Regulation of Juvenile Hormone synthesis in the blowfly Lucilla cuprina

Abstract. Juvenile Hormone III bisepoxide synthesis by ring gland complexes from third-instar larvae of the blowfly Lucilia cuprina Weidemann (Diptera, Calliphoridae) was measured using a radiochemical assay in vitro. Hormone synthesis is regulated by three distinct mechanisms during development of the final larval instar up to the time of pupariation. The first type of regulation is detected as a rapid decline in hormone release coinciding with the final phase of commitment to pupariation. The second is a neurally mediated inhibition by the brain that acts at all stages of development in third-instar larvae. A protease-sensitive factor from brains of third-instar larvae causes dose-dependent reversible inhibition of Juvenile Hormone III bisepoxide synthesis. The third regulatory signal is a neural inhibition, observed in brain-ring gland complexes of prepupal stages. The first two levels of regulation appear to act early in the synthetic pathway for Juvenile Hormone (JH), whereas the third acts on the final steps of bisepoxide synthesis.     

Conditions for estimation of corpus allatum activity in the blowfly, Phormia regina, in vitro

ABSTRACT. Incubation conditions have been established for the corpus cardiacum-corpus allatum (CC-CA) complex of female Phormia regina (Meigen), which will support CC-CA biosynthetic activities in vitro as measured by the incorporation of a labelled methyl group with L-[methyl-³H]methionine as the methyl donor. After incubation, radioactivity in the organic extract of the medium was determined by scintillation counting. Analysis of the organic extract with reverse phase high-performance liquid chromatography (HPLC) revealed that a compound which has similar retention time (UV absorbance) with synthetic JH III was synthesized by the CC-CA complexes of liver-fed females. By using this short-term, in vitro , radiochemical assay for CA activity, it was shown that a protein diet significantly increases the activity of the CA compared with females fed only a sugar-water diet. Furthermore, use of HPLC separation, in conjunction with scintillation counting of time-collected fractions, demonstrated the existence of a moiety containing incorporated radiolabeled methyl group (from the methionine) which did not co-elute with JH I or JH III standards. These results suggest that in P. regina use of the incorporation of a radiolabeled methyl group to measure JH biosynthesis (CA activity) can be misleading if the compounds which do not co-elute with JHs are not considered.     

Waning of repetitive hopping in the blowfly Phormia regina Meigen (Diptera: Calliphoridae)

Wingless blowflies (Phormia reginaMeigen)released on a flat uniform substrate hop repeatedly. The rate of hopping decreases with time. Decremental responsiveness resembles behavioral habituation. It can be reestablished by tactile stimulation and is due neither to muscle fatigue nor to sensory receptor adaptation. No effects of age or circadian rhythms were observed. The initial rate of hopping increases with increasing food deprivation, as does the time for waning to be completed. Decapitated flies and isolated wingless thoraxes hop almost as well as control flies and wane at the same rates.     

The development of the sensory organs of the legs in the blowfly,Phormia regina

Summary The development of the sensory neurons of the legs of the blowfly,Phormia regina has been described from the third instar larva to the late pupa using immunohistochemical staining. The leg discs of the third instar larva contain 8 neurons of which 5 come to lie in the fifth tarsomere of the developing leg. Whereas 2 neurons persist at least to the late pupa, the other cells degenerate. The first neurons of gustatory sensilla arise in the fifth tarsomere at about 1.5 h after formation of the puparium. Most of these sensilla, however, appear within a short time period beginning at about 18 h. The femoral chordotonal sensory neurons first appear at the time of formation of the puparium, as a mass of cells situated in the distal femur. During later pupal development 2 groups of these cells come to lie at the femur-trochanter border, where they become the proximal femoral chordotonal organ of the adult; the remaining cells become the distal femoral chordotonal organ. Other scolopidial neurons appear later in development. The nerve pathways of the late pupal leg are established either by the axons of the cells that are present in the larval leg disc or by new outgrowing processes of sensory neurons. In the tibia, the initial direction of new outgrowth differs in different regions of the segment: proximal tibial neurons grow distally, while distal tibial neurons grow initially proximally.     

