Cockroach Diploptera Punctata Research Articles

Enhanced gut microbiome supplementation of essential amino acids in Diploptera punctata fed low-protein plant-based diet.

Building on our previous work, we investigate how dietary shifts affect gut microbial essential amino acid (EAA) provisioning in the lactating cockroach Diploptera punctata. To that end, we fed cockroaches three distinct diets: a plant-only Gari diet composed of starchy and granulated root tuber Yucca (Manihot esculenta), a dog food diet (DF), and a cellulose-amended dog food (CADF) diet. We anticipated that the high carbohydrate, low protein Gari would highlight increased microbial EAA supplementation to the host. By day 28, we observed distinct profiles of 14 bacterial families in the insect gut microbiomes of the three dietary groups. CADF-fed insects predominantly harbored cellulolytic and nitrogen-fixing bacteria families Streptococcaceae and Xanthomonadaceae. In contrast, Gari-fed insects were enriched in anaerobic lignocellulolytic bacteria families Paludibacteraceae andDysgonomonadaceae, while DF-fed insects had a prevalence of proteolyticanaerobes Williamwhitmaniaceae and sulfate-reducing bacteria Desulfovibrionaceae. Furthermore, we confirmed significantly higher EAA supplementation in Gari-fed insects than in non-Gari-fed insects based on δ13C-EAA offsets between insect and their diets. The δ13C-EAA offsets between DF and CADF were nearly indistinguishable, highlighting the relevance of using the plant-based Gari in this experiment to unequivocally demonstrate this function in this insect. These results were underscored by lower standard metabolic rate (SMR) relative to the DF insect in Gari-fed (intermediate SMR and dietary quality) and CADF (least SMR and dietary quality) insects. The influence of diet on EAA provisioning and SMR responses in insects underscores the need for further exploration into the role of gut microbial functions in modulating metabolic responses.

DNA methylation patterns respond to thermal stress in the viviparous cockroach Diploptera punctata

ABSTRACT It is increasingly recognized that epigenetic mechanisms play a key role in acclimatization and adaptation to thermal stress in invertebrates. DNA methylation and its response to temperature variation has been poorly studied in insects. Here, we investigated DNA methylation and hydroxymethylation patterns in the viviparous cockroach Diploptera punctata at a global and gene specific level in response to variation in temperature. We specifically studied methylation percentage in the heat shock protein 70 (Hsp70), whose function is linked to thermal plasticity and resistance. We found high levels of DNA methylation in several tissues but only low levels of DNA hydroxymethylation in the brain. Hsp70 methylation patterns showed significant differences in response to temperature. We further found that global DNA methylation variation was considerably lower at 28°C compared to higher or lower temperatures, which may be indicative of the optimal temperature for this species. Our results demonstrate that DNA methylation could provide a mechanism for insects to dynamically respond to changing temperature conditions in their environment.

Female clustering in cockroach aggregations—A case of social niche construction?

AbstractIndividuals in groups can suffer costs through interactions with adversarial or unknown conspecifics. Social niche construction allows individuals to buffer such potential costs by only engaging in preferred associations. This may be particularly beneficial in insect aggregations, which are often large and highly fluid. However, little is known regarding the structuring of such aggregations. Here we use social network analyses to test for fine‐scale social structure in resting aggregations of the sub‐social cockroach Diploptera punctata and to explore the social pressures that contribute towards such structure. We showed that females were significantly more gregarious than males and formed the core of the proximity network, thus demonstrating a higher level of social integration. This fine‐scale structure is likely to result from females displacing males; females initiated most displacements whilst males received the majority. We explain this behaviour in terms of social niche construction by showing that females received significantly fewer approaches and investigations at more female‐biased local sex ratios. We therefore suggest that female social clustering occurs in this, and presumably other, species to reduce potential costs associated with male harassment. This demonstrates how social niche construction can lead to higher level social structure; we suggest this approach could be used across a range of species in order to improve our understanding of the evolution of sociality.

Personality in the cockroach Diploptera punctata: Evidence for stability across developmental stages despite age effects on boldness.

