone and one control group and 30 received iso-octane alone. Shameach received 0.5 nl FME dissolved in 0.5 pl operated controls similarly received iso-octane iso-octane. The remaining P-ecdysone group only. At 60 h post feed, all the insects were M colchicine and control group received applications of injected with 2 p1 of 2 x 0.5 p1 iso-octane only. The insects were re- in distilled water. Twenty-four hours later, all turned to the incubator, removed 24 h later and the insects were removed to the incubator at held at 4 ° C pending dissection. Each insect 4 "C until dissection and preparation of right was dissected, the right testis removed, and testis squashes by the aceto-orcein method. The counts from this experiment yielded consquashed in hot aceto-carmine. The results (Table 1) clearly demonstrate a doubling of the sistent and highly significant results (Table 2). mitotic index in the presence of ecdysterone, an In the allatectomized individuals, in which effect which is entirely abolished by the simul- feeding presumably led to ecdysone secretion taneous application of FME. FME showed no in the absence of juvenile hormone, there was


TABLE 2 The effect of allatectomy and FME treatment on the mitotic index in the testis of fed fourth-instar Rhodnius Group

No. of insects

Surgical treatment

Hormonal treatment

Mitotic index

9 7 11

Sham Allatectomy Allatectomy

iso-octane iso-octane FME

9 . 2 f 2.6* 16.0k3.7 9.2f2.3

-

A B C


NOTE:B vs. A, C ; p < 0.001. 'Standard error.

TABLE 3 The effect of ecdysone injection on the mitotic index of fed, decapitated fourth-instar Rhodnius Group A

B C

No. of insects

Treatment

Mitotic index

7 7 6

Water Ecdysone Intact, unfed

1.0+0.3* 2.9k1.3 1 .0 _+ 0 . 6

NOTE:B VS. A. C ; p x 0 1 . *Standard error.

a 75% increase in mitotic index over the shamoperated animals, which were activated in the presence of naturally occurring juvenile hormone. Moreover, applications of the synthetic analogue FME totally abolished this increase. There was, however, no FME-related depression of the mitotic index below the basal level demonstrated by the controls in spite of the rather arbitrary dose of the analogue. In an attempt to standardize and simplify the above procedures, a series of experiments was carried out using insects in which the neural and retrocerebral endocrine axis had been removed by decapitation rather than surgery. Fed fourth-instar insects were decapitated as outlined in Materials and Methods. Ten days later, these insects were injected either with 1.0 pg B-ecdysone in 0.5 p1 distilled water, or with water alone. Unfed, intact siblings were left uninjected. None of the insects received colchicine. Samples were prepared as before at 24 h, and the results obtained (Table 3) demonstrate the effectiveness of ecdysterone in these preparations as well. Further, the mitotic index demonstrated by the fed, decapitated control insects did not differ significantly from the basal mitotic index shown by the unmanipulated insects during the prefeed endocrinological diapause.

In an attempt to provide greater reliability of counting, colchicine was again introduced into the procedure. Decapitated insects were prepared as in the previous experiment. On the assumption that alterations in the mitotic index resulted from changes in the duration of the G1 phase of the cell cycle, ecdysone was injected 12 h before the injection of colchicine. Not bound by a circadian input in these decapitated insects, exposure to colchicine (2 p1 of M) was reduced to 10 h. This experiment involved two groups only : those receiving 0.5 pg P-ecdysone in 0.5 p1 distilled water, and those receiving water alone. The experiment consisted of two trials 2 weeks apart. In the first trial, eight animals receiving ecdysone exhibited a mean mitotic index of 7.2 f 2.6 SE compared to the eight control animals where the mitotic index was 4.6 1.7 SE (p < 0.05). In the second trial, the mean mitotic index of nine ecdysoneinjected animals was 4.3 1.2 SE while that of seven controls was 3.5 1.1 SE (p < 0.1). While these results agree qualitatively with the earlier experiments, they are quantitatively different not only from the earlier data, but from each other as well.

