Nuria Pascual

Differential expression of two RXR/ultraspiracle isoforms during the life cycle of the hemimetabolous insect Blattella germanica (Dictyoptera, Blattellidae).

In insects, the molecular basis of ecdysteroid action has been analysed in great detail in flies and moths, but rarely in primitive orders. Using the primitive hemimetabolous insect Blattella germanica, the German cockroach, as a model, we isolated two cDNAs of RXR/USP, a component of the heterodimeric ecdysone receptor. These two cDNAs correspond to two isoforms, named BgRXR-S (short form) and BgRXR-L (long form). Both are identical except for a 23-amino acid deletion/insertion located in the loop between helices H1 and H3 of the ligand-binding domain. Pattern expression studies show that the two isoforms are differentially expressed throughout the life cycle of B. germanica. During embryogenesis, BgRXR-L occurs in early embryos, whereas BgRXR-S is highly expressed in middle and late embryogenesis. In the penultimate and last larval instars, BgRXR-S mRNA is the predominant form in the fat body and in the prothoracic gland. In the adult female, BgRXR-S mRNA predominates in the fat body, whereas BgRXR-L mRNA predominates in the ovary. Experiments performed with fat body and embryo cells incubated in vitro showed that the expression of BgRXR-S and BgRXR-L is not affected by 20-hydroxyecdysone or by juvenile hormone III.

The nuclear hormone receptor BgE75 links molting and developmental progression in the direct-developing insect Blattella germanica

Ecdysteroid hormones regulate key developmental processes throughout the life cycle of insects. 20-Hydroxyecdysone (20E) acts upon binding to a heterodimeric receptor formed by the nuclear receptors EcR and USP. The receptor, once 20E bounds to it, elicits cascades of gene expression that mediate and amplify the hormonal signal. The molecular characterization of the 20E-mediated hierarchy of transcription factors has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in more basal hemimetabolous species. Using the hemimetabolous species Blattella germanica (German cockroach) as model, we have cloned and characterized five isoforms of B. germanica E75, a member of the nuclear receptor family participating in the 20E-triggered genetic hierarchy. The five isoforms present characteristic expression patterns during embryo and nymphal development, and experiments in vitro with fat body tissue have shown that the five isoforms display specific 20E responsiveness. RNAi experiments in vivo during the penultimate and last nymphal instars of B. germanica revealed that BgE75 is required for successfully complete nymphal-nymphal and nymphal-adult transitions. Detailed analysis of knockdown specimens during the last nymphal instar showed that BgE75 is required for the rise of circulating ecdysteroids that occurs towards the end of the instar. The main cause of ecdysteroid deficiency in BgE75 knockdowns is the premature stage-specific degeneration of the prothoracic gland. As a consequence, BgE75 knockdown nymphs do not molt, live for up to 90 days and start the adult developmental program properly, in spite of remaining as nymphs from a morphological point of view. Finally, RNAi of specific isoforms during the last nymphal instar of B. germanica has showed that they are functionally redundant. Furthermore, it also revealed the occurrence of a complex regulatory relationship among BgE75 isoforms, which is responsible of their sequential expression.

Juvenile Hormone Titer Versus Juvenile Hormone Synthesis in Female Nymphs and Adults of the German Cockroach, Blattella germanica

Abstract Patterns of juvenile hormone have been intensively studied in the cockroach Blattella germanica under different physiological situations. However, data have been mainly obtained in vitro, and refer to hormone synthesized by isolated corpora allata, whereas information available on hormone concentration in the hemolymph is restricted to adult females. In order to complement our studies in vitro, we have measured juvenile hormone titer in the hemolymph of B. germanica females in four characteristic physiological situations: penultimate and last instar nymphs, adults during the first vitellogenic cycle, and adults transporting egg cases (ootheca). In general, a significant positive correlation between rates of hormone synthesis and concentration in the hemolymph is observed. The main disparities appear in the penultimate day of the period of ootheca transport, where titer is high whereas synthesis is low, and on day 6 of the first vitellogenic cycle, where synthesis increases whereas titer decreases. At these stages, the observed disparities between synthesis and titer might be explained by differential action of degradation enzymes.

Allatostatin gene expression in brain and midgut, and activity of synthetic allatostatins on feeding-related processes in the cockroach Blattella germanica.

Allatostatins of the YXFGLamide group were discovered in cockroaches through their capacity to inhibit juvenile hormone biosynthesis. Here, we assess the occurrence of preproallatostatin (preproAST) mRNA in the brain and midgut of adult females of the cockroach Blattella germanica, and estimate brain and midgut preproAST mRNA levels during the first reproductive cycle. Reverse transcription polymerase chain reaction (RT-PCR) shows that brain preproAST mRNA levels increase slightly during the gonadotrophic cycle, and remain high during ootheca transport. In the midgut, preproAST mRNA levels decline around the middle of the gonadotrophic cycle. The pattern of allatostatin expression in gut tissues suggests that these peptides play roles related to feeding and nutrition. Our results have shown that synthetic allatostatins inhibit hindgut motility and activate midgut alpha-amylase secretion. In addition, injected allatostatins inhibit food consumption, which might be connected to the above activities.

Synthesis of 25-fluoroponasterone A, a fluorinated analogue of 20-hdyroxyecdysone.

Abstract Very precise reaction conditions, using mild fluorination procedures combined with acetonide deprotection, were developed for preparation of 25-fluoroponasterone A from 20-hydroxyecdysone. Biological activity of the new compound was examined and the possible relationship between structure and biological activity in ecdysteroids was studied using MMP2 calculations of the conformations of the side chain of three related compounds: ecdysone, 20-hydroxyecdysone and 25-fluoroponasterone A.

