Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.     

A stage-specific ovarian factor with stable stimulation of juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata

This is a study of a feedback loop from a stimulated organ to glands that produce the stimulatory hormone in the cockroach Diploptera punctata. In this insect as in many others, juvenile hormone (JH) produced by corpora allata (CA) stimulates vitellogenesis. In our previous studies, transplantations of ovaries at certain stages of development into ovariectomized mated females stimulated JH synthesis within 24h. An in vitro study by other investigators showed that all stages of ovaries release a stimulatory factor into culture medium that was not retained on a solid-phase extraction column but occurred in the aqueous flow-through. The present study is a comparison of the effect of medium conditioned with ovaries from days 1-4 and 8 of the first reproductive cycle, to the effect of the flow-through of that medium on members of a pair of CA from day 3 females. Results provide evidence for an ovarian factor that stimulates JH synthesis by CA in vitro after removal from the conditioning medium (i.e., stable stimulation). Only medium conditioned with ovaries from days 2 or 3 females significantly stimulated CA more than flow-through. Stimulation was dose dependent, sensitive to trypsin, and survived freezing. These results indicate that CA can be directly and stably stimulated by a stage-specific peptidergic ovarian factor.     

Development-activity relationships in nymphal corpora allata of the cockroach, Diploptera punctata

Abstract. In females of Diploptera punctata the corpora allata undergo a gradual increase in volume during most of the second nymphal stadium. In the first half of the stadium, steady growth of the glands results from a progressive increase in the size of constituent cells. Late in the stadium, cell size declines but the volume of the glands continues to rise due to an increase in cell number. Changes in cell size during the stadium displayed a distinct pattern in relation to Juvenile Hormone (JH) synthesis. Both cell size and activity increased during the first two-thirds of the stadium, peaked early in the last third of the stadium, and decreased before the moult. The rise in cell numbers late in the stadium corresponded to a wave of cellular mitosis and occurred after a steep decline in the rate of JH biosynthesis. Exposure of late second instars to fenoxycarb. a JH analogue, depressed mitosis significantly, suggesting autocrine regulation of cell proliferation in the corpora allata. Possible mechanisms modulating sequential cycles of growth and atrophy of cells and cell proliferation in these glands are discussed in relation to temporal patterns of JH and ecdysteroid titres in nymphs.     

Ultrastructural changes in corpora allata during a cycle of juvenile hormone biosynthesis in embryos of the viviparous cockroach, Diploptera punctata

We have observed changes with time in the fine structure of corpora allata (CA) during a known cycle of increasing and decreasing juvenile hormone (JH) synthesis in late embryos of Diploptera punctata. A previous report showed that rates of JH release were relatively low in 28-day-old embryos, but CA activity subsequently rose linearly to a peak on about day 42, and thereafter steadily declined to a low level on day 64 just before birth (Holbrook et al., 1997). We now show that, regardless of rate of JH synthesis, CA cells are large and replete with organelles which nevertheless exhibit variable morphology in embryos of different age. Highly active CA cells on day 40 contain abundant ring-form mitochondria, whereas CA cells of low activity on days 28 and 64 contain mitochondria that are rod-shaped or globular. Mitochondrial cristae were scarce and indistinct on day 28 but numerous and well developed on day 64. Endoplasmic reticula (ER) are rare on day 28 and appear in increasing numbers when CA activity rises. On day 40, ER are abundant and often exhibit a whorl-like appearance which is not observed on day 28. After day 44, when biosynthetic activity is declining, whorls of ER gradually decrease in number and are ultimately replaced by vesicular smooth ER on day 64. Neurosecretions are found only after day 38, by which time rates of JH synthesis have increased substantially from those of day 28. Except for membranous autophagic vacuoles, which are frequently found when ER whorls disintegrate as rates of JH synthesis decline toward birth, most autophagic vesicles such as multivesicular vesicles and dense bodies occur only sporadically among CA cells at all examined ages. We conclude that synchronous autophagy of exhausted organelles, which results in atrophy of CA cells and long-term arrest of JH synthesis in adult females of D. punctata, does not occur in embryos. The slow cyclic change in rate of JH synthesis in embryonic CA is most likely due to asynchronous autophagic activity and to alterations in certain unique features of intracellular organelles.     