+

+ +

Duration of the Meiotic Prophase During the course of the fifth instar, ecdysone alone is secreted, and juvenile hormone is not produced for at least several days following the blood meal (Wigglesworth 1940). By this time as well, spermatocytes have been differentiated (Dumser and Davey 1974). We took advantage of these two facts to investigate the effect of ecdysone on the duration of the lengthy meiotic prophase. A sibling population of Rhodnius males was isolated 8 days following the 4th to 5th molt. Forty-five unfed insects were injected with 1.5 pCi [3H]thymidine in distilled water; 45 insects were fed to repletion,

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and received similar injections of 2.5 pCi [3H]thymidine 24 h following the blood meal. Permanent testis squashes were made from these individuals, two samples being taken from each group at 12-h intervals from day 6 through day 14 after the isotope injection. The first appearance of specific label in meiotic metaphase plates occurred at 123 days following the injection in both fed and unfed insects. It is thus concluded that ecdysone secretion has no appreciable effect on the duration of the meiotic prophase in Rhodnius spermatocytes. Discussion The kinetic hypothesis of germ cell development in Rhodnius (Dumser and Davey 1974) requires that the rate of cell division of the spermatogonial cells must increase in the presence of ecdysone, and that this increase be wholly or partially inhibited by the simultaneous presence of juvenile hormone. Results reported in this paper demonstrate precisely these responses for the mitotic index using a variety of surgically modified preparations with naturally occurring and exogenously supplied ecdysone and juvenile hormone. Mitotic index is an adequate estimate of the number of germ cell cysts which are changing differentiated compartments through cell division, but it is not necessarily a precise estimate of the rate at which this occurs. The mitotic index, as a measure of identifiable division state compared to total cell population, is affected by any changes in the size or activity of that cell population. This principle has its greatest impact in tissues containing a mixture of cells committed to division, and cells which for one reason or another have ceased to divide. In some cases, major .sources of these nonproliferative cells can be identified and avoided; thus, preparations used for these studies were taken from a population of such an age that differentiation of the spermatocytes had not yet occurred, and would not occur during the time course of the experiments (Dumser and Davey 1974). Spermatocytes, with a long obligatory prophase period, would be considered nonproliferative under the terms of the experiments, and their accumulation would produce a decline in mitotic index with time. However, two processes give rise to other potentially serious sources of non-proliferative cells in this system:

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specific autolysis of the most differentiated germ cell element during diapause (Dumser and Davey 1974) and random germ cell differentiation (Roosen-Runge 1973; Dumser 1974). The first of these, specific autolysis, although related in some manner to the presence of ecdysone, occurs both in the presence and absence of juvenile hormone in the intact insect and thus cannot be responsible for the changes in mitotic index demonstrated in this work. The situation with respect to the apparently random nuclear pycnosis and cell degeneration which occurs throughout the Rhodnius testis is not as clear. There is at present no valid technique to identify which cells are on this nonproliferative pathway (Roosen-Runge and Leik 1968), and attempts at quantifying the degree of pycnosis in any preparation have not been successful. There is no firm evidence that the percentage of the cell population engaged in this activity varies with normal alterations in the physiological milieu, but neither is there evidence that it does not. This complex topic will serve as the subject of a subsequent paper in this series. For the present, the argument will be advanced that this phenomenon is likely to have only a negligible effect on the mitotic index. Firstly, if RoosenRunge (1973) is correct in suggesting the phenomenon to be due to an accumulation of genetic deficiencies resulting in abortive division, the frequency of such an accumulation is unlikely to be influenced by hormonal conditions, nor, functioning through catastrophic division, would there be interphase cells lingering in the non-proliferative compartment. Secondly, the degree of increase of the mitotic index would require a pool of cryptic 'suicide' cells which was considerably larger than that estimated to exist in the population at any time. Of course, such an estimation depends in part on the length of the cell cycle compared to the length of time required for the complete degeneration of a cyst, and this information is not available. However, cysts with actively pycnotic nuclei occur at a maximum rate of 5 to 10% of the total cell population. Furthermore, such cysts can often be found in areas where the surrounding cysts are more advanced in terms of numbers of cells per cyst. This indicates that even the actively degenerating phase of the phenomenon is longer than the duration of a normal cell cycle,