Rates of Juvenile Hormone biosynthesis and degradation during reproductive development and diapause in the boll weevil, Anthonomus grandis

Abstract. The role of Juvenile Hormone (JH) during reproductive development and diapause was investigated in the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae). JH sythesized by corpora allata (CA) in vitro of A.grandis was identified as JH-UJ by high-performance liquid chromatography and by conversion to the methoxyhydrin. Optimal conditions for the use of a short-term assay in vitro were established to examine profiles of CA activity. In addition, rates of JH degradation by JH-specific esterase were determined. Patterns of CA and JH-esterase activity during reproductive development and the diapause state were established with laboratory-reared reproductive weevils and diapausing weevils collected as larvae and pupae in the field after the cotton-growing season. The results indicate that JH production is elevated in reproductive females whereas males and winter field-collected females show no CA activity. Vitellogenin concentrations in haemolymph and rates of oviposition were studied in relation to CA activity and JH degradation. An attempt to induce diapause in the laboratory failed.     

Variability in Juvenile Hormone production by locust corpora allata kept in vitro for long periods

Abstract. Juvenile Hormone III (JH-III) production by corpora allata (CA) of sexually mature female locusts (Locusta migratoria migratorioides (R. & F.)) was maintained in vitro for up to 30 days in an agar-solidified medium. Hormone production was measured periodically with a short-term radiochemical assay. Low-activity CA increased their activity significantly after 24–48 h incubation in the long-term medium, but high-activity glands did not. Variations in activity were considerable among glands tested on the same day and among measurements from the same gland on different days. Farnesoic acid-stimulated rates of JH-III production were always higher than the basal rates, suggesting that the CA were not maximally activated. However, freshly excised low-activity CA, whose hormone production increased in the long-term conditions, showed similar farnesoic acid-stimulated rates of JH-III production to those of freshly excised high-activity glands, suggesting that at the time of excision of the corpora allata rate-limiting step(s) preceding farnesoic acid biosynthesis were inhibited or refractory to stimulation in vivo.     

The biosynthesis of Juvenile Hormone, its degradation and titres in females of the true armyworm: a comparison of migratory and non-migratory populations

In a previous study [ McNeil et al. (1996) Archives of Insect Biochemistry and Physiology, 32, 575–584], patterns of sexual maturation and Juvenile Hormone (JH) biosynthesis were compared in virgin females from migratory (North American) and non‐migratory (Azorean) populations of the true armyworm moth, Pseudaletia unipuncta Haworth (Lepidoptera: Noctuidae). Sexual maturation occurred at a significantly earlier age after emergence in the non‐migrant population, and the rates of biosynthesis of JH in vitro suggested that lower titres of JH may be required to initiate the onset of calling behaviour (pheromone emission) and ovarian development in Azorean females. To examine the physiological differences in the reproductive biology of migratory and non‐migratory populations in greater detail, the haemolymph titres of JH and JH esterase activity were compared in virgin females as a function of age. In addition, the effects of mating on JH biosynthesis in vitro, JH titres, JH esterase activity and egg production were measured in the two populations. As expected, JH titres rose more rapidly after emergence in Azorean females than in their North American counterparts but, contrary to our prediction, the maximum levels were also higher in the non‐migrant population. Activity of JH esterase was much higher in Azorean females on the day of emergence. However, by the second day both populations had similar activity levels (about 17 nmol JH/min/ml) and exhibited a similar age‐related decline in subsequent days. Mating did not affect the rate of JH biosynthesis in vitro but resulted in a significant increase in the titres of JH in the haemolymph of both populations. The maximum titre (a five‐fold increase) occurred within 24 h of mating in Azorean females. In North American individuals the increase was greater (seven‐fold) but did not occur until 48 h after mating. No difference in the activity of JH esterase was observed between mated and virgin North American females. By contrast, while there was an age‐related decline in the activity of JH esterase in mated Azorean females, as seen in both North American groups, activity levels in virgin females remained constant with age. In all females, mating resulted in a significant increase in egg production within 24 h. The Azores is a volcanic archipelago, so these non‐migratory populations were probably founded by immigrants originating from migratory continental populations. It is clear from our results that the change from a life history that includes migration to a non‐migratory one involved more than just a temporal shift in the timing of the production of JH. Furthermore, the interpopulation differences in titres of JH and mating‐induced changes reported here cannot be fully explained by the observed differences in the patterns of activity of JH esterase and JH biosynthesis in vitro.     

