Andrea P. Woodhead

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

SummaryA 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.

Two new allatostatins from the brains of Diploptera punctata

Allatostatins VI and VII have been isolated from saline extracts of the brain of the viviparous cockroach Diploptera punctata. Active fractions, obtained by successive reverse-phase high pressure liquid chromatography separations, inhibited juvenile hormone (JH) III production by corpora allata (CA) in vitro. The primary structures, Y-P-Q-E-H-R-F-S-F-G-L-amide (VI) and D-G-R-M-Y-S-F-G-L-amide (VII), clearly identify them as members of a family of D. punctata allatostatins, five of which have been identified previously and shown to have F-G-L-amide at the C terminus. Synthetic allatostatins VI and VII coeluted with the native allatostatins on two successive high pressure liquid chromatography separations. Allatostatins VI and VII have the same capacity to inhibit JH production by CA of 2 day virgin females as does allatostatin I, previously shown to be a potent inhibitor similar in activity to allatostatin V. Allatostatins VI and VII also resemble the other allatostatins in their ability to affect both larval and adult CA, in the reversibility of their effect, and in the abolition of their effect by addition of farnesoic acid (a late precursor of JH) to CA in vitro.

Sensitivity to allatostatins of corpora allata from larval and adult female Diploptera punctata

Abstract Corpora allata from adult and larval Diploptera punctata females were tested for sensitivity to inhibition of juvenile hormone synthesis by allatostatins in vitro . Inhibition by allatostatin 1 was consistently high (60–82%) after larval and adult moults and thereafter changed with corpora allata activity. In 0–8-day virgin females, with relatively constant juvenile hormone synthesis, the sensitivity of the corpora allata remained relatively constant (51–71%). Mated females in both the first and second vitellogenic cycles showed similar changing patterns of inhibition: low inhibitions (35 and 50%) at the peaks of the two cycles of juvenile hormone synthesis and an increase to maxima (83 and 77%) as juvenile hormone synthesis declined. During the 60-day pregnancy between the two cycles, rates of juvenile hormone synthesis remained low, yet the sensitivity to allatostatin 1 declined gradually. This pattern of sensitivity of adult corpora allata was also observed with allatostatin 4. Sensitivity of corpora allata from 2-day virgins and first- and second-cycle females to allatostatin 1 showed a pronounced increase between 10 −9 and 10 −8 M for all ages tested except day 50 in which a marked increase in sensitivity occurred between 10 −8 and 10 −7 M. In penultimate-instar larvae the sensitivity of the glands to inhibition by allatostatin 1 decreased to 29% one day after the midstadium peak of juvenile hormone synthesis, and recovered only slightly as juvenile hormone synthesis subsequently decreased. In final-instar larvae also, the sensitivity of the corpora allata declined after the moult, but this occurred as juvenile hormone synthesis decreased. Inhibition remained low even as juvenile hormone synthesis began to increase at the end of the final stadium. Similar sensitivities to allatostatins 2, 3 and 4 were found for corpora allata from penultimate- and final-instar larvae.

Allatostatins in the haemolymph of Diploptera punctata and their effect in vivo

Abstract Adult female Diploptera punctata haemolymph extract was separated by high-pressure liquid chromatography, and fractions that coeluted with allatostatins I–V inhibited juvenile hormone synthesis by corpora allata in vitro. Saline extract of haemolymph was heated to 100°C, purified by reverse-phase C18 solid phase extraction and then by high-pressure liquid chromatography separation on a reverse-phase C18 column. Fractions corresponding to the elution times of allatostatins I–IV and allatostatin V were collected from each chromatographic separation. Similar fractions were combined and reapplied to the same column. Inhibition was found in fractions with elution times corresponding to allatostatins IV, I–III and V. Percent inhibition in these fractions from haemolymph of 13–14-day mated females was 44±7, 57±3 and 55±3, and 70±3, respectively (∼1.2 ml haemolymph equivalents/CA). To determine whether allatostatins in the haemolymph can inhibit the corpora allata, D. punctata allatostatins I and V and a related active peptide, callatostatin 5 from Calliphora vomitoria, were injected into mated females on days 1–3 after ecdysis at 12 h intervals. As controls, luteinizing hormone releasing hormone, truncated allatostatin I (N-terminal 1–7, unamidated) and water were injected. On day 4, length of basal oocytes and/or juvenile hormone synthesis by the corpora allata were significantly lower in animals injected with active allatostatins compared with water-injected controls, whereas, they were not affected by injection of inactive peptides.

Larval development of Diploptera punctata reared alone and in groups

Abstract Larvae of the cockroach Diploptera punctata were reared in isolation, in pairs, or in groups of 8–10. Duration of larval development, age at each ecdysis, weights at birth and ecdyses, and adult head-capsule width were measured. Duration of larval development was longer and adult size was larger in isolated animals than in animals reared in pairs and groups. The effect of isolation on development was more pronounced in males. All females had 4 larval instars, whereas males had 3 or 4 instars. The proportion of males with 4 larval instars was higher among animals reared in isolation. There was no difference in the duration of larval development or adult size between pair- and group-reared animals. The sex of animals in the group did not affect adult size or the duration of larval development. Males which underwent 3 or 4 larval instars had different schedules of moulting. Rates of growth of males of both instar types reared in isolation and pairs were similar. Greater adult weight of isolated animals and 4-instar-type males was a result of their longer duration of larval development. Both a higher rate of growth and longer duration of larval development contribute to the larger adult size of females than males.

