David Martín

Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity

Elucidation of molecular mechanisms underlying stage- and tissue-specific expression of genes activated by a blood meal is of great importance for current efforts directed towards utilizing molecular genetics to develop novel strategies of mosquito and pathogen control. Regulatory regions of such genes can be used to express anti-pathogen effector molecules in engineered vectors in a precise temporal and spatial manner, designed to maximally affect a pathogen. The fat body is a particularly important target for engineering anti-pathogen properties because in insects, it is a potent secretory tissue releasing its products to the hemolymph, an environment or a crossroad for most pathogens. Recently, we have provided proof of this concept by engineering stable transformant lines of Aedes aegypti mosquito, in which the regulatory region A. aegypti vitellogenin (Vg) gene activates high-level fat body-specific expression of a potent anti-bacterial factor, defensin, in response to a blood meal. Further study of the Vg gene utilizing Drosophila and Aedes transformation identified cis-regulatory sites responsible for state- and fat body-specific activation of this gene via a blood-meal-triggered cascade. These analyses revealed three regulatory regions in the 2.1-kb upstream portion of the Vg gene. The proximal region, containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh, is required for the correct tissue- and stage-specific expression at a low level. The median region, carrying sites for early ecdysone response factors E74 and E75, is responsible for a stage-specific hormonal enhancement of the Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic to the Vg gene. Furthermore, our study showed that several transcription factors involved in controlling the Vg gene expression, are themselves targets of the blood meal-mediated regulatory cascade, thus greatly amplifying the effect of this cascade on the Vg gene. This research serves as the foundation for the future design of mosquito-specific expression cassettes with predicted stage- and tissue specificity at the desired levels of transgene expression.

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.

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.

bantam miRNA Promotes Systemic Growth by Connecting Insulin Signaling and Ecdysone Production

During the development of multicellular organisms, body growth is controlled at the scale of the organism by the activity of long-range signaling molecules, mostly hormones. These systemic factors coordinate growth between developing tissues and act as relays to adjust body growth in response to environmental changes [1]. In target organs, long-range signals act in concert with tissue-autonomous ones to regulate the final size of a given tissue. In Drosophila, the steroid hormone ecdysone plays a dual role: peaks of secretion promote developmental transitions and maturation, while basal production negatively controls the speed of growth. The antagonistic action of ecdysone and the conserved insulin/insulin growth factor (IGF) signaling pathway regulate systemic growth and modulate final body size [2, 3]. Here we unravel an unexpected role of bantam microRNA in controlling body size in Drosophila. Our data unveil that, in addition to its well-characterized function in autonomously inducing tissue growth [4-9], bantam activity in ecdysone-producing cells promotes systemic growth by repressing ecdysone release. We also provide evidence that the regulation of ecdysone production by insulin signaling relies on the repression of bantam activity. These results identify a molecular mechanism that underlies the crosstalk between these two hormones and add a new layer of complexity to the well-characterized role of bantam in growth control.

Patterns of haemolymph vitellogenin and ovarian vitellin in the German cockroach, and the role of Juvenile Hormone

Abstract. The concentrations of fat body and haemolymph vitellogenin and ovarian vitellin during the first gonadotropic cycle of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) have been studied. For these purposes, a polyclonal antibody against B. germanica vitellogenin and vitellin has been obtained, and an ELISA to quantify these proteins has been developed. Ovarian vitellin levels follow a pattern which parallels those of basal oocyte growth and Juvenile Hormone production by the corpora allata. This suggests that Juvenile Hormone regulates vitellogenin uptake into oocytes. Fat body and haemolymph vitellogenin levels give cyclic and parallel patterns. However, the cycle of Juvenile Hormone appears delayed with respect to that of vitellogenin. We suggest that the production of Juvenile Hormone, although cyclic in profile, does not modulate alone the cycle of vitellogenin. At least a supplementary mechanism, apparently independent of Juvenile Hormone, may be involved in the decline of vitellogenin production at the end of the vitellogenic cycle.

Nuclear receptor BgFTZ‐F1 regulates molting and the timing of ecdysteroid production during nymphal development in the hemimetabolous insect Blattella germanica

Postembryonic development of holometabolous and hemimetabolous insects occurs through successive molts triggered by 20‐hydroxyecdysone (20E). The molecular action of 20E has been extensively studied in holometabolous insects, but data on hemimetabolous are scarce. We have demonstrated that during the nymphal development of the hemimetabolous insect Blattella germanica, 20E binds to the heterodimeric receptor formed by the nuclear receptors BgEcR‐A and BgRXR activating a cascade of gene expression, including the nuclear receptors BgE75 and BgHR3. Herein, we report the characterization of BgFTZ‐F1, another nuclear hormone receptor involved in 20E action. BgFTZ‐F1 is activated at the end of each instar, and RNAi has demonstrated that BgHR3 is needed for BgFTZ‐F1 activation, and that BgFTZ‐F1 has critical functions of during the last nymphal instar. Nymphs with silenced BgFTZ‐F1 cannot ecdyse, arrest development, and show structures of ectodermal origin duplicated. BgFTZ‐F1 also controls the timing of the ecdysteroid molting pulse. Developmental Dynamics 237:3179–3191, 2008.

Redundant ecdysis regulatory functions of three nuclear receptor HR3 isoforms in the direct-developing insect Blattella germanica

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.

Ketomethylene and Methyleneamino Pseudopeptide Analogues of Insect Allatostatins Inhibit Juvenile Hormone and Vitellogenin Production in the Cockroach Blattella germanica

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino psi [CH2NH] and ketomethylene psi [COCH2] peptide bond surrogates have been introduced at the position Leu3-Tyr4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu3-Phe4 of [Phe4]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.