Modulation of juvenile hormone release and oocyte growth following (RS)-hydroprene treatment in virgin female Diploptera punctata

ABSTRACT. The effect of (flS)-hydroprene treatment (2, 20, 200 μ g) on JH release was assessed in virgin females of D. punctata (Eschscholtz) during the first 10 days of adult life as was basal oocyte length and number of cells in the CA. At a dose of 2 μ g hydroprene, JH release was stimulated slightly and, on days 4 and 6, oocyte growth was significantly greater than that of acetone-treated controls. A similar but more striking enhancement of JH release and basal oocyte growth was observed at a dose of 20 μ g and a significant inhibition of JH release, in concert with a rapid growth of basal oocytes, was observed at a dose of 200 μ g. During the observation period, the mean number of cells in the CA decreased in a dose-dependent fashion, with a highly significant reduction in numbers in 20 and 200 μ g-treated animals. Reimplantation of vitellogenic ovarioles (three or six) into ovariectomized virgin females also resulted in an enhancement of JH release; this indicates that virgin female CA can respond to the stimulatory action of the ovary and is consistent with a model for ( RS )-hydroprene action in which the 'positive feedback' effect (stimulation of JH release) observed with low doses of the analogue occurs as a consequence of the action of the analogue on the ovary. ( RS )-hydroprene treatment stimulates basal oocyte growth to the point at which the previously unstimulatory virgin oocytes are able to enhance JH release by a feedback loop involving the CA and probably the brain.     

Effects of social conditions on Juvenile Hormone mediated reproductive development in Bombus terrestris workers

Abstract. During the annual life cycle of the bumble bee Bombus terrestris (L.) colony, there is a stage characterized by worker reproduction in the presence of the queen. It has been proposed that this is a result of a decrease in queen inhibition. This hypothesis was examined by studying the effects of queens taken from colonies at different stages of development on several aspects of worker physiology and behaviour: rates of Juvenile Hormone (JH) release in vitro , ovary development, and behaviour associated with reproduction. After optimizing and validating the radiochemical assay for JH release for bumble bee workers, we found that queenless workers had significantly more developed ovaries and higher rates of release of JH than did queenright workers, confirming and extending previous findings that suggest that bumblebee ovarian development is under JH control. Mated queens, separated from their colony and brood, can have the same inhibitory effect on the reproductive development of callow workers. In contrast, workers confined with virgin queens or in queenless groups demonstrated a significantly higher rate of release of JH, overt aggression and threatening behaviours. However, there were no differences in rates of release of JH between workers confined in groups in the laboratory with queens taken from colonies either before or after the onset of worker reproduction. Furthermore, overt aggression and threatening behaviours were similar and low in both types of groups. These results gave no support to the hypothesis that a decrease in queen inhibition is associated with the onset of worker reproduction. We also show that young workers reared in colonies either before or after worker reproduction occurs, or in queenless colonies, all demonstrated similar, low rates of release of JH. These results suggest that older workers may inhibit the corpora allata of younger workers in queenless colonies.     

Morphogenetic and gonadotrophic effects of a juvenile hormone analogue ((7S)-hydroprene) in last instar female larvae of Diploptera punctata

ABSTRACT. The effect of treatment of last instar female larvae of Diploptera punctata with a Juvenile Hormone (JH) analogue, (7 S )-hydroprene, has been determined with respect to the ability of the analogue to alter the duration of the stadium and the nature of the ensuing ecdysis. We have also investigated the effects of the analogue on JH release, the growth of the basal oocytes, as well as ecdysteroid titres during the fourth stadium. Analogue treatment prior to day 10 of the stadium results in prolongation of the stadium and desynchronization of ecdysteroid release. Thereafter, treatment with the analogue has little effect. Analogue treatment results also in the formation of supernumerary larvae and intermediates, in a dose-dependent fashion, provided that animals are treated on day 10 or earlier. Thus, the 'critical' period for metamorphosis in last instar D. punctata is between days 0 and 10.
Treatment with (7 S )-hydroprene produces profound effects also on both JH release, and basal oocyte growth. At a dose of 500μg administered on day 1, JH release is stimulated significantly at a time when JH release is normally undetectable. Significant growth of basal oocytes is observed in such treated animals, and appears to precede the peak in JH release. We suggest that the growth of the basal oocytes, as a result of analogue treatment, stimulates the production of JH by CA in these last instar larvae.     

