Smads and insect hemimetabolan metamorphosis.

In contrast with Drosophila melanogaster, practically nothing is known about the involvement of the TGF-ß signaling pathway in the metamorphosis of hemimetabolan insects. To partially fill this gap, we have studied the role of Smad factors in the metamorphosis of the German cockroach, Blattella germanica. In D. melanogaster, Mad is the canonical R-Smad of the BMP branch of the TGF-ß signaling pathway, Smox is the canonical R-Smad of the TGF-ß/Activin branch and Medea participates in both branches. In insects, metamorphosis is regulated by the MEKRE93 pathway, which starts with juvenile hormone (JH), whose signal is transduced by Methoprene-tolerant (Met), which stimulates the expression of Krüppel homolog 1 (Kr-h1) that acts to repress E93, the metamorphosis trigger. In B. germanica, metamorphosis is determined at the beginning of the sixth (final) nymphal instar (N6), when JH production ceases, the expression of Kr-h1 declines, and the transcription of E93 begins to increase. The RNAi of Mad, Smox and Medea in N6 of B. germanica reveals that the BMP branch of the TGF-ß signaling pathway regulates adult ecdysis and wing extension, mainly through regulating the expression of bursicon, whereas the TGF-ß/Activin branch contributes to increasing E93 and decreasing Kr-h1 at the beginning of N6, crucial for triggering adult morphogenesis, as well as to regulating the imaginal molt timing.

CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis.

BACKGROUND: CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-ß signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway. METHODS AND RESULTS: Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-ß, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-ß pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described here for the first time. CONCLUSIONS: Studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling. GENERAL SIGNIFICANCE: Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.

Embryonic development of Eucorydia yasumatsui Asahina, with special reference to external morphology (Insecta: Blattodea, Corydiidae).

As the first step in the comparative embryological study of Blattodea, with the aim of reconstructing the groundplan and phylogeny of Dictyoptera and Polyneoptera, the embryonic development of a corydiid was examined and described in detail using Eucorydia yasumatsui. Ten to fifteen micropyles are localized on the ventral side of the egg, and aggregated symbiont bacterial "mycetomes" are found in the egg. The embryo is formed by the fusion of paired blastodermal regions, with higher cellular density on the ventral side of the egg. This type of embryo formation, regarded as one of the embryological autapomorphies of Polyneoptera, was first demonstrated for "Blattaria" in the present study. The embryo undergoes embryogenesis of the short germ band type, and elongates to its full length on the ventral side of the egg. The embryo undergoes katatrepsis and dorsal closure, and then finally, it acquires its definitive form, keeping its original position on the ventral side of the egg, with its anteroposterior axis never reversed throughout development. The information obtained was compared with that of previous studies on other insects. "Micropyles grouped on the ventral side of the egg" is thought to be a part of the groundplan of Dictyoptera, and "possession of bacteria in the form of mycetomes" to be an apomorphic groundplan of Blattodea. Corydiid embryos were revealed to perform blastokinesis of the "non-reversion type (N)", as reported in blaberoid cockroaches other than Corydiidae ("Ectobiidae," Blaberidae, etc.) and in Mantodea; the embryos of blattoid cockroaches (Blattidae and Cryptocercidae) and Isoptera undergo blastokinesis of the "reversion type (R)," in which the anteroposterior axis of the embryo is reversed during blastokinesis. Dictyopteran blastokinesis types can be summarized as "Mantodea (N) + Blattodea [= Blaberoidea (N) + Blattoidea (R) + Isoptera (R)]".

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.

Cuticular hydrocarbon synthesis and its maternal provisioning to embryos in the viviparous cockroach Diploptera punctata.

Embryos of the viviparous cockroach Diploptera punctata accumulate large amounts of hydrocarbon (HC) of either maternal or embryonic origin. HC synthesis and its accumulation in maternal and embryonic tissues were measured over the course of gestation. Female abdominal integument was the only tissue that synthesized appreciable amounts of HC in vitro, and did so at an increasing rate from the time of mating to mid-pregnancy, when rates of synthesis declined. The embryos synthesized HC at rates <1% those of the female, showing that the majority of HC detected in and on embryos was of maternal origin. The brood sac that houses the developing embryos did not synthesize HC in vitro, indicating that HC must be transported from the female abdominal integument to the embryos. The mass of female epicuticular HC was constant at approximately 183 microg, while her internal HC increased fourfold from mating to mid-pregnancy, then declined until parturition. The decline in internal HC reflected both declining HC synthesis in the female and greater export to the embryos, as embryonic internal HC increased 250-fold prior to parturition. An external HC coating over the oothecal covering and chorion of the embryos increased to mid-pregnancy, then declined. Unlike oviparous cockroaches, D. punctata females fed throughout the reproductive cycle, reflecting the nutritional demands of continuously provisioning the developing embryos.

Formation of cockroach interganglionic connectives: an in vitro analysis.

