Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach Diploptera punctata.

The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternatively spliced variants in the cockroach Diploptera punctata. This is the first report of NR1 paralogs in insects. The tissue distributions and expression profiles of DpNR1A, DpNR1B and DpNR2 in different tissues were also investigated. Previous studies have demonstrated NMDA-stimulated biosynthesis of juvenile hormone (JH) in the corpora allata through the influx of extracellular Ca(2+) in Diploptera punctata. However, our data show that the transcript levels of DpNR1A, DpNR1B and DpNR2 were low in the corpora allata. MK-801, a high-affinity antagonist of NMDAR, did not show any effect on JH biosynthesis in vitro. In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-801 resulted in any significant change in JH biosynthesis or basal oocyte growth. Injection of animals with a high dose of MK-801 (30 µg per animal per injection), which paralyzed the animals for 4-5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata.

Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development.

The Pacific beetle cockroach, Diploptera punctata, has proven to be a valuable model insect in the study of the dynamics regulating juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymphal development and reproduction. This stems in part from its unusual mode of reproduction, adenotrophic viviparity, in which females give birth to live young that have been nourished throughout embryonic development by a protein-rich 'milk' secreted by the wall of the brood sac or uterus. In this animal, as in most insects, JH regulates both vitellogenin production and its uptake by developing oocytes. However, JH has an antagonistic effect on embryonic development and following oviposition of the fertilized oocytes into the brood sac, JH production halts, in part through the action of a peptide family, the FGLa allatostatins. JH production remains at a low level throughout pregnancy and is only reinstated at the end of gestation, at which time, the next wave of oocytes begins to develop and enter vitellogenesis. Thus, JH production in this species is precisely regulated, since the appearance of JH at inappropriate times would result in abortion of the embryos. Numerous factors are responsible for the regulation of JH biosynthesis, including peptides, biogenic amines, neurotransmitters, ecdysteroids and second messenger effectors. In this review, we discuss these factors and highlight potentially fruitful areas of future research. Although several of the enzymes of the biosynthetic pathway have been cloned, the precise points of rate limitation remain uncertain. The dissection of the biosynthetic pathway and its control awaits the completion of the genome and transcriptome of this important model insect.

Farnesoic acid and methyl farnesoate production during lobster reproduction: possible functional correlation with retinoid X receptor expression.

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.

Reconstruction of ancestral FGLamide-type insect allatostatins: a novel approach to the study of allatostatin function and evolution.

Allatostatins (ASTs) are a class of regulatory neuropeptides, with diverse functions, found in an array of invertebrate phyla. ASTs have complex gene structure, in which individual ASTs are cleaved from a precursor peptide. Little is known about the molecular evolution of AST structure and function, even in extensively studied groups such as cockroaches. This paper presents the application of a novel technique for the analysis of this system, that of ancestral reconstruction, whereby ancestral amino acid sequences are resurrected in the laboratory. We inferred the ancestral sequences of a well-characterized peptide, AST 7, for the insect ancestor, as well as several cockroach ancestors. Peptides were assayed for in vitro inhibition of JH production in Diploptera punctata and Periplaneta americana. Our results surprisingly, indicate a decrease in potency of the ancestral cockroach AST7 peptide in comparison with more ancient ones such as the ancestral insect peptide, as well as more recently evolved cockroach peptides. We propose that this unexpected decrease in peptide potency at the cockroach ancestor may be related to the concurrent increase in peptide copy number in the lineages leading to cockroaches. This model is consistent with current physiological data, and may be linked to the increased role of ASTs in the regulation of reproductive processes in the cockroaches.

Characterization of the juvenile hormone pathway in the viviparous cockroach, Diploptera punctata.

Juvenile hormones (JHs) are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase (HMGR) and Juvenile hormone acid methyltransferase (JHAMT). As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

RXR/USP and EcR are critical for the regulation of reproduction and the control of JH biosynthesis in Diploptera punctata.