Antifeeding properties of myosuppressin in a generalist phytophagous leafworm, Spodoptera littoralis (Boisduval)

Insect myosuppressins are a family of peptides with a characteristic HV/SFLRFamide carboxy terminus. They are expressed in brain, neurohemal organs, stomatogastric nervous system, and in midgut endocrine cells. From a functional point of view, myosuppressins inhibit contractions of different visceral muscles, stimulate certain skeletal muscles and activate enzyme secretion from the gut. Moreover, in the omnivorous cockroach Blattella germanica, myosuppressin inhibits food intake. Based on these results, we studied the antifeeding activity of myosuppressin in the phytophagous leafworm Spodoptera littoralis. Firstly, we isolated the cDNA corresponding to the S. littoralis myosuppressin precursor encoding the typical myosuppressin peptide of lepidopterans: pQDVVHSFLRFamide. Then, we determined the expression patterns (in terms of mRNA and peptide) of myosuppressin in brain and midgut, and peptide levels in the haemolymph. Myosuppressin patterns in the brain and haemolymph were similar, and symmetrical to that of food consumption, thus suggesting that myosuppressin might inhibit feeding in S. littoralis. Moreover, synthetic myosuppressin effectively inhibited food intake in non-choice antifeeding tests. Taken together, the obtained results point to the hypothesis that myosuppressin represses feeding in S. littoralis.

Juvenile hormone and allatostatins in the German cockroach embryo

Levels of juvenile hormone III (JH), FGLamide allatostatin peptides (ASTs), ASTs precursor (preproAST) mRNA and methyl farnesoate epoxidase (CYP15A1) mRNA were measured in embryos of the cockroach Blattella germanica. JH starts to rise just after dorsal closure, reaches maximal levels between 60% and 80% of embryogenesis, and decrease subsequently to undetectable levels. ASTs show low levels during the first two thirds of embryogenesis, increase thereafter and maintain high levels until hatching. PreproAST mRNA shows quite high levels during the two days following oviposition, thus behaving as a maternal transcript, the levels then become very low until mid embryogenesis, and increase afterwards, peaking towards the end of embryo development. CYP15A1 transcripts were detected around 25% embryogenesis and the levels tended to increase through embryogenesis, although differences amongst the days studied were not statistically significant. The opposite patterns of JH and AST towards the end of embryo development, along with the detection of AST immunoreactivity in corpora allata from late embryos, suggest that JH decline is caused by the increase of AST. Moreover, the uncorrelated patterns of JH concentration and CYP15A1 mRNA levels suggest that CYP15A1 expression does not modulate JH production.

Ecdysteroid depletion by azadirachtin in Tenebrio molitor pupae.

Abstract The injection of 1 μg of azadirachtin into freshly ecdysed pupae of Tenebrio molitor induced a significant depletion of levels of immunoreactive ecdysteroids, suppressing the ecdysteroid peak that normally appears at the middle of the instar. Combined high-performance liquid chromatography-enzyme immunoassay studies have revealed that this depletion mainly affects 20-hydroxyecdysone levels, whereas ecdysone levels remain practically unchanged in comparison with controls.

Identification of a tachykinin-related peptide with orexigenic properties in the German cockroach.

A number of evidences suggest that tachykinin-related peptides (TRPs) of insects can stimulate food consumption after being released from the midgut to the hemolymph. The idea of the present work has been to test this hypothesis in the anautogenous cockroach Blattella germanica. First, we have identified the peptide LemTRP-1 (APSGFLGVR-NH(2)) from brain extracts, by means of an ELISA developed with a polyclonal antibody against this peptide. ELISA studies have also shown that, whereas brain LemTRP-1 levels were fairly constant, midgut levels increase to a maximum on day 3 after adult emergence, falling thereafter until the end of the gonadotrophic cycle. Interestingly, maximum values of food consumption are concomitant with the decrease of LemTRP-1 immunoreactivity in the midgut. Furthermore, starvation decreases LemTRP-1 immunoreactivity in midgut, whereas in the hemolymph it increases. Finally, injection of synthetic LemTRP-1 to adult females significantly stimulates food consumption. The whole observations suggest that LemTRP-1 is released from the midgut to the hemolymph when sustained food consumption is required to maintain vitellogenesis at the highest levels, and that LemTRP-1 in the hemolymph stimulates food consumption in these days.

A microdialysis study of allatostatin degradation in Blattella germanica (L.) (Dictyoptera, Blattellidae)

Allatostatins with a typical C‐terminal sequence YXFGL‐NH2 are insect neuropeptides with inhibitory properties upon Juvenile Hormone production in the corpora allata, vitellogenin release by the fat body, and gut and dorsal vessel motility. All these biological effects are rapidly reversible, suggesting the occurrence of effective mechanisms for inactivation of the peptides. We have studied the degradation of DRLYSFGL‐NH2 (BLAST‐2), one of the allatostatins of Blattella germanica, in the internal milieu of adult females of this cockroach. The experimental approach combined the use of the radioiodinated derivative [125I‐Tyr4]BLAST‐2, microdialysis techniques and HPLC analysis with a radioisotope detector. Under these experimental conditions, the half‐life of BLAST‐2 in the internal milieu of the adult female of B. germanica was between 3 and 6 min. Such a short half‐life explains the high doses of allatostatins required to obtain the expected biological effects when tested in vivo, and suggests that circulating allatostatins are subject to rapid rates of synthesis and degradation in order to be operative physiologically.