Unusual functions of insect vitellogenins: minireview.

Insect vitellogenins are an intriguing class of complex proteins. They primarily serve as a source of energy for the developing embryo in insect eggs. Vitellogenesis is a complex hormonally and neurally controlled process that command synthesis of vitellogenin molecules and ensures their transport from the female fat bodies or ovarial cells into eggs. The representatives of all insect hormones such as juvenile hormones, ecdysteroids, and neurohormones participate in vitellogenesis, but juvenile hormones (most insect species) and ecdysteroids (mostly Diptera) play the most important roles in the process. Strikingly, not only insect females, but also males have been reported to synthesize vitellogenins indicating their further utility in the insect body. Indeed, it has recently been found that vitellogenins perform a variety of biological functions in the insect body. They participate in defense reactions against entomopathogens such as nematodes, fungi, and bacteria, as well as against venoms such as the honeybee Apis mellifera venom. Interestingly, vitellogenins are also present in the venom of the honeybee itself, albeit their exact role is unknown; they most likely increase the efficacy of the venom in the victim's body. Within the bee's body vitellogenins contribute to the lifespan regulation as anti-aging factor acting under tight social interactions and hormonal control. The current minireview covers all of these functions of vitellogenins and portrays them as biologically active substances that play a variety of significant roles in both insect females and males, and not only acting as passive energy sources for developing embryo.

Role of allatostatin-like factors from the brain of Tenebrio molitor females.

The effect of brain extract from females of freshly emerged Tenebrio molitor on ovary, oocyte development, total protein content of hemolymph, and ovary was studied in 4-day-old adult mealworm females. Injections of extracts of 2-brain equivalents into intact (unligatured) Tenebrio females did not affect ovarian and oocyte development. Injections of ligated females, however, with 2-brain equivalents on day 1 and 2 after adult emergence strongly inhibited ovarian growth and oocyte development. At day 4, ligated and injected females did not develop their ovaries and pre-vitellogenic oocytes were not found. The changes in ovarian development correlated with an increase in the concentration of soluble proteins in the hemolymph as compared with the saline-injected controls. Additionally, a strong reduction of total protein content in ovarian tissue was observed. Reverse phase HPLC separation of a methanolic brain extract of T. molitor females showed that fraction 5 has a similar retention time to synthetic cockroach allatostatin. Fraction 5 was eluted at 12.88 min, which was closest to the internal standard Dippu-AST I, which eluted at 12.77 min. An ELISA of fraction 5 from the methanolic brain extract using antibodies against allatostatins Grybi-AST A1 and Grybi-AST B1 from cricket Gryllus bimaculatus showed that fraction 5 cross-reacted with Grybi-AST A1 antibodies. The cross-reactivity was similar to the synthetic allatostatin from D. punctata, which was used as a positive control. These observations demonstrate a possible role for allatostatin-like brain factor(s) in regulating the reproductive cycle of Tenebrio molitor.

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum).

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p-nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

Developmental and diel changes of adipokinetic hormone in CNS and haemolymph of the flightless wing-polymorphic bug, Pyrrhocoris apterus (L.).

We have used an enzyme-linked immunoassay (ELISA) for determination of the AKH content in CNS (brain + corpora cardiaca + corpora allata) and haemolymph of adult macropterous and brachypterous females of the bug Pyrrhocoris apterus. The tests revealed that the AKH content fluctuates between 1 and almost 4 pmol/CNS during the first 14 days of adult life and significantly increased in order diapausing brachypters

A quantitative study of adipokinetic hormone of the firebug, Pyrrhocoris apterus.

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/&mgr;l.

Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor.

Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteins, and contains a few small peptides of hitherto unknown functions. We demonstrate that two of these peptides inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These 'silk proteinase inhibitors' 1 and 2 (SPI 1 and 2) are produced in the middle section of the silk-secreting glands prior to cocoon spinning and their production is controlled at transcription level. The full length cDNA of pre-SPI 1 contains 443 nucleotides and encodes a peptide of 76 amino-acid residues, of which 20 make up a signal sequence. The mature SPI 1 (6056.7 Da, 56 residues) is a typical thermostable Kunitz-type proteinase inhibitor with Arg in P1 position. The cDNA of pre-SPI 2 consists of 260 nucleotides and yields a putative secretory peptide of 58 amino-acid residues. The functional SPI 2 (3993 Da, 36 residues) is a single-domain Kazal-type proteinase inhibitor with unique structural features: free segment of the N-terminus is reduced to a single amino-acid residue, lack of CysI and CysV precludes formation of the A-ring and provides increased flexibility to the C-ring, and absence of several residues around the normal position of CysV shortens and changes the alpha helix segment of the protein. The structure reveals that the length and arrangement of the B-ring, including exposure of the P1 residue, and the position of the C-terminus relative to the B-loop, are essential for the activity of the Kazal-type inhibitors.