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata

Summary A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H₂O₂-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.     

Ultrastructure of corpora allata of known activity during the vitellogenic cycle in the cockroach Diploptera punctata

Summary Ultrastructure was correlated with rates of juvenile hormone synthesis in corpora allata from females of the viviparous cockroach Diploptera punctata at seven daily intervals during the first vitellogenic cycle. Synthetic activity of the glands was determined by in vitro radiochemical assay before the glands were fixed for electron microscopic analysis. The cycle in rates of juvenile hormone synthesis progressed from about 20 pmol h^-1 per gland pair (oocytes 0.60 mm long) to a maximum mean rate of 140 pmol h^-1 per pair (oocytes 1.40–1.47 mm long) and declined to about 20 pmol h^-1 per pair at ovulation (oocytes about 1.65 mm long). Conspicuous ultrastructural changes occurred with changing synthetic rates. In glands with increasing rates of synthesis, mitochondria showed less electron-dense matrix, greater diameter and more irregular shape. Smooth endoplasmic reticulum changed from easily seen to obscure tubules, networks, and vesicles. Rough endoplasmic reticulum appeared in longer, more curved segments. Newly formed autophagic vacuoles appeared in all glands of highest activity rates. In glands with decreasing rates of synthesis, the mitochondrial matrix became denser, width smaller, and shapes less irregular. Smooth endoplasmic reticulum again appeared tubular and distinct. Golgi complexes were more conspicuous. Rough endoplasmic reticulum in whorls and large numbers of autophagic vacuoles continued to be present.This work was supported by USPH Grant AI 15230. We thank Kuen-Kuen Chan for skillful and thoughtful technical assistance     

Endocrine and reproductive differences and genetic divergence in two populations of the cockroach Diploptera punctata

The viviparous cockroach, Diploptera punctata, has been a valuable model organism for studies of the regulation of reproduction by juvenile hormone (JH) in insects. As a result of its truly viviparous mode of reproduction, precise regulation of JH biosynthesis and reproduction is required for production of offspring, providing a model system for the study of the relationship between JH production and oocyte growth and maturation. Most studies to date have focused on individuals isolated from a Hawaiian population of this species. A new population of this cockroach was found in Nakorn Pathom, Thailand, which demonstrated striking differences in cuticle pigmentation and mating behaviours, suggesting possible physiological differences between the two populations. To better characterize these differences, rates of JH release and oocyte growth were measured during the first gonadotrophic cycle. The Thai population was found to show significantly earlier increases in the rate of JH release, and oocyte development as compared with the Hawaiian population. Breeding experiments to determine the degree of interfertility between the two populations demonstrated greatly reduced fertility in crosses between the two populations. Additionally, levels of genetic divergence between the two populations estimated by sequencing a fragment of the mitochondrial 16S rRNA gene were surprisingly high. The significant differences in physiology and mating behaviours, combined with the reduced interfertility and high levels of sequence divergence, suggest that these two populations of D. punctata are quite distinct, and may even be in the process of speciation. Moreover, these studies have important implications for the study of JH function in the reproductive cycle of insects, as differences in timing of rates of JH biosynthesis may suggest a process of heterochrony in reproduction between the two populations.     

In vitro growth of corpora allata fromDiploptera punctata

Summary An in vitro organ culture system was established to support growth of corpora allata from the cockroachDiploptera punctata. During a 1-wk incubation in L-15B medium supplemented with 10% fetal bovine serum (FBS) and 10% cockroach hemolymph, adult male corpora allata exhibited a cycle of de novo DNA synthesis followed by cell division. The number of S-phase cells and metaphase cells per corpus allatum were counted from whole-mount monolayers after labeling in vitro with 5′-bromo-2′-deoxyuridine and exposure to colchicine, respectively. While both FBS and cockroach hemolymph were essential for proliferation of allatal cells, the growth-promoting effect of insect hemolymph was not species-specific and adult female hemolymph was more potent than hemolymph from adult males. Furthermore, DNA synthesis of corpus allatum cells was stimulated in vitro by 20-hydroxyecdysone. This sensitive assay system will be of immense utility in the search for allatal growth factors.     