ALLATOSTATIN IN HEMOCYTES OF THE COCKROACH DIPLOPTERA PUNCTATA

Abstract.Allatostatins are neuropeptides that inhibit the production, by the corpora allata, of a major insect hormone, juvenile hormone. These peptides are produced by cells of the brain and ganglia as well as by midgut endocrine cells. Transport from these sites may contribute to the allatostatin content in the hemolymph (insect blood). Using a monoclonal antibody against Diploptera punctata allatostatin I (A-P-S-G-A-Q-R-L-Y-G-F-G-L-NH2) and in situ hybridization with a digoxigenin-labeled cRNA probe generated from a portion of the allatostatin gene, it is demonstrated that allatostatin is present in and synthesized by granular hemocytes of D. punctata. About 5% of the hemocytes react with anti-allatostatin antibody and a similar number hybridize with a cRNA probe that detects allatostatin-specific mRNA. Electron micrographs showed that allatostatin-immunoreactive material occurs in membrane-bound, uniformly dense granules that frequently fill fusiform-shaped cells. Allatostatin in cell and plasma fractions of hemolymph quantified by enzyme-linked immunosorbent assay and by bioassay for inhibition of juvenile hormone synthesis in vitro indicated that about equal quantities (0.1–0.2 fmol/μl) are present in cell and plasma fractions. The production of allatostatin by hemocytes suggests that allatostatins may function as regulatory peptides in hemolymph activities in addition to their other known functions.

Release of neurosecretory granules within the corpus allatum in relation to the regulation of juvenile hormone synthesis in Diploptera punctata

The release of neurosecretory granules within the corpora allata (CA) of the viviparous cockroach Diploptera punctata has been compared in glands with intact nerves from the brain (Brain-CA) and those detached from the brain. Measurements of juvenile hormone (JH) synthesis in vitro, comparing these two conditions of the CA at several stages of vitellogenesis in adult females, showed lower production of hormone in Brain-CA complexes than in CA alone. Glands treated with tannic acid to trap exocytotic granules before fixation for electron microscopical examination showed, in sample sections, 10 times more exocytotic profiles in the glands with intact nerves to the brain than in the isolated glands. Sections treated with antibody against allatostatin I (Dip 7), a member of the neuropeptide family that inhibits JH synthesis by CA in vitro, showed neurosecretory granules in allatostatin immunoreactive nerves to be 75+/-4% of the granules in the sample of sections of CA. Because the total quantity of allatostatin in CA was found by ELISA not to vary significantly with changes in JH synthesis, it is concluded that the lower rates of JH synthesis by glands with intact nerves to the brain are most likely due to the release of small amounts of allatostatin within the CA.

Neural inhibition of corpora allata in protein-deprived Diploptera punctata

Abstract Protein-deprived adults of the cockroach Diploptera punctata were produced by feeding last-instar larvae on a diet without protein. Fat bodies of protein-deprived females (fed a 0%-protein diet) contained less protein (0.5 mg) at the adult moult than fat bodies of females fed a 20%-protein diet (1.5 mg) or a standard lab-chow diet (2.2 mg). Protein-deprived females released the spermatophore later (day 6) and oviposited later (day 9) than females fed a 20%-protein diet (day 5, day 8) or a lab-chow diet (day 4.5, day 7.5). Although length of oviposited eggs and vitellin content per egg were similar for females on the three diets, fewer eggs developed in protein-deprived females (10.5) compared with 20%-protein (11.9) and lab-chow females (12.4). The maximal rate of juvenile hormone synthesis was lower in protein-deprived females (73 pmol h−1 pair−1) than in 20%-protein (116 pmol h−1 pair−1) and lab-chow females (109 pmol h−1 pair−1). Denervation of corpora allata on day 1 in protein-deprived females led to an increase in the rate of juvenile hormone synthesis on day 4 to the level found in control animals, with a corresponding increase in vitellin content of the ovary. Thus, neural inhibition of the corpora allata occurs in protein-deprived females.

Male age: effect on mating behaviour and success in the cockroach Diploptera punctata

Abstract Older cockroach, Diploptera punctata , males (28 days old) transferred more sperm and larger spermatophores to females than 8-or 14-day-old males. In one male: one female behavioural tests, no significant correlations were found between either how rapidly males initiated courtship with females (response time), or intensity or duration of courtship, and the number of sperm or size of spermatophore subsequently transferred. Eight-day males were slower to initiate courtship than 28-day males. There was no significant differences in the duration of courtship for 8-, 14- and 28-day males, even though the mean duration of courtship was more than twice, as long for 8-day males compared with 14- and 28-day males. In competitive mating tests with one female and three males, one of each age, 28-day males were significantly more successful than 8- and 14-day males. It is concluded that the greater success of 28-day males is due to differences in male behaviour rather than to female discrimination.

Effect of duration of larval development on sexual competence in young adult male Diploptera punctata

ABSTRACT. The duration of larval development was 27% longer, adult weight was 10% greater, and adult head capsule was 2% wider in Diploptera punctata (Eschscholtz) males with four larval stadia compared with those with three larval stadia. Four‐instar males transferred significantly more sperm than three‐instar males 8, 14 and 28 days after adult eclosion, but there was no difference in the length of sperma‐tophore transferred by three‐ and four‐instar males at these ages. Eight‐day‐old four‐instar males were more successful than 8‐day‐old three‐instar males in fertilizing complete batches of eggs. For both three‐ and four‐instar males, males that mated when they were older (measured in days after adult eclosion) transferred more sperm and larger spermatophores than younger males. Body size did not have a significant effect on the number of sperm or size of spermatophore transferred by males.