Lluïsa Vilaplana

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.

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.

Density-related variation in vertical transmission of a virus in the African armyworm

Larvae of the African armyworm, Spodoptera exempta, are darker and more resistant to baculovirus infection when reared in groups (gregarious form) compared to being reared singly (solitary form). Lepidoptera that survive virus challenge as larvae could potentially retain a sublethal virus infection which is then transmitted vertically to the next generation. Here we examine whether gregarious and solitary forms of the armyworm differ in the costs of surviving virus infection and in their capacity to transmit an active baculovirus infection to their offspring. Pupae of larvae reared gregariously that survived virus challenge weighed significantly less than uninfected individuals, but this was not so for those reared solitarily. This did not, however, translate into differences in fecundity, at least under laboratory conditions. As found in previous studies, pre-oviposition period was shorter for solitary than gregarious insects, and it was also shorter for females that had been challenged with virus as larvae. Both the prevalence of egg batches containing larvae that died from nucleopolyhedrovirus (NPV) infection and the proportion of infected larvae within each egg batch were significantly increased (approximately doubled) when parental moths were previously challenged with the virus during their larval state. This demonstrates that horizontal transmission in one generation can elevate vertical transmission to the next generation. Moreover, prevalence of overt infection in the offspring generation was two to three times greater when parental moths were reared solitarily as larvae than when reared gregariously. Disease prevalence and proportional infection were both independent of the sex of the infected parent and whether or not the egg batch was surface-sterilized to remove potential contaminants. This suggests that the eggs are infected internally (transovarial) rather than externally (transovum). These results help to shed light on the observed temporal pattern of virus epizootics in eastern Africa.

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.

Determination of allatostatin levels in relation to the gonadotropic cycle in the female of Blattella germanica (L.) (Dictyoptera, Blattellidae)

A polyclonal antibody against the allatostatin BLAST‐3 (AGSDGRLYSFGL‐NH2) of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) has been raised and characterized, and an ELISA (enzyme‐linked immunosorbent assay) for allatostatin quantification has been developed. Allatostatin contents in brain, midgut and haemolymph have been measured in females of B. germanica during the first gonadotropic cycle. Brain allatostatin content increases steadily from adult emergence to the formation of the first ootheca. The values range from 2 ng/brain on the day of adult emergence to 25 ng/brain when the insect forms the ootheca 8 days later. In the midgut, the pattern is similar but the values are about half those of the brain. Allatostatin concentrations in the haemolymph after HPLC separation are in the nanomolar range. The occurrence of allatostatins in the haemolymph suggests that these peptides can act through a humoral pathway, as well as via nerves. The allatostatin content of both brain and midgut are high while the female is transporting the ootheca, which suggests that these peptides could be related to the low metabolic status characterising the period of oothecal transport.

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.

INHIBITION OF JUVENILE HORMONE DURING THE FORMATION OF THE SPERMATOPHORE IN BLATTELLA GERMANICA (L.) (DICTYOPTERA, BLATTELLIDAE)

Allatectomy showed that Juvenile Hormone (JH) induced the accumulation of proteins in the accessory reproductive glands (ARC) of male Blattella germanica. During mating, the formation of the spermatophore and its transfer to the female was observed, and a selective depletion of most ARG proteins occurred in parallel, as shown by electrophoretic studies carried out at selected moments of the process. The synthetic activity of corpora allata incubated in vitro, measured at the same times, indicated that JH production is inhibited during the formation and transfer of the spermatophore. It is suggested that this inhibition may be necessary to start a new cycle of ARG maturation. @ 1996 LVliey-Liss, Inc.

The conglobate gland of Blattella germanica (L.) (Dictyoptera, Blattellidae). Maturation, juvenile hormone dependency and changes during spermatophore formation

Summary The conglobate gland of male cockroaches is a reproductive organ anatomically close to the accessory glands. From a functional point of view it has been postulated that the conglobate gland is involved in the formation of the spermatophore, although no clear demonstration of this role has been published so far. In addition, previous endocrinological studies have suggested that juvenile hormone might influence conglobate gland maturation. In the present work, taking the conglobate gland of the cockroach Blattella germanica as a model, we have studied the protein pattern during maturation, the effects of allatectomy, and the depletion of gland proteins during the formation of the spermatophore. Taken together, the results suggest that juvenile hormone stimulates the accumulation of proteins in the conglobate gland and that these proteins contribute to the formation of the spermatophore.