Effect of precocene II on the endocrine regulation of development and reproduction in the bug, Oncopeltus fasciatus.

Large milkweed bugs, Oncopeltus fasciatus, were exposed to precocene II for different times as larvae or adults. Short-term contact during the stages in which the corpora allata are active (1 hr in the second instar, 12 hr in the young adults), inactivated the gland completely. Treatment during the last instar, in which the activity of corpus allatum is limited did not injure the gland which was subsequently activated in the young adults. The inactivation of the corpora allata resulted in precocious metamorphosis and degeneration of the prothoracic glands in younger larval instars and inhibition of vitellogenesis in adult females. However, the males with inactivated corpora allata were fertile. The corpora allata of treated insects remained inactive even when transplanted into an untreated insect. The effects of precocene II can be counteracted by subsequent application of juvenile hormone 1. The irreversibility of changes induced by precocene II holds promise for the practical application of compounds of this type as insect growth regulators if substances with a broad spectrum of activity and good persistency can be found.     

An ultrastructural study of neurosecretory fibers within the corpora allata of Manduca sexta

Two areas of the corpora allata of last instar Manduca sexta larvae contain neurosecretory axons; the gland proper which consists of typical stellate glandular cells and the sheath in which groups of neurosecretory axons are separated by the lamellated stromal sheath. In midinstar larvae the neurosecretory axons within the gland are quite large in comparison with those in the pupa. This size fluctuation during development and the proximity of these axons to gland cells suggest that these fibers may influence juvenile hormone synthesis and/or secretion. The population of neurosecretory axons in the sheath, on the other hand, does not change throughout postembryonic development. The products of these fibers have easy access to the general circulation. Thus, the corpus allatum sheath may serve as a neurohemal area for prothoracicotropic hormone (PTTH) since this tropic hormone is released from the corpora allata during development (Agui et al., 1980). Synaptoid release sites are found in fibers from both areas.     

Nervous and humoral inhibition of C16 juvenile hormone synthesis in last instar females of the viviparous cockroach, Diploptera punctata

The control of the decline in C₁₆ juvenile hormone (C₁₆JH) synthesis in the final larval stadium of the viviparous cockroach Diploptera punctata Eschscholtz was investigated to establish how nervous and humoral signals affect the activity of the corpora allata (CA) before metamorphosis. Denervation of the CA in young last instars caused an increase in the rate of JH biosynthesis as monitored by an in vitro radiochemical assay and resulted in a supernumerary larval form at the following ecdysis. However, when larval CA were transplanted into corpus allatectomized adults, they exhibited a further 5- to 10-fold stimulation in synthetic rate and supported host oocyte development at near normal rates. Conversely, adult CA implanted into young and old last instars were inhibited in comparison to those implanted into penultimate instars and adults. These experiments suggest that in D. punctata, an unfavorable humoral environment as well as nervously transmitted inhibitory signals maintain low rates of JH synthesis prior to the imaginal molt.     

Inhibition of corpora allata by larval brain extract in the cockroach diploptera punctata

Extract of brains from final-instar males inhibited juvenile hormone synthesis by corpora allata from adult females and larval males in vitro. The inhibition was dose-dependent; maximal response was 70% reduction in activity elicited by 0.5 protocerebrum. Inhibition occurred within 1 h as did recovery. The inhibitory factor is trypsin-sensitive. Inhibitory activity was found in extract of brains from males of the three larval instars and adults, and was not localized to a particular region of the brain. Larval corpora allata showed nerve endings containing neurosecretory granules; extracts of larval corpora allata inhibited juvenile hormone synthesis in vitro. The hypothesis that an inhibitory factor is released from these nerve endings was supported by the observation that corpora allata treated with high concentrations of potassium were inhibited, but not in the presence of magnesium.     

The synthesis of neurosecretory protein during the fifth instar of Locusta migratoria migratorioides

The synthesis of a neurosecretory protein during the fifth instar of female locusts has been estimated by incorporation of [³H]cystine and rocket immunoelectrophoresis. Highest specific activity of neurosecretory protein was found both in the brain and corpora cardiaca on Day 6 of the instar. Eightfold as much radiolabelled cystine is incorporated in neurosecretory protein (of the brain) than into other proteins of the brain on this day. An earlier, though less pronounced, peak of synthetic activity by the median neurosecretory cells during the fifth instar closely parallels changes in previously established ecdysone titres. The two ecdysone peaks which occur in fifth-instar Locusta (data of A. Bouthier, J.-L. Pennetier, B. Mauchamp, and R. Lafont, 1975, C. R. Acad. Sci. Paris280, 1837–1840) follow the peaks of neurosecretory synthesis within 24 hr. Such parallels between neurosecretory synthesis and ecdysone titres again raise the possibility of a prothoracotropin being synthesised by the median neurosecretory cells of Locusta.     