Despite a recent surge in the popularity of animal personality studies and their wide-ranging associations with various aspects of behavioural ecology, our understanding of the development of personality over ontogeny remains poorly understood. Stability over time is a central tenet of personality; ecological pressures experienced by an individual at different life stages may, however, vary considerably, which may have a significant effect on behavioural traits. Invertebrates often go through numerous discrete developmental stages and therefore provide a useful model for such research. Here we test for both differential consistency and age effects upon behavioural traits in the gregarious cockroach Diploptera punctata by testing the same behavioural traits in both juveniles and adults. In our sample, we find consistency in boldness, exploration and sociality within adults whilst only boldness was consistent in juveniles. Both boldness and exploration measures, representative of risk-taking behaviour, show significant consistency across discrete juvenile and adult stages. Age effects are, however, apparent in our data; juveniles are significantly bolder than adults, most likely due to differences in the ecological requirements of these life stages. Size also affects risk-taking behaviour since smaller adults are both bolder and more highly explorative. Whilst a behavioural syndrome linking boldness and exploration is evident in nymphs, this disappears by the adult stage, where links between other behavioural traits become apparent. Our results therefore indicate that differential consistency in personality can be maintained across life stages despite age effects on its magnitude, with links between some personality traits changing over ontogeny, demonstrating plasticity in behavioural syndromes.

Synthesis and biological activity of FGLamide allatostatin analogs with Phe(3) residue modifications.

A FGLamide allatostatin neuropeptide mimic (H17) is a potential insect growth regulator which inhibits the production of juvenile hormone by the corpora allata. To find more evidence to reveal the structure-activity relationships of the Phe(3) residue in the C-terminal conserved pentapeptide and search for novel analogs with high activity, a series of Phe(3) residue-modified analogs were designed and synthesized using H17 as the lead compound. Bioassay using juvenile hormone (JH) production by corpora allata of the cockroach Diploptera punctata indicated that analogs 4, 11, and 13 showed strong ability to inhibit JH production in vitro, with IC50 of 38.5, 22.5, and 26 nM, respectively. As well, the activity of analog 2 (IC50 : 89.5 nM) proved roughly equivalent to that of H17. Based on the primary structure-activity relationships of Phe(3) residue, we suggest that for analogs containing six-membered aromatic rings, removing the methylene group of Phe(3) or an o-halogen or p-halogen-substituted benzene ring could increase the ability to inhibit biosynthesis of JH. This study will be useful for the design of new allatostatin analogs for insect management. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

RXR/USP and EcR are critical for the regulation of reproduction and the control of JH biosynthesis in Diploptera punctata

During development and reproduction the response to ecdysteroids is mediated by a heterodimeric receptor complex comprising the retinoid X receptor/ultraspiracle (RXR/USP) and the ecdysone receptor (EcR). Here, the role of these receptors in the endocrine control of reproduction is examined in the cockroach Diploptera punctata. We report the sequence of four DpRXR and three DpEcR splice variants, including the first description of a Drosophila EcRB2-like isoform in a hemimetabolous insect. DpRXR and DpEcR are broadly expressed in the tissues of adult females, with relatively high transcript levels in the corpora allata (CA), nervous tissue and ovary. Developmental profiling revealed an inverse correlation between DpRXR and DpEcR expression and the activity of the CA. RNAi-mediated depletion of DpRXR and DpEcR did not affect oocyte growth, but inhibited oviposition and impaired chorion formation. Retained oocytes exhibited a degenerating follicular epithelium and were slowly resorbed. Treated animals showed significantly higher rates of JH biosynthesis and a decrease in ecdysteroid titers at the end of vitellogenesis. Reduction of DpRXR and DpEcR expression resulted in an upregulation of genes involved in JH production and a downregulation of allatostatin receptor mRNA in the CA. Treatment with dsRNA also affected the expression of genes downstream of JH in target tissues including vitellogenin and Krüppel-homolog 1 as well as Broad-Complex, an early ecdysone response gene. Overall, results suggest that DpRXR and DpEcR are not required early in the reproductive cycle when events are JH-dependent, but do mediate critical ecdysteroid feedback to the CA late in the gonadotropic cycle.

