Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata,and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.     

Myotropic effect of helicokinins,tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae)

Different insect neuropeptides (helicokinins, tachykinin-related and allatoregulating peptides) were investigated with regard to their myostimulatory effects using whole-gut preparations isolated from fifth instar Heliothis virescens larvae. The experiments demonstrated that representatives of all three peptide families are able to induce and amplify gut contractions in this species in a dose-dependent manner. Structure-activity studies (alanine scan, D-amino acid scan and truncated analogues) with the helicokinin Hez-K1 supported the finding, that the core sequence for biological activity of kinins is the amidated C-terminal pentapeptide (FSPWG-amide). Similar investigations with insect tachykinin isolated from Leucophaea madera (Lem-TRP1) revealed that the minimum sequence evoking a physiological gut response in H. virescens is the amidated hexapeptide (GFLGVR-amide), which represents the conserved amino acid sequence for Leucophaea TRPs in general. The peptide concentration causing a half-maximal gut contraction (EC(50)) for Lem-TRP1 was about 26 nM. Although the potency of Lem-TRP1 was 9-fold lower compared with Hez-KI (EC(50): 3 nM), the maximal tension of the gut obtained with Lem-TRP1 was 1.7-fold higher compared with Hez-KI. The EC(50) of Manduca sexta allatotropin (Mas-AT; 79 nM) was of lowest potency among all three peptides tested. In a pharmacological study, co-incubation experiments with Lem-TRP1, Hez-KI or Mas-AT and compounds interfering with signal transduction pathways were employed to investigate the mode of action of the myotropic effects of these peptides. Cadmium and the protein kinase C (PKC) inhibitor tamoxifen attenuated the contractile effects of all three peptides tested. The data suggest that in the gut muscle of H. virescens the myotropic peptides bind to G-protein-coupled receptors that cause contraction by promoting the entry of extracellular calcium mediated by a PKC involved pathway.     

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.     

Allatostatins and allatotropin in the corpus cardiacum/corpus allatum complex of larval and adult lepidopterans studied by confocal laser scanning microscopy: correlation to juvenile hormone biosynthesis

Peptidergic innervation of the corpus cardiacum/corpus allatum (CC/CA) retrocerebral complex, and neurosecretory areas of the brain of the lepidopterans Lacanobia oleracea, Heliothis virescens and Manduca sexta was studied by immunocytochemistry linked to confocal laser scanning microscopy. The patterns of immunostaining resulting from the simultaneous application of fluorochrome-conjugated antibodies against Manduca sexta allatostatin (Mas-AS), M. sexta allatotropin (Mas-AT), and a representative of the –Y/FXFGL-NH₂ superfamily of allatostatins was correlated with the physiological effects of these putative allatoregulatory peptides on juvenile hormone (JH) biosynthesis by the corpora allata. Whereas the two types of allatostatin immunoreactivity are present in both larval and adult CA of the three species, allatotropin immunoreactivity occurs only in the adult gland. The conclusion that withdrawal of the stimulatory effect of allatotropin is unlikely to be involved in the downregulation of CA activity prior to the onset of metamorphosis, but that an inhibitory influence of at least Mas-AS is important, is borne out in physiological experiments on JH biosynthesis in M. sexta larvae (Mas-AS inhibitory, Mas-AT without effect). Immunoreactivity to the Y/FXFGL-NH₂ allatostatins is present in both larval and adult CA and CC, frequently co-localised with Mas-AS. The function of this peptide family in the retrocerebral complex remains enigmatic since experiments on JH biosynthesis, either when the peptide is administered alone, or together with Mas-AS, show no effect on JH biosynthesis.Financial support was provided by The Wellcome Trust (063367/Z/00) (to A.T.) and by the Pesticide Safety Directorate of the Department for Environment, Food and Rural Affairs (to N.A. and R.J.W.)     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.     

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6-8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.     

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.     

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.