and the frequency of occurrence is thus less than the 5 to 10% figure estimated. In the light of such observations, it would seem likely that the changes in mitotic index noted represent a real shortening of the duration of the germ cell cycle. However, any more concrete statement must await a direct measurement of this parameter under the appropriate hormonal conditions. I t is perhaps pertinent to note here that a definite shortening of cell cycle duration has been noted in the larval wing discs of Ephestia kuhniella during a period of known ecdysone presence (Lobbecke 1969). Similar preparations in Drosophila exhibit a sequential differentiation (Mindek and Nothiger 1973) not unlike that demonstrated for the Rhodnius male germ cell system (Dumser and Davey 1975). It is possible that the observed effects on division activity simply reflect the overall increase in metabolic activity of the insect which is known to be under hormonal influence. Before the blood meal in each instar, Rhodnius exhibits a period of developmental arrest. This is terminated, and the next molt cycle initiated, by the secretion of brain hormone and ecdysone production subsequent to the act of gorging (Wigglesworth 1934). The temporal contiguity of the blood meal stimulus and initiation of development places this phenomenon outside the classical definition of diapause (Lees 1955). It is perhaps more relevant, however, that the process is equivalent to the endocrine scheme proposed for pupal diapause by Williams (1952). As in these developing lepidopteran pupae, cessation of the endocrinological larval diapause in Rhodnius results in a marked increase in the level of oxidative metabolism to 4 or 5 times the diapause level (Zwicky and Wigglesworth 1957), and it is conceivable that this nonspecific metabolic stimulation, initiated by the secretion of ecdysone, may be responsible ultimately for the stimulation of division activity in the testis. However, Zwicky and Wigglesworth (1957) note that no unusual demands seem to be made on oxidative metabolism during the period of epidermal cell mitosis in Rhodnius: the curve of oxygen utilization seems rather to reflect the increasing demands of protein synthesis. Similarly, although inhibitory effects of juvenile hormone on oxidative metabolism have been described for other species (Firstenberg and

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Silhacek 1973), this appears not to be the case in Rhodnius. The ratio of the 0, consumption between the fourth and fifth instars (i.e., in the presence and absence of juvenile hormone) is 1 :2.3 (Zwicky and Wigglesworth 1957), a ratio which is virtually identical to that between their weights (1 :2.4 (Dumser 1974)). Thus, juvenile hormone, effective in inhibiting the ecdysone stimulation of germ cell division activity, is without effect on normal oxidative metabolism, suggesting a more specific role for these hormones in the dynamic control of spermatogenesis. Finally, certain phases of the germ cell cycle, for instance the duration of the meiotic prophase, are unaffected by ecdysone presence and attendant metabolic acceleration. The apparent antagonism of ecdysone and juvenile hormone in the system described, though somewhat unusual, is not without precedent. Juvenile hormone abolishes ecdysone-stimulated RNA synthesis and protein synthesis in Samia cynthia wing discs in vivo (Pate1 and Madhavan 1969), and RNA synthesis in Calliphora erythrocephala fat body in vitro (Congote et al. 1969). The growth of Tenebrio molitor ovaries in vitro is also enhanced by ecdysone, an effect which is abolished in the presence of the juvenile hormone analogue farnesol (Laverdure 1970). Hormonal interactions in insects are as yet imperfectly understood. It has been conclusively demonstrated, however, that ecdysone exerts its effects on spermiation in lepidopteran testes through a second bioactive substance termed "macromolecular factor" (Williams and Kambysellis 1969; Kambysellis and Williams 1971). In vivo preparations used for the experiments reported in this paper do not permit the recognition of such macromolecular factors; however, the introduction of intervening steps such as this in the hormonal stimulus-response framework could provide a ready explanation for the somewhat non-classical interactions found for the morphogenetic hormones. That is, for division rate as well as spermiation, the germ cells themselves may not be the primary target tissues of the morphogenetic hormones. Experiments on fed, decapitated preparations yielded results which, though statistically significant at the 0.5 to 1.0% level and in agreement with results obtained in other preparations, showed a less-pronounced hormonal stimulation and a large variation between trials.


