IDENTIFICATION OF THE FGL-AMIDE ALLATOSTATIN GENE OF THE PRIMITIVE TERMITE Mastotermes darwiniensis AND THE WOODROACH Cryptocercus darwini.

Allatostatins with the C-terminal ending Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide (FGLa/ASTs) are widespread neuropeptides with multiple functions. The gene encoding the FGLa/AST polypeptide precursor was first isolated from cockroaches and since then could be identified in many insects and crustaceans. With its strictly conserved regions in combination with variable regions the gene seems to be a good candidate for phylogenetic analyses between closely and distantly related species. Here, the structure of the FGLa/AST gene of the most primitive termite, the giant northern termite Mastotermes darwiniensis Froggatt, was identified. The FGLa/AST gene of the woodroach Cryptocercus darwini was also determined. Precursor sequences of both species possess the general organization of dictyopteran FGLa/AST precursors containing 14 putative FGLa/AST peptides. In M. darwiniensis, only 11 out of the 14 FGLa/AST-like peptides possess the C-terminal conserved region Y/FXFGL/I/V/M and four of the putative peptide structures are not followed by a Gly residue that would lead to nonamidated peptides. Phylogenetic analyses show the high degree of similarity of dictyopteran FGLa/AST sequences. The position of termites, nested within the Blattaria, confirms that termites have evolved from primitive cockroaches.

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.

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

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.

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.

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.

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus.

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.

Functional analysis of the allatostatin-A type gene in the cricket Gryllus bimaculatus and the armyworm Spodoptera frugiperda.

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence specific manner. Here we show, that dsRNA targeting the allatostatin (AS)-A type (FGL/I/V-amide) gene of Gryllus bimaculatus (Ensifera, Gryllidae) and Spodoptera frugiperda (Lepidoptera, Noctuidae) injected into freshly moulted larvae or adult crickets and moths produced a rapid and long-lasting reduction in the mRNA levels in various tissues. The effect lasted up to 7 days. Following dsRNA injection, the juvenile hormone (JH) titers in the hemolymph were clearly raised in both species. AS-dsRNA injection induced a reduced body weight in larval and adult crickets and the imaginal moult was incomplete. Silencing allatostatin type-A expression also reduced the egg and testes development in crickets, and the oviposition rate was drastically diminished in both species.

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.

Type-A allatostatins from the fall armyworm, Spodoptera frugiperda: molecular cloning, expression and tissue-specific localization.

The gene encoding the Spodoptera frugiperda allatostatin type-A peptide family (Y/FXFGL-amides) was isolated from S. frugiperda brain cDNA. The gene encodes a precursor of 231 amino acids containing nine (or ten) Y/FXFGL-a peptides that are tandemly arranged in three blocks. The comparison of the Spofr-AST A precursor with the respective precursor genes from two other lepidopteran species, Helicoverpa armigera and Bombyx mori, shows high homology in size, sequence (84 and 57%, respectively), and organisation of the allatostatins. One-step RT-PCR analysis using a Spofr-AST A-6 to A-9 probe shows that the gene is not only expressed as one transcript in the brain and midgut of larvae and adults in a time- and tissue-specific manner, but also in the reproductive tissues of adult S. frugiperda. Data confirm the nature of the allatostatin type-A peptides as brain/gut myoregulatory hormones, whereas their function(s) in ovaries, oviduct, and testes still have to be resolved. The cell-specific localization of the preprohormone expression, as demonstrated by whole mount in situ hybridization, confirms the overall distribution of the Spofr-AST A preprohormone as shown by RT-PCR and supports the pleiotropic functions of the peptides.

Expression and localization of the Spodoptera frugiperda allatotropin (Spofr-AT) and allatostatin (Spofr-AS) genes.

Manduca sexta allatotropin and allatostatin were the first corpora allata (CA) regulating neuropeptides identified from Lepidoptera. Recently, we cloned the allatotropin (Spofr-AT) and the allatostatin (Spofr-AS) genes from the fall armyworm Spodoptera frugiperda. Using one-step RT-PCR for semi-quantification of the gene expression, we now demonstrate that three mRNA isoforms of the Spofr-AT gene and the Spofr-AS gene are expressed in brain, digestive tract, and reproductive organs of larvae, pupae, and adults in a time- and tissue-specific manner. Expression rates in the brain and in various parts of the digestive tract prove the dual role of the peptides as brain/gut (neuro)peptides. The functional meaning of ovarian and testes expression of the genes is not yet clear, although myoregulatory properties of the peptides are probable. The tissue-specific localization of the prohormone expression, as demonstrated by whole mount in situ hybridization, confirms the overall distribution of the prohormones as shown by RT-PCR and supports the pleiotropic functions of the peptides.