Apparent inhibition of in vitro juvenile hormone biosynthesis by the corpus cardiacum of adult crickets (Gryllus bimaculatus and Acheta domesticus) is due to juvenile hormone esterase

When measuring the in vitro JH III-biosynthesis by corpora allata (CA) from adult female crickets in the presence of corpora cardiaca (CC), the amount of JH III in the medium decreased in a dose dependent manner. The CC of a 4-day-old female Gryllus bimaculatus contain 42 pmol.pair CC⁻¹ Grb-AKH, 0.62 pmol.pair CC⁻¹ octopamine, and a JH-esterase activity of 9.8 pmol JH.h⁻¹.pair CC⁻¹. Comparable values for Acheta domesticus are 21 pmol.pair CC⁻¹ Grb-AKH, 0.53 pmol.pair CC⁻¹ octopamine, and 6.5 pmolJH.h⁻¹.pair CC⁻¹ of JH-esterase activity. Even if the entire octopamine content of the CC were released into the medium, the concentration would be below the 10⁻⁵ M threshold for octopamine inhibition of JH synthesis. An in vitro AKH inhibition of JH III synthesis was observed, but only at a relatively high concentration (10⁻⁵ M). If the entire AKH content (10⁻⁶ M) of the CC were released into the medium, the AKH concentration would approach JH synthesis inhibiting levels. However, the rate of release of AKH in vitro was very low, and, therefore, AKH from the CC could not affect JH synthesis. In contrast, a specific JH-esterase, released by isolated CC into the medium, was sufficiently high in both cricket species to account for the observed decrease in JH III present. OTFP-sulfone (10⁻⁵ M) restored apparent JH synthesis of the CA to the control level. There was no reduction in the amount of JH released when CA were incubated with heat treated CC. The CA themselves contained almost no JH-esterase activity. © 1997 Wiley-Liss, Inc.     

Effects of diet and mating status upon corpus allatum activity, oocyte growth, and salivary gland size in the ring-legged earwig

The effect of diet on mating behavior and the subsequent effects of diet and mating status on the biosynthesis of juvenile hormone III, basal follicle length, salivary gland size and total body weight were assessed in the ring-legged earwig, Euborellia annulipes (Lucas) (Family Carcinophoridae; subfamily Carcinophorinae) during the first 15 days of adult life (the first gonadotrophic cycle of those fed presumably near-optimal diets of catfood) and again on day 25 (late vitellogenesis of the second gonadotrophic cycle of those fed catfood). Diets of catfood, honey, fructose and total starvation, respectively, imposed on 0-day adult females did not affect sexual receptivity, mating success or duration of mating as assessed on day 7. With the addition of a group of virgin, catfood fed females, we noted that only those females maintained on catfood oviposited within 25 days; enforced virginity virtually abolished oviposition. Total food deprivation of females as well as diets of honey or fructose abolished the cycles in total body weight, basal follicle length, salivary gland size and juvenile hormone production. Thus, starvation decreased the reproductive success of these insects, and carbohydrates only (fructose) or in combination with trace amounts of nutrients and protein (honey) were not sufficient to promote reproduction and associated cycles in this insect. Furthermore, virgins failed to undergo the decreases in salivary gland size that were characteristic of mated females. Among mated, catfood-fed females, the second cycle in juvenile hormone production appeared to be smaller than the first.     

Patterns of haemolymph vitellogenin and ovarian vitellin in the German cockroach, and the role of Juvenile Hormone

Abstract. The concentrations of fat body and haemolymph vitellogenin and ovarian vitellin during the first gonadotropic cycle of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) have been studied. For these purposes, a polyclonal antibody against B. germanica vitellogenin and vitellin has been obtained, and an ELISA to quantify these proteins has been developed. Ovarian vitellin levels follow a pattern which parallels those of basal oocyte growth and Juvenile Hormone production by the corpora allata. This suggests that Juvenile Hormone regulates vitellogenin uptake into oocytes. Fat body and haemolymph vitellogenin levels give cyclic and parallel patterns. However, the cycle of Juvenile Hormone appears delayed with respect to that of vitellogenin. We suggest that the production of Juvenile Hormone, although cyclic in profile, does not modulate alone the cycle of vitellogenin. At least a supplementary mechanism, apparently independent of Juvenile Hormone, may be involved in the decline of vitellogenin production at the end of the vitellogenic cycle.     

