Juvenile hormone signaling in short germ-band hemimetabolan embryos.

The role of juvenile hormone (JH) in insect embryos is far from understood, especially in short germ-band hemimetabolan species. To shed light on this issue, we depleted the mRNA levels of Krüppel homolog 1, Methoprene-tolerant and JH acid O-methyltransferase, key elements of JH signaling, in embryos of the short germ-band hemimetabolan species Blattella germanica This precluded the formation of the germ-band anlage in a group of embryos. Hatchability was also reduced, which might have been caused by premature upregulation of laccase 2, a promoter of cuticle tanning. In other cases, development was interrupted in mid embryogenesis, involving defects related to dorsal closure and appendage formation. These phenotypes possibly result from the low levels of Broad-complex (BR-C) produced under JH-depleted conditions. This contrasts with holometabolan species, in which JH does not promote BR-C expression, which remains low during embryo development. Possibly, the stimulatory role of JH on BR-C expression and the morphogenetic functions of BR-C in hemimetabolan embryos were lost in holometabolan species. If so, this might have been a key driver for the evolution of holometabolan metamorphosis.

SPARC preserves follicular epithelium integrity in insect ovaries.

The importance of juvenile hormone regulating insect oogenesis suggests looking for genes whose expression is regulated by this hormone. SPARC is a calcium-binding glycoprotein that forms part of the extracellular membranes, which in vertebrates participates in bones mineralization or regulating cell proliferation in some cancer types. This large number of functions described for SPARC in different species might be related to the significant differences in its structure observed when comparing different species-groups. Indeed, these structural differences allow characterizing the different clades. In the cockroach Blattella germanica, a SPARC homolog emerged from ovarian transcriptomes that were constructed to find genes responding to juvenile hormone. In insects, SPARC functions have been studied in oogenesis and in embryo development of Drosophila melanogaster. In the present work, using RNAi approaches, novel functions for SPARC in the B. germanica panoistic ovaries are described. We found that depletion of SPARC does not allow to the follicular cells to complete mitosis, resulting in giant follicular cells nuclei and in a great alteration of the ovarian follicle cytoskeleton. The SPARC contribution to B. germanica oogenesis occurs stabilizing the follicular cell program and helping to maintain the nuclear divisions. Moreover, SPARC is necessary to maintain the cytoskeleton of the follicular cells. Any modification of these key processes disables females for oviposition.

Diversity of piRNA expression patterns during the ontogeny of the German cockroach.

The Piwi-interacting RNA (piRNA) system is an evolutionarily conserved mechanism involved in the control of transposable elements and maintenance of genomic stability, especially in germ line cells and in early embryo stages. However, relevant particularities, both in mechanism and function, exist across species among metazoans and even within the insect class. As a member of the scarcely studied hemimetabolan group, Blattella germanica can be a suitable reference model to study insect evolution. We present the results of a stringent process of identification and study of expressed piRNAs for B. germanica across 11 developmental stages, ranging from unfertilized egg to nymphs and adult female. Our results confirm the dual origin of piRNA in this species, with a majority of them being generated from the primary pathway, and a smaller but highly expressed set of sequences participating in the secondary ("ping-pong") reamplification pathway. An intriguing partial complementarity in expression is observed between the piRNA of the two biogenesis pathways, with those generated in the secondary pathway being quite restricted to early embryo stages. In addition, many piRNAs are exclusively expressed in late embryo and nymphal stages. These observations point at piRNA functions beyond the role of transposon control in early embryogenesis. Our work supports the view of a more complex scenario, with different sets of piRNAs acting in different times and having a range of functions wider than previously thought.

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germ-band types.

BACKGROUND: Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. RESULTS: We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. CONCLUSION: miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

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.

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.

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.

The structural basis for water exchange between the female cockroach (Blattella germanica) and her ootheca.

Female German cockroaches usually carry their oothecae until they hatch. The success of embryogenesis may be dependent on the water-balance relationship between females and their developing oothecae. Oothecae detached from females early in embryogenesis often fail to develop, especially in low-humidity environments. Experiments reported here using tritiated water have confirmed the transport of a significant amount of water to the ootheca from the female during embryogenesis; 18% of the tritiated water injected into gravid females was recovered in their oothecae after 24 h. We describe a structural basis of water absorption by the oothecae. An area located on the proximal end of the ootheca (adjacent to the 'escutcheon-shaped vaginal imprint') contains small pores that penetrate the oothecal covering to access regions of the chorion lying beneath these pores. Experiments using microparabiotic chambers to examine transport of tritiated water and water-soluble materials across the escutcheon region of the oothecal covering, along with dark-field birefringent microscopy of the chorion, support the hypothesis that this chorionic network is capable of conducting water throughout the interior of the ootheca. Furthermore, the structural arrangement and intimate association of the female vestibulum with the oothecal pore field contained in the region of the escutcheon-shaped vaginal imprint appear to provide an efficient conduit to the chorion. The overall structural relationship might be a means for maintaining water balance between females and their oothecae during embryonic development. Evidence presented here supports observations that this species represents an important link in the transition from oviparity to ovoviviparity by internalization of cockroach oothecae.

Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body.

A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.