Martina Meyering-Vos

Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides.

Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing. All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity. Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.

RNA interference with the allatoregulating neuropeptide genes from the fall armyworm Spodoptera frugiperda and its effects on the JH titer in the hemolymph

The juvenile hormone (JH) titer was measured by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). Three JH homologs, the JH I-III were detected in various amounts in larvae, prepupae and virgin adult females of Spodoptera frugiperda. In penultimate larvae, the JH II and III titers were relatively high, but decreased continuously during the 3 days of that stage, whereas JH I was detectable at low amounts only on the first 2 days. At the beginning of the last larval stage almost no JH could be detected but thereafter, a consistent low amount of JH III was present until the prepupal stage. In adult virgins, the JH titer peaked on the 2nd and 6th day after the imaginal molt. The measured hormone titers well agree with general lepidopteran physiology, because in larvae the JH titer should be high to prevent premature metamorphosis, but decrease in last instar larvae before pupation, whereas in adults JH returns to control various aspects of reproduction. JH biosynthesis is thought to be the main factor influencing the JH titer in the hemolymph and there is evidence that neuropeptides either act stimulatory (allatotropins) or inhibitory (allatostatins) on this process. After silencing of either the allatostatin AS-C-type (Spofr/Manse-AS) or the allatotropin AT 2 (Spofr-AT 2) gene the transcript level was reduced in brain and gut of last instar larvae as well as of adult S. frugiperda. This suppression led to an increased JH titer in larvae, suggesting an allatostatic activity of both the peptides in this stage. As a result of the elevated hormone titer, the last larval stage was prolonged. In prepupae, the JH titer was decreased, but the animals pupated and molted normally. In adult female virgin moths the effect on the JH titer was inversely dependent on the age of the moths and varied among the JH homologs, indicating that the peptides act either allatostatic or allatotropic. For both peptides, gene silencing clearly reduced the oviposition rates of adult females.

The allatostatin gene of the cricket Gryllus bimaculatus (Ensifera, Gryllidae)

The gene encoding allatostatins (AST) of the FGLamide family from the cricket Gryllus bimaculatus is expressed in the brain. The mRNA, which contains four polyadenylation signals, encodes a hormone precursor that is split into at least 14 putative hormones. Five of them have been previously found in the cricket, six to seven others, or their close homologues, are known from other insects. Hormone AST 2 contains an internal cleavage site and may exist in a shorter version 2b. The hormones AST 3 and 4 are identical. The cDNA sequence revealed that a single point mutation and a single deletion eliminated an additional hormone between AST 12 and 13. The deduced hormone precursor is very similar to that in cockroaches, but is different from a shorter precursor in locusts, indicating that the gene evolved very fast in the latter. Regions conserved between cockroaches and crickets include parts of the acidic spacers that separate clusters of hormones, suggesting that these spacers may have additional functions.

Effects of temperature on reproductive output, egg provisioning, juvenile hormone and vitellogenin titres in the butterfly Bicyclus anynana

Environmentally induced phenotypic plasticity is common in nature. Hormones, affecting multiple traits and signaling to a variety of distant target tissues, provide a mechanistic link between environments, genes and trait expression, and may therefore well be involved in the regulation phenotypic plasticity. Here, we investigate whether in the tropical butterfly Bicyclus anynana temperature-mediated plasticity in egg size and number, with fewer but larger eggs produced at lower temperatures and vice versa, is under control of juvenile hormone, and whether different temperatures cause differences in egg composition. Female B. anynana butterflies showed the expected response to temperature, however, we found no evidence for an involvement of juvenile hormone. Neither haemolymph JH II and JH III titres nor vitellogenin levels differed across temperatures. The smaller eggs produced at the higher temperature contained relatively higher amounts of water, free carbohydrates and proteins, but relatively lower amounts of lipids. While these smaller eggs had a lower absolute energy content, total reproductive investment was higher at the higher temperature (due to a higher fecundity). Overall, our study indicates that temperature-mediated plasticity in reproduction in B. anynana is mechanistically related to a biophysical model, with oocyte production (differentiation) and oocyte growth (vitellogenesis) having differential temperature sensitivities.

Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus

Abstract In Gryllus bimaculatus, more digestive enzymes (amylase, trypsin, aminopeptidase) are secreted in the caecum of fed crickets than in unfed crickets, but the enzymes are released continuously at a basal rate in unfed animals. The rate of synthesis of the enzymes appears to parallel their rate of release. Digestive enzymes are released in response to a specific ratio of nutrients, although a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Rinsed flat‐sheet preparations of the caecum are incubated with specific nutrients (carbohydrates and proteins) and various concentrations of a neuropeptide (type‐A allatostatin), which affects generally the basal rates of secretion. Both maltose and glucose increase the release of amylase in vitro, but starch produces an inhibition of amylase release at lower concentrations. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase or carboxypeptidase, and only low concentrations of peptone increase trypsin release. High concentrations of both BSA and peptone strongly inhibit trypsin activity, perhaps by excess substrate binding to the trypsin active site. The allatostatin Grybi‐AST 5 elevates the release of amylase in vitro, but not of trypsin or aminopeptidase, in 2‐day‐old fed females. In the caeca from 1‐day‐old unfed crickets, both amylase and the trypsin release are stimulated in the presence of AST 5. The paracrine AST 5 is probably released from the gut endocrine cells and binds to the enzyme‐producing caecal cells.