Synthesis, biological activity, and conformational study of N-methylated allatostatin analogues inhibiting juvenile hormone biosynthesis.

An allatostatin (AST) neuropeptide mimic (H17) is a potential insect growth regulator, which inhibits the production of juvenile hormone (JH) by the corpora allata. To determine the effect of conformation of novel AST analogues and their ability to inhibit JH biosynthesis, eight insect AST analogues were synthesized using H17 as the lead compound by N-methylation scanning, which is a common strategy for improving the biological properties of peptides. A bioassay using JH production by corpora allata of the cockroach Diploptera punctata indicated that single N-methylation mimics (analogues 1-4) showed more activity than double N-methylation mimics (analogues 5-8). Especially, analogues 1 and 4 showed roughly equivalent activity to that of H17, with IC50 values of 5.17 × 10(-8) and 6.44 × 10(-8) M, respectively. Molecular modeling based on nuclear magnetic resonance data showed that the conformation of analogues 1 and 4 seems to be flexible, whereas analogues 2 and 3 showed a type IV β-turn. This flexible linear conformation was hypothesized to be a new important and indispensable structural element beneficial to the activity of AST mimics.

Characterization of the juvenile hormone pathway in the viviparous cockroach, Diploptera punctata.

Juvenile hormones (JHs) are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase (HMGR) and Juvenile hormone acid methyltransferase (JHAMT). As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach Diploptera punctata.

The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternatively spliced variants in the cockroach Diploptera punctata. This is the first report of NR1 paralogs in insects. The tissue distributions and expression profiles of DpNR1A, DpNR1B and DpNR2 in different tissues were also investigated. Previous studies have demonstrated NMDA-stimulated biosynthesis of juvenile hormone (JH) in the corpora allata through the influx of extracellular Ca(2+) in Diploptera punctata. However, our data show that the transcript levels of DpNR1A, DpNR1B and DpNR2 were low in the corpora allata. MK-801, a high-affinity antagonist of NMDAR, did not show any effect on JH biosynthesis in vitro. In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-801 resulted in any significant change in JH biosynthesis or basal oocyte growth. Injection of animals with a high dose of MK-801 (30 µg per animal per injection), which paralyzed the animals for 4-5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata.

Methoprene-tolerant (Met) knockdown in the adult female cockroach, Diploptera punctata completely inhibits ovarian development.

Independent of the design of the life cycle of any insect, their growth and reproduction are highly choreographed through the action of two versatile hormones: ecdysteroids and juvenile hormones (JH). However, the means by which JH can target tissues and exert its pleiotropic physiological effects is currently still not completely elucidated. Although the identity of the one JH receptor is currently still elusive, recent evidence seems to point to the product of the Methoprene-tolerant gene (Met) as the most likely contender in transducing the action of JH. Studies on the role of this transcription factor have mostly been focused on immature insect stages. In this study we used the viviparous cockroach Diploptera punctata, a favorite model in studying JH endocrinology, to examine the role of Met during reproduction. A tissue distribution and developmental profile of transcript levels was determined for Met and its downstream partners during the first gonadotropic cycle of this cockroach. Using RNA interference, our study shows that silencing Met results in an arrest of basal oocyte development; vitellogenin is no longer transcribed in the fat body and no longer taken up by the ovary. Patency is not induced in these animals which fail to produce the characteristic profile of JH biosynthesis typical of the first gonadotropic cycle. Moreover, the ultrastructure of the follicle cells showed conspicuous whorls of rough endoplasmic reticulum and a failure to form chorion. Our study describes the role of Met on a cellular and physiological level during insect reproduction, and confirms the role of Met as a key factor in the JH signaling pathway.

Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins

Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata from the cockroach Diploptera punctata showed that the activity of analog I (IC 50: 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr–Xaa–Phe–Gly–Leu–NH 2, IC 50: 0.13 μM) it mimicked and the activity of the analog II (IC 50: 0.13 μM) proved roughly equivalent to the C-terminal pentapeptide. The results indicate that a new simple mimicry for Tyr–Xaa–Phe–Gly has been discovered; analog I may be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST analogs for insect management.

Suppression of JH biosynthesis by JH analog treatment: Mechanism of suppression and roles of allatostatins and nervous connections in the cockroach Diploptera punctata

Juvenile hormone analogs are known to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). However, the mechanism of this inhibition remains undefined. We have used two JH mimics, fenoxycarb and pyriproxyfen, to examine the mechanism of suppression in the cockroach, Diploptera punctata. Denervation experiments demonstrated the importance of nervous connections between the brain and CA for the inhibition of JH biosynthesis by fenoxycarb. Fenoxycarb treatment alters the sensitivity of CA to allatostatin treatment in vitro. Suppression of JH biosynthesis by fenoxycarb following denervation of the CA showed that innervation was in part responsible for the inhibition. Similarly, maximal inhibition by Dippu-AST7 requires intact nervous connections between the brain and CA, particularly during rapid vitellogenesis. qPCR analysis of brain, CA, ovary and midgut extracts revealed that both allatostatin and its receptor Dippu-ASTR2 show increased levels of expression following topical fenoxycarb treatment, particularly in brain tissue on days 4 and 5 of the first gonadotrophic cycle and in CA on day 4. The correlation between inhibition of JH biosynthesis and increased expression of AST and ASTR2 in brains and CA, together with increased sensitivity of CA to allatostatin in vitro, suggests that allatostatin may be one of the effectors by which fenoxycarb inhibits JH biosynthesis.

Identification of Phe-Gly-Leu-amide type allatostatin-7 in Reticulitermes flavipes: Its localization in tissues and relation to juvenile hormone synthesis

The allatostatins (ASTs), with a Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide C-terminus, are neuropeptides that occur in many orders of insects, but are known to inhibit juvenile hormone (JH) synthesis by corpora allata (CA) only in cockroaches, crickets, and termites. 5 AST peptides with similar sequences to those of 6 species of cockroaches have been isolated and sequenced from extract of brain tissue of the termite Reticulitermes flavipes. The amino acid sequence of a 6th peptide, R. flavipes AST-7, determined by LC–MS/MS following HPLC fractionation of brain extract, is S-P-S-S-G-N-Q-R-L-Y-G-F-G-L-NH 2. The 8 terminal amino acids are identical to AST-7 of the cockroach Diploptera punctata. R. flavipes and D. punctata AST-7s inhibited JH synthesis by CA of both species equally and their affinity for antibody against D. punctata AST-7 is similar. Immunoreactivity of termite tissue with this antibody indicates neuro- and myomodulatory activity of the peptide in addition to its demonstrated allatostatic function. The density of AST immunostaining in axons within the CA of R. flavipes and the rate of JH synthesis by similar glands were negatively correlated. This is evidence that when AST is abundant in the glands it is being released in vivo to limit JH production.

Effects of the NMDA receptor antagonist MK-801 on female reproduction and juvenile hormone biosynthesis in the cricketGryllus bimaculatusand the butterflyBicyclus anynana

Apart from regulating insect development, juvenile hormones (JHs) play an important role in insect reproduction, where they initiate vitellogenin synthesis and regulate the uptake of yolk by the ovary. JH synthesis is a tightly regulated process controlled by neurons and peptidergic neurosecretory cells. One of the known stimulatory regulators of JH biosynthesis is glutamate, and its N-methyl-d-aspartate (NMDA) receptor has been recently found in the cockroach Diploptera punctata. In this study we demonstrate a strong reduction in reproductive output in the tropical butterfly Bicyclus anynana and the Mediterranean field cricket Gryllus bimaculatus caused by the NMDA receptor antagonist MK-801. Such inhibiting effects on reproduction could be overruled by the application of JH mimics. In G. bimaculatus, MK-801 inhibits in vitro JH biosynthesis in the corpora allata and reduces in vivo JH haemolymph titres in a dose-dependent manner. These results suggest that JH biosynthesis in the corpora allata is at least in part controlled by an NMDA receptor with Ca2+ as a second level messenger. Based on our findings we consider NMDA receptor antagonists as important tools for manipulating juvenile hormone biosynthesis and therefore for gaining a better understanding of the mechanistic basis of reproduction.

Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus

Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH(31)) family of insect peptides. Previously we have demonstrated the presence of DH(31)-like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH(31) from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH(31) from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH(31) in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH(31) and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH(31)-like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH(31) increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut.

Molecular cloning and characterization of an allatostatin-like receptor in the cockroach Diploptera punctata

Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH 2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a λZAP-cDNA library prepared from the corpora allata of the D. punctata. As an approach to testing the function of this receptor in vivo, the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.

Probing the conformation and dynamics of allatostatin neuropeptides: A structural model for functional differences

Allatostatins are a family of related neuropeptides that play an important role in development, reproduction, and digestion in insects. The cockroach Diploptera punctata has 13 allatostatin neuropeptides, with pleiotropic functions, two of which are: inhibition of juvenile hormone (JH) production and inhibition of gut muscle contraction. In this study, the conformation and dynamics of D. punctata allatostatin 5 (Dippu-AST 5) and allatostatin 8 (Dippu-AST 8) are investigated by CD, NMR, and molecular dynamics simulations. These peptides contain eight and nine residues, respectively, and the identical six-residue C-terminal motif. Yet Dippu-AST 5 and Dippu-AST 8 affect juvenile hormone production and hindgut contraction with different potencies. Dippu-AST 5 is one of the most potent inhibitors of juvenile hormone production and one of the least potent inhibitors of gut contraction, whereas Dippu-AST 8 has the opposite potencies with respect to these tissues. From the NMR structure, it is clear that Dippu-AST 5 has a 3 10 helix involving three of its residues and a “γ” turn at the end of its C-terminal motif. In contrast Dippu-AST 8 has an open “π” turn among five of its central residues. In addition, the orientation preferences within the membrane of the two peptides were simulated. Our simulation results show that the C-terminal segment of Dippu-AST 5 orients in the membrane surface with an average angle of 17.5°, whereas Dippu-AST 8 orients with an average angle of 5.1°. Taken together, from the structures and orientation preferences of these peptides within the membrane, it appears that these peptides may interact with the receptor very differently.

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.

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 24 h. 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.

Allatostatin-like immunoreactivity in the abdomen of the locust Schistocerca gregaria

A polyclonal antibody against allatostatin 1 (AST-1) of cockroach Diploptera punctata was used to investigate the distribution of AST-like immunoreactivity within the abdomen of the locust, Schistocerca gregaria. In the abdominal ganglia, AST-like immunoreactivity was found in both cell bodies and neuropile. In ganglia 6 and 7, staining was found in serial homologous cell bodies in anterior dorsolateral and dorsomedial, and posterior ventrolateral and ventromedial locations. In the terminal ganglion, the numerous immunoreactive somata were smaller in size than those in the unfused ganglia. The combination of backfill experiments with immunocytochemistry showed that, in abdominal ganglion 7, one neuron of the ventromedian cell body cluster and two of the ventrolateral cluster innervated the oviduct, which itself was covered with a dense mesh of AST-immunoreactive varicosities. Combining electron microscopy with immunohistochemistry revealed AST-like immunoreactivity in dense-core vesicles located in neurohaemal-like terminals lacking structures normally found in synapses. A mesh of AST-immunoreactive varicosities was also found on the muscles of the spermatheca and the spermathecal duct. In addition, a mesh of strongly stained varicosities was present in the distal perisympathetic organs (neurohaemal organs in the abdomen) and on the lateral heart nerves (a putative neurohaemal release zone). These data indicate that AST is an important neuroactive substance that is probably involved in multiple tasks in the control of the locust abdomen.