Ultrastructural changes induced by precocene II and 3,4-dihydroprecocene II in the corpora allata of Blattella germanica

Summary Ultrastructural studies on corpora allata (CA) from different stages during the first gonadotropic cycle of the cockroach Blattella germanica have shown well defined changes which have a correspondence with oocyte length, CA volume and juvenile hormone (JH) biosynthesis. The most significant variations concern the mitochondria and the endoplasmic reticulum. Topically applied precocene II (P II) at a dose of 200 g induced a transient arrest of CA function, although cytotoxic effects were occasionally observed. When CA were maintained in vitro with 10^-3 M of P II, a relationship between the time of treatment (3, 6 or 9 h) and the intensity of the effects was apparent. The 9-h treatment led to an irreversible inhibition of JH production which parallels the severe damages observed in the CA (membrane lysis, nuclear pyknosis, vacuolization). Equivalent studies performed with the chroman derivative 3,4-dihydroprecocene II (DHP II) showed that it is less active than P II. Only treatments as severe as 12 h of incubation with a 10^-3 M concentration elicited cytotoxic effects which could be due to radical species involved in the in situ oxidative bioactivation of DHP II. Thus, this compound could be regarded as a new type of pro-allatocidin.     

Improved conditions for culture of biosynthetically active cockroach corpora allata

Summary Currently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.     

Allatostatin in ovaries, oviducts, and young embryos in the cockroach Diploptera punctata

The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.     

Activity of the corpora allata of adult female Aedes aegypti: effects of mating and feeding

The synthesis of juvenile hormone III (JH III) by the isolated corpora allata (CA) of Aedes aegypti adult female was studied using an in vitro radiochemical assay. We dissected the corpora allata-corpora cardiaca (CA-CC) complex attached to a piece of aorta. The complex was left connected to the intact head capsule to facilitate the visualization and transfer of the glands. A linear increase in the cumulative amount of biosynthesized JH III was found for at least the first 6 h of incubation; approximately 45% of the synthesized JH III was present in the medium. There was a dependence of JH III synthesis on exogenous methionine supply. Using reversed phase high performance liquid chromatography two major labeled products biosynthesized by the CA were separated. They co-migrated with JH III and methyl farnesoate (MF). The identity of the biosynthesized JH III was confirmed by gas chromatography-mass spectrometry. JH III synthesis was only 2.0 fmol/pair gland/h immediately after adult emergence, but increased to 32.6 fmol/ pair gland/h 18 h later in sugar-fed females. Two days after emergence, the CA biosynthetic activity slowly started to decrease, and reached values of around 5.3 fmol/pair gland/h by one week after emergence. Synthesis of JH was similar from either sugar-fed females mated or unmated. A blood meal resulted in a decrease of JH III synthesis in CA from mated females by 12 h after feeding and from virgin females by 24 h after feeding. JH III biosynthesis remained low for at least 96 h in mated females, but was back to higher levels 72 h after feeding in virgin females. Rates of JH III biosynthesis closely reflected the hemolymph levels of JH III both after emergence and after a blood meal described by Shapiro et al. (1986). The activity of the CA in Aedes aegypti females seems to be regulated by developmental changes and nutritional signals, and to be independent of mating stimulus.     

The effects of octopamine on juvenile hormone biosynthesis,electrophysiology, and cAMP content of the corpora allata of the cockroach Diploptera punctata