Individual ganglia of the cockroach embryo (Periplaneta americana) were explanted on clean glass coverslips immersed in a chemically defined liquid medium and incubated for periods up to eight weeks. Substantial, straight interganglionic connections were formed between: (1) rows of ganglia arranged in the normal in vivo configuration; (2) rows of ganglia placed in abnormal orders; (3) rows of ganglia which never form connections in vivo because they occur singly in the embryo; and (4) rows of ganglia in natural sequences but which have had their rostro-caudal axes rotated 90 degrees in relation to the line of the row. Therefore fascicles and interganglionic connectives were formed without regard to normal in vivo relationships. Daily observations with a Nomarski microscope indicated that several processes are involved in connective formation. (1) Initial outgrowth is in a random, radial pattern. (2) Intersecting fibers from adjacent ganglia are deflected toward each others' perikarya. (3) Initially bowed fiber connectives are straightened, perhaps by increases in fiber tension or by fiber shortening which may be brought about by neuronal or extraneuronal (glial) processes. (4) Outgrowing fibers follow already established fiber pathways. The present results indicate that fiber-fiber and fiber-target interactions play a significant role in the formation of interganglionic connectives. In this system, the spatial relationships between ganglia determine the patterns and varieties of permissible neuronal connections. Thus, major, straight nerve trunks may be formed between adjacent ganglia which are growing out fibers on a glass surface submerged in a liquid medium which offers minimal orientation cues and provides a growth substrate vastly different and simpler than that encountered by outgrowing fibers in vivo.

Cytochrome c oxidase subunit I from the cockroach Blattella germanica: cloning, developmental pattern and tissue expression.

The nucleotide sequence of a cDNA corresponding to the transcription product of the mitochondrial structural gene cytochrome c oxidase subunit I was determined. A polypeptide of 508 residues was deduced from the reading frame established by the nucleotide sequence. TGA codes tryptophan, as in most other mitochondrial systems. From the comparison of the amino acid sequence of the putative Blattella germanica cytochrome c oxidase with those of Drosophila, we conclude that ATA and AGA codons specify methionine and serine respectively, instead of isoleucine and arginine. The sequence proposed for cytochrome c oxidase subunit I of B. germanica is largely homologous to that of other species. From the alignment of cytochrome c oxidase subunit I protein sequences we have found that 125 residues (positional identity of 22.3%) have remained invariant in this enzyme for more than one billion years of divergence. There is a developmental pattern of gene expression affecting the embryo stages. Northern blot analysis of RNA samples from different adult female tissues shows high cytochrome c oxidase subunit I mRNA levels in gut and fat body, and lower levels in ovary and colleterial glands.

Overexpression of the cytochrome c oxidase subunit I gene associated with a pyrethroid resistant strain of German cockroaches, Blattella germanica (L.).

A cytochrome c oxidase subunit I gene (COXI) was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains using PCR-selected subtractive hybridization and cDNA array techniques. The cDNA sequence of COXI has an open reading frame of 1533 nucleotides encoding a putative protein of 511 amino acid residues. Northern blot analysis indicated that levels of COXI expression were similar in three life stages (eggs, nymphs, and adults) of the susceptible ACY strain. The expression of COXI in the resistant Apyr-R strain was developmentally regulated, with low expression in eggs, an increase (approximately 1.4-fold) in nymphs, and rose to a maximum (approximately 3-fold) in both adult females and males. Comparison of COXI expression between ACY and Apyr-R strains indicated that there was no difference in the eggs of the two strains, but expression was higher (approximately 1.5-fold) in nymphs and much higher (approximately 3- to 4-fold) in adult males and females of the Apyr-R strain. The levels of COXI mRNA showed about 1.4- and 1.7-fold increase in the abdomen tissues compared with the head+thorax tissues of ACY and Apyr-R strains, respectively. Although expression patterns of COXI in head+thorax and abdomen tissues were similar (i.e. lower in the head+thorax tissues and higher in the abdomen tissues) in both the ACY and Apyr-R strains, the expression of COXI was about 2.5-fold higher in the head+thorax and approximately 3-fold higher in the abdomen tissues of the Apyr-R strain compared with the corresponding ACY samples. The overexpression of COXI in resistant German cockroaches merits the investigation of the importance of the gene in insecticide resistant German cockroaches.

Methyl farnesoate and juvenile hormone production in embryos of Diploptera punctata in relation to innervation of corpora allata and their sensitivity to allatostatin.

Corpora allata (CA) of embryos of Diploptera punctata have been previously shown to produce JH III. We have re-examined sesquiterpenoid biosynthesis throughout embryonic development and have found that early embryos produce both methyl farnesoate (MF) and JH III; as development proceeds, less MF and more JH is produced. The cockroach allatostatin peptide Dippu-allatostatin (AST) 7 inhibits sesquiterpenoid production by CA of mid to late embryos whereas it exerts a dose-dependent stimulatory effect in early embryos. This stimulatory effect is particularly apparent on MF biosynthesis. CA become innervated by allatostatin-containing nerves in early embryos (35% development). Shortly thereafter, the allatostatin-containing innervation of the CA appears complete.