A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera).

A new member of the AKH/RPCH family was isolated and identified from the corpora cardiaca of the firebug Pyrrhocoris apterus. The peptide was isolated in a single step by reversed phase HPLC and the structure deduced from the multiple MS (MS(N)) electrospray mass spectra and amino acid analysis as that of an octapeptide with the sequence pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH(2): this sequence was confirmed by synthesis. The synthetic peptide induced lipid mobilisation and stimulated locomotory activity in macropterous females. This peptide, designated as Pyrrhocoris apterus adipokinetic hormone (Pya-AKH), is the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera.

Identification of a novel type of silk protein and regulation of its expression.

The silk of lepidopteran insects has been studied extensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the middle section. We now describe a third category that is named seroins to accentuate the fact that both the sericin- and the fibroin-producing cells participate in seroin secretion. Using a probe derived from the N-terminal sequences of a 23-kDa components of Galleria mellonella silk, we isolated silk gland-specific cDNA encoding 167 amino acids, of which 17 constitute the signal peptide. The following 14 residues match the N-terminal sequences of the 23- and 22.5-kDa silk proteines. The reaction of these proteins with concanavalin A and the presence of two glycosylation sites in the seroin peptide sequence indicate that seroin is secreted in two forms that both contain a mannose-rich sugar moiety. Seroin is distinguished from other silk proteins by high proline content (34 residues or 20.26% by weight), lack of cysteines, and the presence of two kinds of short amino acid repeats. The seroin gene is expressed in both the posterior and middle silk gland sections. The expression fluctuates during development in correlation with the feeding regime and the changes in hormone titers: seroin mRNA is high in the silk glands of feeding larvae, declines at ecdysis, reaches a maximum during cocoon spinning, and thereafter rapidly drops to an undetectable level. In vivo and in vitro experiments showed that the drop is caused by ecdysteroid hormones and is prevented by juvenile hormones. N-terminal sequencing of several silk proteins of Bombyx mori revealed that the 8- and 13-kDa proteins share 5 or 6 out of 10 identified amino acids with the N terminus of Galleria seroin and obviously represent seroin homologues. The result suggests that seroin-type proteins are a general component of lepidopteran silk.

The P25 component of Galleria silk.

The water-insoluble core of lepidopteran silk is composed of four major proteins, but only three genes have been identified. This study demonstrates that the 29- and 30-kDa components of Galleria mellonella silk are derived from a single gene designated P25. The gene is expressed exclusively in the posterior section of the silk glands as a 2-kb mRNA, which accumulates in the feeding larvae and declines at molting. The mRNA encodes a peptide of 24,864 Da that exhibits 51% identity with the putative product of the P25 gene of Bombyx. The conservation of several amino acid stretches, including the relative positions of all 8 cysteines in the mature polypeptide, implies that the P25 proteins play similar, and apparently significant roles in silk formation in the two species. A Galleria P25 cDNA yields a peptide of about 25 kDa when translated in vitro; the 29- and 30-kDa forms present in the silk are derived from this primary translation product by differential glycosylation.

Light-chain fibroin of Galleria mellonella L.

The posterior section of Galleria mellonella silk glands contains two abundant mRNAs that are identical except for the non-coding tail, which includes either two (1.1 kb mRNA) or three (1.2 kb mRNA) consensus sequences for polyadenylation sites. The transcripts are 40% homologous in the coding as well as non-coding regions with the mRNA encoding light-chain fibroin (L-fibroin) in Bombyx mori; the deduced translation product shows 43% identity with the Bombyx L-fibroin peptide, with all three cysteines conserved. Amino acid analysis of the N-termini of Galleria silk proteins revealed that L-fibroin (25 kDa) occurs in two isoforms, the shorter one lacking the Ala-Pro dipeptide residue at its N-terminus. The 29 and 30 kDa Galleria silk proteins appear to be homologs of Bombyx silk component P25. The results suggest that evolutionary diversification of Galleria and Bombyx L-fibroins involves alternative polyadenylation and proteolytic processing sites.