Summary Juvenile hormone production by the corpora allata of the adult female cockroach, Diploptera punctata, can be modulated by treatment with the biogenic amine, octopamine. Endogenous octopamine has been identified within the CA, using HPLC and electrochemical detection. Treatment with octopamine results in a sinusoidal, dose-dependent inhibition of JH biosynthesis by CA from day 2 virgin females, with maximal inhibition occurring at 10^-10 M and 10^-4 M. In day 4 and day 8 mated female corpora allata octopamine inhibited JH biosynthesis at 5·10^-5 M. Although the elevation of either cAMP or cGMP within the CA is known to be associated with an inhibition of JH biosynthesis, treatment with high concentrations of octopamine results in an increase in the level of cAMP but not cGMP. This effect is both dose- and time-dependent.Octopamine treatment also initiates changes in the passive membrane responses of the CA. Superfusion of CA with octopamine results in a pronounced hyperpolarization of CA cells and an increase in the electrotonic potential (indicative of the degree of electrical coupling between CA cells). This effect could be blocked by the octopamine receptor blocker phentolamine. Treatment with octopamine or phentolamine also blocked the hyperpolarization of CA cells normally associated with electrical stimulation of the axon tracts innervating the CA.We hypothesize that octopamine may be a natural neuromodulator of JH production by CA, regulating ion channels in CA cells themselves as well as release of the inhibitory neuropeptide, allatostatin, from the terminals within the CA.Abbreviations 4-AP 4-aminopyridine - CA corpora allata - CC corpora cardiaca - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid - HPLC high pressure liquid chromatography - IBMX 3-isobutyl-1-methylxanthine - JH juvenile hormone - ms millisecond - nA nanoampere - NCA I nervi corporis allati I - OCT octopamine - TEA tetraethyl ammonium     

Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8

Summary Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum/corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the cap regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimophism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.     

Neurons projecting from the brain to the corpora allata in orthopteroid insects: anatomy and physiology

Retrograde and orthograde labeling of neurons projecting to the corpus allatum was performed in locust, grasshopper, cricket, and cockroach species in order to identify brain neurons that may be involved in the regulation of juvenile hormone production. In the acridid grasshopper Gomphocerus rufus L., and the locusts Locusta migratoria (R.&F.) and Schistocerca gregaria Forskal, the corpora allata are innervated by two morphologically distinguishable types of brain neurons. One group of 9–13 neurons (depending on species) with somata in the pars lateralis extend axons via the nervus corporis cardiaci 2 and nervus corporis allati 1 to the ipsilateral corpus allatum, whereas two cells in each pars lateralis have bilateral projections and innervate both glands. No direct connection between the pars intercerebralis and corpus allatum has been found. In contrast, neurons with paired axons innervating both glands are not present in Periplaneta americana (L.) and Gryllus bimaculatus de Geer. Instead, two cells in each pars lateralis project only to the gland contralateral to their somata. Electrophysiological experiments on acridid grasshoppers have confirmed the existence of a direct conduction pathway between the two glands via the paired axons of four cells that have been identified by neuroanatomy. These cells are not spontaneously active under experimental conditions. Ongoing discharges in the left and right nerves are unrelated, suggesting that the corpora allata receive independent neuronal inputs from the brain.     

Cuticular hydrocarbon synthesis and its maternal provisioning to embryos in the viviparous cockroach Diploptera punctata

Embryos of the viviparous cockroach Diploptera punctata accumulate large amounts of hydrocarbon (HC) of either maternal or embryonic origin. HC synthesis and its accumulation in maternal and embryonic tissues were measured over the course of gestation. Female abdominal integument was the only tissue that synthesized appreciable amounts of HC in vitro, and did so at an increasing rate from the time of mating to mid-pregnancy, when rates of synthesis declined. The embryos synthesized HC at rates <1% those of the female, showing that the majority of HC detected in and on embryos was of maternal origin. The brood sac that houses the developing embryos did not synthesize HC in vitro, indicating that HC must be transported from the female abdominal integument to the embryos. The mass of female epicuticular HC was constant at approximately 183 microg, while her internal HC increased fourfold from mating to mid-pregnancy, then declined until parturition. The decline in internal HC reflected both declining HC synthesis in the female and greater export to the embryos, as embryonic internal HC increased 250-fold prior to parturition. An external HC coating over the oothecal covering and chorion of the embryos increased to mid-pregnancy, then declined. Unlike oviparous cockroaches, D. punctata females fed throughout the reproductive cycle, reflecting the nutritional demands of continuously provisioning the developing embryos.     

