Maria-Dolors Piulachs

The vitellogenin of the honey bee, Apis mellifera : structural analysis of the cDNA and expression studies

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

Allatostatic neuropeptides from the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Identification, immunolocalization and activity

Four allatostatic neuropeptides were isolated from extracts of the brain of the cockroach Blattella germanica. The primary structures of these peptides were assigned as Leu-Tyr-Asp-Phe-Gly-Leu-NH2 (BLAST-1), Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-2), Ala-Gly-Ser-Asp-Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-3) and Ala-Pro-Ser-Ser-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (BLAST-4). Each of the peptides showed C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The four peptides inhibited in vitro juvenile hormone production by corpora allata from virgin females of B. germanica. Immunoreactivity against allatostatins was seen in the lateral neurosecretory neurons and in the axonal pathway leading to the corpora allata.

Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant

During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)‐mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre‐adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.

Inhibition of vitellogenin production by allatostatin in the German cockroach

Allatostatins with a typical YXFGL-amide C-terminus constitute a neuropeptide family, which was discovered because of its inhibitory action on insect juvenile hormone synthesis. In the search for possible new functions for allatostatins we focused our attention on the fat body. Our previous studies on the cockroach Blattella germanica suggested the occurrence of factors terminating vitellogenesis, and the hypothesis here was that allatostatins might be one of these factors. Our experiments have shown that allatostatin impaired vitellogenin release in fat bodies incubated in vitro, and that this effect appears to be mediated by the inhibition of vitellogenin glycosylation. Fluvastatin also inhibited vitellogenin release, and mevalonolactone counteracted the inhibitory effects of allatostatin. These results suggest that allatostatin acts upon the mevalonate pathway and synthesis of dolichol, which would explain the inhibition of vitellogenin glycosylation. We finally conclude that allatostatins may effectively contribute to the termination of the vitellogenic cycle in B. germanica.

Deep Sequencing of Organ- and Stage-Specific microRNAs in the Evolutionarily Basal Insect Blattella germanica (L.) (Dictyoptera, Blattellidae)

Background microRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs. Methodology/Principal Findings Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. Conclusions/Significance Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

In vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica (L)

Abstract A radiochemical assay which fulfills the required validation criteria has been used for quantification of the in vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica throughout the 7 days of the first reproductive cycle. The presence of JH III has been independently confirmed by HPLC and mass spectrometry. Results indicate that rates of JH release increase repidly from day 3 to day 6, which is correlated with oocyte growth. The highest levels of JH release (2.58 ± 1.11 pmol/hr per pair) were obtained from day-6 females. The time course of JH production by CA from day-6 females showed that CA released JH at a linear rate for at least 9 hr. From these results, it can be concluded that titers at high production ages and linearity ranges are satisfactory enough to be used in studies on the regulation of JH production in this species.

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.

Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae)

The present paper describes the effect of juvenile hormone III (JH III) upon vitellogenin (Vg) gene expression in cardioallatectomized females of Blattella germanica. Northern blot analyses of time course studies showed that Vg mRNA can be detected 2 h after the treatment with 1 microgram of JH III. Western blot analyses revealed that Vg protein is detectable 4 h after the same treatment. The study of the influence of the age showed that 48-h-old females seem more sensitive than 24-h-old females, whereas differences were less apparent between 48- and 72-h-old females. Dose-response studies indicated that 0.01 microgram of JH III is ineffective, whereas the doses of 0.1, 1 and 10 micrograms induced the synthesis of Vg in a dose-dependent fashion. Finally, the administration of three successive doses, of 0.01 microgram of JH III each, did not result in detectable Vg production, whereas two doses of 0.01 microgram followed by one of 1 microgram of JH III induced a greater response than that resulting from a sole dose of 1 microgram of JH III, which suggests that sub-effective doses of JH III elicit a priming effect on Vg production.

Induction of vitellogenin gene transcription in vitro by juvenile hormone in Blattella germanica.

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10(-7) or 10(-8) M), partially inhibited the response to JH III.