Brain neurosecretion during ovarian development and after ovariectomy in adult Acheta domesticus L

Paraldehyde fuchsin (PAF) stained serial sections of the brain were examined for neurosecretory material (NSM) during adult ovarian development and in 10-day-old ovariectomized adult Acheta domesticus L. Type I neurosecretory cells of the pars intercerebralis were relatively low in NSM for the first 3 days of adult life, including the period during which vitellogenin synthesis and uptake are initiated. As mature oocytes accumulate after Day 4, NSM accumulates in the brain, reaching a peak by about Day 10. Ten-day-old adults ovariectomized as last instar larvae contained dramatically less brain NSM than intact controls, and injection of 0.48 μg of ecdysterone into ovariectomized crickets partially restored the brain to a state resembling that of intact animals. Peptides from the brains and corpora cardiaca-corpora allata complexes of intact and ovariectomized animals were separated by SDS-polyacrylamide gel electrophoresis and their PAF stainability was analyzed. Ovariectomy resulted in a decrease in the amounts of 12 brain peptides, but these showed little or no preferential staining with PAF. The possibility of regulatory interactions between the brain and ovaries is discussed.     

Adrenocortical ultrastructure in the squaliform elasmobranch (Ginglymostoma cirratum Bonnaterre): cell death, a postulate for holocrine secretion.

Allatectomy reduces the synthetic activity of the cerebral neurosecretory system of Locusta migratoria migratorioides. Since the median neurosecretory cells are thought to produce an allatotrophic factor it is possible that a close interrelationship exists between the two endocrine centres. This possibility was investigated further by inactivating the cerebral neurosecretory system by allatectomy or starvation and subsequently implanting supernumerary active corpora allata. After corpus allatum implantation into allatectomized L. migratoria females a significant stimulation of neurosecretory synthesis, transport, and release occurs. Starved female locusts implanted with active corpora allata show elevated rates of synthesis and transport of neurosecretion.It is concluded that a positive feedback of corpus allatum hormone on the median neurosecretory A-cells occurs. It is suggested that such a feedback system facilitates the continuous production of batches of eggs under favorable conditions. Such a relationship may affect the interpretation of the results of endocrine surgery.     

Cellular localization of the insect prothoracicotropic hormone: In vitro assay of a single neurosecretory cell

The distribution of prothoracicotropic hormone in the pupal brain of Manduca sexta has been determined by an in vitro assay for prothoracic gland activation. Prothoracicotropic activity was observed in both the brain and retrocerebral complex, but predominantly in the dorosolateral regions of the protocerebrum. Of the two groups of neurosecretory cells present in this area of the brain, only the two lateral type III neurosecretory cells exhibited significant prothoracicotropic hormone activity. Further analysis revealed that the neurohormone was localized in only one of the two type III cells, suggesting that a single neurosecretory cell in each hemisphere is the source of the hormone at the stage examined (day 0). Prothoracicotropic hormone activity was detected in both the corpora allata and the corpora cardiaca, but the corpora allata contained 2 to 9 times the activity of the corpora cardiaca, depending on developmental stage. The significantly higher level of activity in the corpora allata suggests that they may be the neurohemal organs through which the prothoracicotropic hormone of Manduca is released.     

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor--allatinhibin (AI)--from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 microliters medium. CA must be exposed to AI for 12-16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.     

Endocrine control by neurosecretory cells of the pars intercerebralis and the corpora allata during the earlier phases of vitellogenesis in Locusta migratoria migratorioides R and F (Orthoptera)

In the adult female Locusta migratoria, the neurosecretory cells (A,B,C) of the pars intercerebralis and the corpora allata are essential for the onset of the second phase of vitellogenesis, which is characterized by the uptake of yolk proteins by the oocyte. Electrocoagulation of the neurosecretory cells inhibits vitellogenesis only if this operation is performed during the first five days of adult life (oocyte length from 0.4 to 1 mm). On the other hand, the presence of the corpora allata is essential for vitellogenesis until the beginning of the second phase of vitellogenesis (oocyte length from 0.4 to 4 mm). Implantation of one pair of corpora allata or an injection of juvenile hormone (JH) (10 μg per insect; JH₁, C₁₈) in allatectomized females at first stimulates the differentiation of the follicular cells, which occurs normally during the first phase of vitellogenesis; the different structures observed in such oocytes are the same as in untreated insects.     