Allatostatin in ovaries, oviducts, and young embryos in the cockroach Diploptera punctata.

The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.

Oenocyte differentiation correlated with the formation of ectodermal coating in the embryo of a cockroach.

The first signs of 'embryonic membrane' deposition could be observed at the 11th/12th stage of the embryonic development, while serosal apolysis occurs, and the first signs of oenocyte differentiation could be detected at the 15th stage. When pleuropodial cuticle deposition occurs, at the 16th stage, there is a rapid increase in the number of differentiating oenocytes. At the 19th stage there are some fully differentiated oenocytics, whereas, just before the cuticulin layer of the embryonic cuticle is laid down, another wave of oenocyte differentiation could be observed. The differentiation process of oenocytes and of vertebrate cells with a rapid cell membrane biogenesis (steroid secreting cells and hepatocytes) are compared. The correlation of oenocyte differentiation with ectodermal coating deposition, with molting hormone titer and with prothoracic gland differentiation is discussed.

Some aspects of intracellular symbiosis during embryo development of Mastotermes darwiniensis (Isoptera: Mastotermitidae).

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.

[Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models]. / Basi ultrastrutturali delle interazioni tra procarioti ed eucarioti in differenti modelli simbiotici.

This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach. In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life. In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism. In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement. Intracellular symbiotic bacteria are also described in nematodes. In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium. The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes. The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used. In addition, also freeze-fracture and deep-etching techniques were employed. The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body. These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell. Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M. darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP). The symbiotic bacteria are transmitted transovarially and, during embryogenesis, they are integrated into the morphogenetic processes. In particular, we were able to demonstrate that the origin of the bacteriocyte should be looked for in the cells of the haemocyte line (embryonic plasmatocytes). The eggs are infected by the bacteria emerging from the bacteriocytes of the ovaric fat body and, at the end of the vitellogenesis, they are actively phagocytized by the egg membrane. In filarial nematodes, intracellular bacteria belonging to the genus Wolbachia have been described: they have evolved an obligatory mutualistic association with their host. In fact, antibiotic treatments lead to the clearance of bacteria and this loss produces a negative impact on reproduction and survival of the filarial host. We evidenced, by TEM, the degenerative events occurring during the embriogenesis of Brugia pahangi and Dirofilaria immitis after tetracycline treatment. The data suggest that the Wolbachia play a direct role in worm metabolism. Finally, a new additional model of the prokaryote-eukaryote interaction has been described: we have recently discovered a new intracellular alpha-proteobacterium, named Iric ES1, which resides in the ovarian tissues of the tick Ixodes ricinus. The intriguing characteristic of this bacterium is its ability to invade and consume the ovaric mitochondria. From an evolutionary perspective, it is interesting to note that Iric ES1 enters mitochondria in a similar way to that employed by the "predatory" bacterium Bdellovibrio bacteriovorus.

RNAi reveals the key role of Nervana 1 in cockroach oogenesis and embryo development.

Na(+), K(+)-ATPases is a heterodimer protein consisting of α- and ß-subunits that control the ion transport through cell membranes. In insects the ß-subunit of the Na(+), K(+)-ATPase, known as Nervana, was characterized as a nervous system-specific glycoprotein antigen from adult Drosophila melanogaster heads. Nervana is expressed ubiquitously in all insect tissues, and in epithelial cells appeared located in a basolateral position as part of the septate junctions. Herein we study two Nervana isoforms from Blattella germanica, a cockroach species with panoistic ovaries. The sequencing and the phylogenetic analysis results suggest that these two isoforms are orthologs of D. melanogaster Nervana 1 and Nervana 2, respectively. Nervana 1 is highly expressed in the ovary of B. germanica, and depleting its expression results in changes in oocyte shape that do not impair oviposition. However, the resulting embryos show different defects and never hatch. These findings highlight the importance of this type of membrane pump in insect oogenesis as well as in embryo development, and its possible regulation by juvenile hormone.

Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply.

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera+Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.

Key roles of the Broad-Complex gene in insect embryogenesis.

The Broad-Complex (BR-C) is a primary response gene in the ecdysteroid signaling pathway which encodes a family of transcription factors that play a key role in metamorphic processes. With the aim of studying the possible functions of BR-C in the embryogenesis of a short germ band insect, we cloned BR-C cDNAs in the cockroach Blattella germanica (BgBR-C) and obtained the full coding region of six BR-C isoforms (Z1-Z6) of which, Z1-Z5 have homologous sequences in other insects, whereas Z6 is new. Temporal-expression patterns indicate that BgBR-C isoforms are present throughout the embryogenesis of B. germanica, although with weak fluctuations. Silencing all BgBR-C isoforms in the embryo through parental RNAi elicited a diversity of phenotypes. These phenotypes suggest roles for BgBR-C in different embryogenesis processes of B. germanica, mainly in the formation of the germ band, in the transition from 30% to 35% of total development, when the amnion and serosa rupture and the secondary dorsal organ is formed, and in the hatching process, which involves neural, endocrine and muscular coordination.

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.