Structure of the sulfakinin cDNA and gene expression from the Mediterranean field cricket Gryllus bimaculatus

The sulfakinins are multifunctional insect neuropeptides displaying sequence similarities with the gastrin/ cholecystokinin (CCK) peptide family. In vertebrates, the peptides gastrin and CCK are involved in the regulation of digestion and food‐intake. In this study sulfakinin cDNA was cloned and sequenced from the Mediterranean field cricket Gryllus bimaculatus. The cDNA encodes two peptides flanked by endoproteolytic processing sites, designated GrybiSKI (QSDDYGHMRFG) and GrybiSKII (EPFDDYGHMRFG). The peptides include the characteristic amino acid Tyr, which is potentially sulphated, and a Gly, as a recognition site for amidation yeilding the common C‐terminal amino acid sequence of the sulfakinin peptide family. RT‐PCR studies indicate an expression of the gene restricted to the brain, with a constant level of expression throughout the last larval stage, but showing an age‐dependent decrease of expression in adult females.

Characterization of a novel peptide with allatotropic activity in the fall armyworm Spodoptera frugiperda

A cDNA that encodes 53 amino acids, including one copy of the RVRGNPISCF-OH peptide, was cloned from Spodoptera frugiperda. This peptide strongly stimulates the synthesis and release of juvenile hormone (JH) in vitro by the corpora allata (CA) of S. frugiperda and was code-named Spofr-AT 2. Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that the preprohormone is expressed as one transcript in the brain, midgut (Mg) and ovary (Ov) in a tissue- and developmental-specific manner. Whole-mount in situ hybridization confirmed the gene expression in the suboesophageal ganglion (SOG) and in the ovary of adult females. Treating the CA with the synthetic peptide caused an up to tenfold increase in the release of JH. The stimulation was dose-dependent with an apparent EC(50) of ca. 10(-7) M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of synthetic allatostatin type-C from Manduca sexta (Manse-AS). This is the first report on the presence and function of two different peptides with allatotropic activity in an insect species.

Expression of allatostatins in the Mediterranean field cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae)

The allatostatin (AST) type A gene of the cricket Gryllus bimaculatus encodes a hormone precursor including at least 14 putative peptides with a common C-terminus Y/FXFGL/Iamide. By RT-PCR we have analyzed the expression of the allatostatin precursor in various tissues of 0-21 days old adult virgin and mated females. In 3-day-old virgin females, the gene is strongly expressed in the brain (oesophageal ganglion), the suboesophageal ganglion and the caecum, but to a lower extent in other parts of the digestive tract (ileum, midgut, colon), and in various other tissues such as the fat body, ovaries and female accessory reproductive glands. In the brain and ovaries of virgin females, the AST expression is rather constant throughout adult life, whereas in brains of mated animals, expression is low until day 7, but increases sharply from day 8 onwards to reach values triple those before day 7. In ovaries of mated animals AST gene expression is also age-dependent, with high expression rates during the first 4 days after imaginable moult, a second but smaller peak from day 15 to 21, and very low values in between. In the fat body of virgin crickets allatostatin expression is high during the first 9 days after ecdysis and declines thereafter, whereas in mated animals two peak values, day 1 and day 6, are observed, and a third peak in older animals.

Putative-farnesoic acid O-methyltransferase (FAMeT) in medfly reproduction.

A gene potentially involved in juvenile hormone (JH) biosynthesis was previously identified in Ceratitis capitata as the putative-farnesoic acid O-methyltransferase (FAMeT). Since JH is involved in insect reproduction, we silenced the putative-FAMeT expression by RNA interference in Ceratitis capitata to evaluate its implication in egg production. FAMeT gene expression was knocked down in females and males after eclosion and in 1- and 2-day-old females. Treated specimens were left to mate with each other or with untreated partners to evaluate the extent of each sex influencing egg production. Gene silencing was investigated by Real-Time PCR. Results unambiguously showed that FAMeT has a measurable role on the fertility of both medfly sexes.

Neuropeptides affecting the transfer of juvenile hormones from males to females during mating in Spodoptera frugiperda

In the polyandric moth, Spodopterafrugiperda, juvenile hormone (JH) is transferred from the male accessory reproductive glands (AG) to the female bursa copulatrix (BC) during copulation (see Hassanien et al., 2014). Here we used the RNA interference technique to study the role of allatoregulating neuropeptides in controlling the synthesis and transfer of JH during mating. Knockdown of S. frugiperda allatostatin C (Spofr-AS type C) in freshly emerged males leads to an accumulation of JH in the AG beyond that in the control and mating results in a higher transport of JH I and JH II into the female BC. Knockdown of S. frugiperda allatotropin 2 (Spofr-AT2) significantly reduces the amount of JH in the AG as well as its transfer into the female BC during copulation. Knockdown of S. frugiperda allatostatin A (Spofr-AS type A) and S. frugiperda allatotropin (Spofr-AT; Hassanien et al., 2014) only slightly affects the accumulation of JH in the AG and its transfer from the male to the female. We conclude that Spofr-AS type C and Spofr-AT2 act as true allatostatin and true allatotropin, respectively, on the synthesis of JH I and JH II in the male AG. Moreover, both peptides seem to control the synthesis of JH III in the corpora allata of adult males and its release into the hemolymph.