Neuroendocrine regulation of oocyte maturation in the imported fire ant Solenopsis invicta

Extirpation of the corpus allatum and brain neurosecretory cells and application of juvenile hormone demonstrated that the neuroendocrine regulation of oocyte maturation in the imported fire ant Solenopsis invicta closely resembles the general insect pattern of neuroendocrine regulation where both the brain and corpora allata are required. Evidence is presented that the brain has a role in oocyte maturation in addition to the generally accepted role in regulating secretion of juvenile hormone by the corpora allata.     

Induced changes in neurosecretory activity of adult female Schistocerca gregaria in relation to feeding

Experimentally induced changes in the activity of the neurosecretory system have been followed in adult female Schistocerca gregaria using autoradiographic techniques. In females starved for 5 days the neurosecretory system is full of stainable material and in such females the rates of synthesis, transport, and release of neurosecretion are low.Starved animals which are allowed to recommence feeding show an immediate change in neurosecretory activity that is not entirely discernible from studies on the changes in amounts of stainable neurosecretory material in the system. Some 70% of the neurosecretion stored within the corpora cardiaca is released into the haemolymph within 10 min of the onset of feeding. The neurosecretory cells discharge half their products into the NCCI between 10 min and 1 hr of feeding, and the rate of synthesis of new material is increased. The rate of transport of neurosecretory material down the NCCI to the corpora cardiaca is at least doubled in response to feeding.     

Hormonal control of prepupal diapause in Monema flavescens (Lepidoptera)

Injection of β-ecdysone into the abdomen of diapausing prepupae of Monema flavescens induced an extra ecdysis, resulting in prepupal-pupal intermediates instead of normal pupae. If diapausing prepupae were allatectomized, diapause soon broke and pupation took place frequently. Implantation of corpora allata from diapausing donors into early post-diapause or allatectomized prepupae induced diapause again in the hosts. Hemolymph levels of juvenile hormone rose during early diapause period and fell gradually toward the end of diapause. Changes in volume of corpora allata occurred in parallel to rise and fall in hemolymph level of juvenile hormone. Brains from diapausing prepupae implanted into decapitated diapausing prepupae caused pupation in the hosts within a short period. By contrast, implantation of brain-corpora allata complexes from diapausing prepupae did not result in quick pupation of the recipients. Implantation of corpora allata into diapausing prepupae gave rise to a significant decrease in nuclear volume in neurosecretory B cells in the pars intercerebralis, while extirpation of corpora allata resulted in a significant increase. The increase in nuclear volume following allatectomy was suppressed by implantation of corpora allata from diapausing donors. Prepupal diapause in Monema flavescens seems to be initiated and maintained by an inhibitory action of juvenile hormone on the release of neurosecretory materials from B cells of the pars intercerebralis. When the period of active secretion of corpora allata comes to an end, the brain-prothoracic gland system is activated and diapause is broken.     

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.     

Activation of Manduca sexta corpora allata in vitro by a cerebral neuropeptide

When an in vitro assay system and radioimmunoassays specific for juvenile hormones (JH) I and III were used to probe the effect of co-incubating pupal brains with last instar larval corpora allata (CA) from the tobacco hornworm, Manduca sexta, a selective activation of JH III synthesis by the CA was observed. This homolog-specific activation suggested the presence of an allatotropic factor for the synthesis of JH III (JH III ATF), and its presence was demonstrated by the ability of a postmicrosomal supernatant of a day 0 pupal brain homogenate to activate the CA in vitro in a dose-dependent manner. This moiety appears to be a protein, based on its heat lability and protease sensitivity, and has an apparent molecular size of 40 kD and an isoelectric point of 5.5 JH III ATF activity is localized in specific neural tissues of the day 0 pupa, the brain and first three abdominal ganglia, with the brain containing 4 times the activity in the ganglia. The existence of this factor suggests that JH III synthesis by the CA of Manduca is regulated by a neuropeptide.     

Tracheolization and the effects of implantation of corpora allata on the invagination of tracheoblasts into the developing flight muscle fibers of Locusta migratoria

The process of tracheolization of the flight muscle fibers of Locusta, essential for the specific metabolic properties of the adult muscles, occurs during the last part of the fifth larval instar. Several ultrastructural criteria of activity were recognized in the cytoplasm of the tracheoblasts which are concerned with the formation of both small tracheae and interfibrillar tracheoles. The small tracheae appear to originate from an extracellular lumen enclosed by intruding and invaginating tracheoblasts which probably secrete amorphous material used in the formation of an intima bordering the tracheal lumen. Cytoplasmic protrusions of tracheoblasts invaginate into the muscle fibers, enclosing the interfibrillar tracheoles; the exact mode of formation of the latter, however, remains as yet unknown. Connecting links between the small tracheae and the interfibrillar tracheoles probably emerge from evaginations of the plasma membrane of the tracheoblast(s) enclosing the developing small trachea. Implantation of active corpora allata into female locusts on the first day of the fifth larval instar results in a delay in the invagination of tracheoblasts into the muscle fibers till the end of an extra (sixth) larval stage. The relation between tracheoblast development and differentiation of flight muscle enzyme pattern, as well as influences of juvenile hormone and ecdysone on the developmental processes, are discussed.     

Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm Manduca sexta

The hemolymph juvenile hormone (JH) titers of Manduca sexta larvae parasitized by Apanteles congregatus were determined using the black Manduca bioassay and found to be significantly higher than titers of normally fed or starved unparasitized fifth instar larvae. In vitro measurements of hemolymph esterase activity indicated that JH-specific esterase activity was reduced in parasitized larvae, contributing to the high JH titer. Analysis of host ecdysteroid titers by radioimmunoassay showed the presence of an ecdysteroid peak during parasite emergence from the host that was similar to the prewandering peak seen in unparasitized larvae. Host prothoracic gland activity is apparently necessary for emergence, as indicated by the failure of parasites to emerge from hosts that were neck or abdomen ligatured early in the fifth instar. The onset of emergence in intact hosts was accelerated by the infusion of ecdysone prior to the normal expected time of emergence. In addition to causing in vivo endocrinological changes, parasitism also markedly affected host epidermal responsiveness to exogenous 20-hydroxyecdysone (20-HE). When terminal stage fourth instar larvae were neck ligatured and injected with 20-HE prior to parasite emergence, the host epidermis failed to synthesize new cuticle. Sensitivity increased after the onset of emergence, and larval molting was elicited with large doses of 20-HE similar to molting of preterminal stage hosts with first instar parasites. These findings were interpreted as indicating that parasitism interferes with the normal molting response to exogenous ecdysteroids.     

In vitro biosynthesis of juvenile hormone by the larval corpora allata of Manduca sexta: Quantification by radioimmunoassay

An in vitro method has been developed for the investigation of the regulation of juvenile hormone biosynthesis by insect corpora allata. Glands were maintained in Marks medium 19AB and JH synthesis quantified by a modified radioimmunoassay for juvenile hormone I. The radioimmunoassay is specific for JH I and exhibits ~12.6% cross reactivity with JH II and no cross reactivity with JH III. The assay directly measures the JH present in culture medium and has a maximum sensitivity of 50 pg JH I equivalents. Corpora allata from day 5 last instar Manduca sexta larvae were used to define the kinetics parameters of the in vitro system, including a demonstration that small groups of right and left glands synthesize equivalent amounts of juvenile hormone. The juvenile hormones synthesized were identified as juvenile hormones I and II in a ratio of 1 : 4, respectively. Juvenile hormone III could not be excluded as a product of the corpora allata owing to the low cross reactivity of this homolog (1.7%) in the radioimmunoassay. Corpora allata from different developmental stages exhibited synthesis rates generally consistent with predicted activity based on in vivo hormone titers with the exception of day 5 of the last instar. The variation in gland activity relative to the control of juvenile hormone titer in vivo is discussed.     

In vitro inactivation of Tenebrio molitor corpora allata by a synthetic precocene analog

Tenebrio molitor (Còleoptera: Tenebrionidae) is an insect insensitive to treatment with the antiallatal compound ethoxy-precocene (EP; 7-ethoxy-6-methoxy-2,2-dimethylchromene). However, when isolated T. molitor corpora allata (CA) are incubated in vitro in the presence of EP and then subsequently assayed for juvenile hormone (JH) activity, they are inactivated in a time-dependent, dose-dependent manner. These results indicate that sensitivity of an insect species to precocene in vivo is not solely dependent on the intrinsic sensitivity of the CA of that insect.     

In vitro activation of neurosecretory brain cells in Mamestra brassicae by beta-ecdysone

The effect of ecdysone or of the prothoracic gland on the neurosecretory cells of brains taken from diapausing pupae of the cabbage armyworm, Mamestra brassicae L., was investigated histologically and functionally in vitro. When the diapausing brain was cultured in a medium containing β-ecdysone, the neurosecretory cells of the median group I in the pars intercerebralis increased staining intensity more than those of brains cultured in medium alone. Furthermore, when the prothoracotropic activity of a brain treated with β-ecdysone was compared with that of an untreated brain in vitro, the brain treated with hormone showed higher activity than the untreated one. These results suggest that β-ecdysone acts on the neurosecretory cells of pupal brain in M. brassicae as a positive feedback regulator.