Chilling affects allatal cell proliferation via antennae and protocerebral neurons in the cockroach Diploptera punctata

The corpora allata (CA) cells in a mated female of the cockroach Diploptera punctata undergo numerous mitotic divisions before an increase in juvenile hormone synthesis. A previous study demonstrated that this mitotic wave could be suppressed by exposure of the mated female to melting ice. Herein, we report that chilling suppresses CA mitosis via antennal perception. Cell proliferation-suppressing stimuli from chilling were acquired in proportion to the length of time of exposure to the low temperature and the physical length of the antennae exposed to chilling. Sixty basal antennal annuli should remain exposed to chilling for at least 1.5 h in order to suppress mitotic divisions in CA. Mitotic divisions in corpus allatum are suppressed by stimuli from contralateral antenna, predominantly via pars intercerebralis neurons. Selective disconnection of pars intercerebralis neurons from CA, prior to chilling, restored the mitotic wave in CA. Cellular divisions did not occur in CA of chilled females if either pars lateralis neurons were severed or left intact.     

Evidence for a calcium-dependent outward conductance in endocrine cells of the cockroach, Diploptera punctata

Abstract The cell membranes of the corpora allata of the cockroach Diploptera punctata contain voltage-dependent calcium channels. Depolarizing current injection into cells of the corpora allata in the presence of the calcium channel blockers, cadmium, cobalt or verapamil allows the production of multiple action potentials, as does treatment with the intracellular calcium chelator, BAPTA/AM. These results suggest that calcium currents are involved both in decreasing the excitability and in activating an outward current in cells of the corpora allata. Electrophysiological measurements also suggest a concomitant reduction in outward conductance following the multiple action potentials produced in the presence of the channel blockers or BAPTA/ AM. We hypothesize that the calcium current may play an important role in the regulation of intracellular calcium concentration and Juvenile Hormone biosynthesis.     

Activity of corpora allata,endocrine balance and reproduction in female Labidura riparia (Dermaptera)

Summary The reproductive activity of Labidura riparia females involves, after a 5-day maturation stage, a regular alternation of ovarian cycles and egg-care stages averaging 10 days each. Vitellogenesis is characterized by an increase in the size of the corpora allata (CA) where structured SER bodies appear, and by a rise of juvenile hormone (JH III) content in the hemolymph which is followed by an increase in the level of ecdysteroids. During the egg-care periods, the CA are inactive; structured bodies generate autophagic vacuoles, the titer of JHs and later that of ecdysteroids in the hemolymph decreases and remains stationary. Ovariectomy causes hypertrophy and hyperactivity of the CA for about two months. Subsequently, the titer of JH decreases and old females may display parental behaviour; the level of ecdysteroids falls and remains unchanged. After cauterization of the pars intercerebralis (PI) of the protocerebrum, the ovarian activity stops, the ovary shrinks, the JHs rapidly disappear but ecdysteroids remain at the same or even higher levels than those of normal females of the same age. On the basis of these data, we postulate the existence of a center located in the PI, inhibiting the production of ecdysteroids, and of a stimulating center located outside this area. The PI also exhibits an allatotropic function.

---

Résumé L'activité sexuelle des femelles de Labidura riparia comporte au terme d'une phase de maturation sexuelle de 5 jours une alternance régulière de cycles ovariens et de phases de soins aux oeufs d'une durée moyenne de 10 jours. La vitellogenèse est caractérisée par l'accroissement des CA oú apparaissent des amas structurés de RE, par une élévation du titre hémolymphatique de la JH III, puis des ecdystéroïdes. En période de soins aux oeufs, les CA sont inactifs (rejet des amas de reticulum sous la forme de vacuoles autophagiques), le titre hémolymphatique de l'hormone juvénile puis des ecdystéroïdes diminue puis reste stationnaire. L'ovariectomie détermine une hypertrophie et pendant 2 mois environ une hyperactivité des CA; ultérieurement, le titre de l'hormone juvénile diminue et les femelles âgées peuvent manifester un comportement parental; le titre des ecdystéroïdes ne s'annule pas chez les femelles ovariectomisées. Après parsectomie, l'activité ovarienne cesse, l'ovaire régresse, le titre de l'hormone juvénile s'annule rapidement mais les ecdystéroïdes restent présents à des titres égaux, voire supérieurs à ceux appréciés chez les femelles normales de même âge. Ces données permettent d'envisager l'existence d'un centre inhibiteur de la production des ecdystéroïdes situé dans la PI et d'un centre excitateur situé en dehors de cette région. La PI a également une fonction allatotrope.

     

Quantification of allatostatin receptor mRNA levels in the cockroach, Diploptera punctata, using real-time PCR

The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.