The relationship between parthenogenetic embryonic development and cumulus cell-oocyte intercellular coupling during oocyte meiotic maturation

Intercellular coupling between cumulus cells and oocytes persists after oocyte meiotic maturation has been initiated. The experiments described here focus on the relationship between oocyte-cumulus cell intercellular coupling during maturation and the subsequent embryonic development of spontaneous mouse parthenotes. Several lines of evidence suggest that this coupling during oocyte maturation is required for the acquisition of the capacity for spontaneous mouse parthenotes to develop embryologically. First, the period of time that LT/Sv oocytes remained coupled to cumulus cells during oocyte maturation in vivo corresponded to that required for the acquisition of the capacity for parthenogenetic embryonic development. Second, the longer that cumulus cells were present during Fpontaneous oocyte maturation in vitro, the higher was the percentageofova undergoing subsequent parthenogenetic development. Third, cumulus cell-free oocytes cocultured with cumulus cell-enclosed oocytes during the maturation period in vitro did not develop embryologically. Fourth, intercellular coupling between cumulus cells and oocytes persisted throughout the oocyte maturation period in vitro. Fifth, incubation of oocyte-cumulus cell complexes in medium containing follicle-stimulating hormone (FSH) promoted uncoupling and decreased the percentage of ova undergoing parthenogenetic development. Thus, cell-to-cell communication, mediated via the intercellular coupling pathway between cumulus cells and oocytes, plays an important role during oocyte maturation and relates to subsequent preimplantation development.     

Absence of coupling between release and biosynthesis of peptide hormones in insect neuroendocrine cells

Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. In the present study we address the question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis.     

Juvenile hormone biosynthesis and oocyte development in adult female Blattella germanica: Effects of grouping and mating

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day 8. Three factors were identified as the major contributors to this difference: (1) an experimental artifact of selection for sexually receptive females, (2) a positive effect of grouping on JH synthesis and oocyte maturation, and (3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating is low, as in experiments with isolated females that are exposed to males for a short period of time. Long-term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA–corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.     

De-alation, flight muscle histolysis, and oocyte development in the striped ground cricket, Allonemobius fasciatus

ABSTRACT. Removal of hindwings from long-winged females of the striped ground cricket, Allonemobius fasciatus , DeGeer (Gryllidae), induces flight muscle histolysis and oocyte development. Such females develop oocytes as rapidly as do short-winged forms, while intact long-winged females retain their flight muscles and develop few oocytes.
Flight muscle histolysis occurs in starved long-winged females when they are de-alated. However, such females fail to mature oocytes. Implantation of corpora allata (CA) into long-winged females results in flight muscle histolysis as well as oocyte maturation even if their hindwings remain intact, indicating that flight muscle histolysis can take place without de-alation. It is likely that the CA are responsible for both flight muscle histolysis and oocyte development, and that CA activity is enhanced by de-alation.     

Effects of the corpora allata on sexual receptivity and completion of oocyte development in females of the cricket, Gryllus bimaculatus

Abstract Allatectomy of young penultimate nymphs of Gryllus bimaculatus De Geer (Gryllidae) resulted in prothetelic creatures which exhibited reproductive competence. The same operation performed on young last instar nymphs resulted in moulting to morphologically normal adults. Allatectomized morphologically normal adult females, as well as prothetelic ones, showed the same level of sexual receptivity as untreated control females. Allatectomized morphologically normal and prothetelic females laid viable eggs, but rate of egg laying and number of eggs produced by these females were much reduced in comparison with the controls. Administration of methoprene (a Juvenile Hormone analogue) to allatectomized females restored egg production to a more or less normal rate. Removal of the spermatophore within 10 min of copulation had no effect on subsequent sexual receptivity of the females, nor on the reduced rate of egg laying by the allatectomized females, but did affect the rate of egg laying by control females.
It is suggested that the corpora allata (CA) and the Juvenile Hormone (JH) play no major role in controlling basic sexual receptivity of G.bimaculatus females, and do not have an all-encompassing control on egg production, though they do exert a marked quantitative effect on the rate of egg production.