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Reasons for this variation are not known. Simple physical parameters such as degradation of the P-ecdysone solution employed in both trials, or a less effective delivery of the thoracic injections of hormone to the relevant target tissues resulting from a reduced haemolymph circulation in these preparations, might be the cause. Similarly, while such decapitated preparations might survive for several months, the incidence of mortality is high in comparison to preparations involving specific surgical manipulation. Decapitates are capable of very limited coordinated locomotor activity, and thus the degree of viability of any given insect is difficult to assess. Inclusion of a high and variable number of such physiologically substandard individuals in the sample might mask hormonal responses. A more complex explanation for this reduced and variable response might be linked to the phenomenon of hyperecdysonism (Williams 1968; Mouze et al. 1973). Like many insects, Rhodnius responds to injections of P-ecdysone by an accelerated apolysis and new cuticle deposition (Wigglesworth 1970). Doses employed in the headless preparations were chosen to produce ecdysis in a nearly normal molting span (Dumser 1974) ; however, some endogenous ecdysone secretion can be presumed to have occurred in the 24 h between feeding and decapitation. Apolysis in P-ecdysone-treated decapitates is evident within 24 h of injection of the hormone, a response considerably advanced over that of normally activated insects. Large doses of P-ecdysone in the normal insect produce apolysis and cuticle deposition without the normally preceding mitoses in epidermal cells (Wigglesworth 1970), a phenomenon which might be reflected in demonstrations of mitotic inhibition of epidermal cells in in vitro wing-disc preparations by P-ecdysone (Oberlander 1972). Such inhibition of epidermal cell mitosis may result from a truncated functional differentiation in these cells, in which division is an apparently facultative event (Wigglesworth 1970). However, the possibility of a specific mitotic inhibition by large amounts of ecdysone, either directly or through more complex interactions with other circulating factors, cannot be excluded at this time. Indeed, specific inhibition of mitosis by P-ecdysone has been reported for cultured insect cells (Courgeon 1972).

In summary, in support of the kinetic hypothesis of germ cell development, ecdysone increases the amount of division activity in the testis in Rhodnius. The simultaneous presence of juvenile hormone extinguishes this acceleratory effect, but has no effect on the basal division rate demonstrated by the unfed insect or the fed, decapitated insect. During early larval instars the rate of germ cell division, and hence differentiation (Dumser and Davey 1974), would thus be suppressed in the presence of a high titer of juvenile hormone, despite periodic ecdysone secretion. Ecdysone secretion in the preimaginal instar, in the absence of juvenile hormone, permits the maximal rate of division in the testis and the subsequent production and maturation of large numbers of spermatozoa. Maintenance of the basal division level in vitro, and in the adult, would allow a continuation of spermatogenesis in the absence of any insect hormones. At present, it is not possible to differentiate conclusively between changes in the duration of the germ cell cycle or shifts in population between proliferative and non-proliferative states as responsible for the observed changes in mitotic index. Indeed, the two phenomena may be inextricably linked (Frankfiirt 1971). Like the meiotic prophase of the Rhodnius spermatocyte, the duration of the repetitive cycles of the spermatic epithelium in mammals is unresponsive to hormonal manipulation (Clermont 1972). The more continuous linear sequence of spermatogenesis in Rhodnius, in contrast, would seem to more closely resemble the control of spermatogonial stem cell division recently defined for mammalian testes (Clermont and Mauger 1974).

Acknowledgment Research at the Institute of Parasitology is supported by the National Research Council of Canada. J. B. Dumser was the recipient of a NRCC postgraduate scholarship. CLERMONT, Y. 1972. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev. 52: 198-236. Y . , and A. MAUGER.1974. Existence of a CLERMONT, spermatogonial chalone in the rat testis. Cell and Tissue Kinet. 7: 165-172. L. F . , C . E. SEKERIS, and P. KARLSON. 1969. CONGOTE, On the mechanism of hormone action. XIII. Stimulatory



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