During development and reproduction the response to ecdysteroids is mediated by a heterodimeric receptor complex comprising the retinoid X receptor/ultraspiracle (RXR/USP) and the ecdysone receptor (EcR). Here, the role of these receptors in the endocrine control of reproduction is examined in the cockroach Diploptera punctata. We report the sequence of four DpRXR and three DpEcR splice variants, including the first description of a Drosophila EcRB2-like isoform in a hemimetabolous insect. DpRXR and DpEcR are broadly expressed in the tissues of adult females, with relatively high transcript levels in the corpora allata (CA), nervous tissue and ovary. Developmental profiling revealed an inverse correlation between DpRXR and DpEcR expression and the activity of the CA. RNAi-mediated depletion of DpRXR and DpEcR did not affect oocyte growth, but inhibited oviposition and impaired chorion formation. Retained oocytes exhibited a degenerating follicular epithelium and were slowly resorbed. Treated animals showed significantly higher rates of JH biosynthesis and a decrease in ecdysteroid titers at the end of vitellogenesis. Reduction of DpRXR and DpEcR expression resulted in an upregulation of genes involved in JH production and a downregulation of allatostatin receptor mRNA in the CA. Treatment with dsRNA also affected the expression of genes downstream of JH in target tissues including vitellogenin and Krüppel-homolog 1 as well as Broad-Complex, an early ecdysone response gene. Overall, results suggest that DpRXR and DpEcR are not required early in the reproductive cycle when events are JH-dependent, but do mediate critical ecdysteroid feedback to the CA late in the gonadotropic cycle.

Methoprene-tolerant (Met) knockdown in the adult female cockroach, Diploptera punctata completely inhibits ovarian development.

Independent of the design of the life cycle of any insect, their growth and reproduction are highly choreographed through the action of two versatile hormones: ecdysteroids and juvenile hormones (JH). However, the means by which JH can target tissues and exert its pleiotropic physiological effects is currently still not completely elucidated. Although the identity of the one JH receptor is currently still elusive, recent evidence seems to point to the product of the Methoprene-tolerant gene (Met) as the most likely contender in transducing the action of JH. Studies on the role of this transcription factor have mostly been focused on immature insect stages. In this study we used the viviparous cockroach Diploptera punctata, a favorite model in studying JH endocrinology, to examine the role of Met during reproduction. A tissue distribution and developmental profile of transcript levels was determined for Met and its downstream partners during the first gonadotropic cycle of this cockroach. Using RNA interference, our study shows that silencing Met results in an arrest of basal oocyte development; vitellogenin is no longer transcribed in the fat body and no longer taken up by the ovary. Patency is not induced in these animals which fail to produce the characteristic profile of JH biosynthesis typical of the first gonadotropic cycle. Moreover, the ultrastructure of the follicle cells showed conspicuous whorls of rough endoplasmic reticulum and a failure to form chorion. Our study describes the role of Met on a cellular and physiological level during insect reproduction, and confirms the role of Met as a key factor in the JH signaling pathway.

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata.

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca(2+) and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.

Sequencing and validation of housekeeping genes for quantitative real-time PCR during the gonadotrophic cycle of Diploptera punctata.

BACKGROUND: Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression. RESULTS: Candidate housekeeping genes were sequenced: ß-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expression of these eight genes was analyzed in corpora allata (CA) and ovaries of adult female D. punctata. Both geNorm, as well as Normfinder characterized SDHa, EF1a and Arm as being the most stably expressed in the corpora allata. In the ovary, the geNorm calculation showed Tub, EF1a and RpL32 to be most stable, whereas Normfinder identified Tub, EF1a and Arm as the best. In ovary, the least stable gene was Actin, challenging its usefulness in normalization. As a proof of principle, the expression of follicle cell protein 3c and CYP15A1 was monitored during the first gonadotrophic cycle. CONCLUSION: Arm and EF1a form the most stably expressed combination of two reference genes out of the eight candidates that were tested in the corpora allata. Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata. Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata. The results stress the necessity for validation of reference genes in q-RT-PCR studies in cockroaches.

Molecular evolution of ultraspiracle protein (USP/RXR) in insects.

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d(N)/d(S)), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d(S) is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand.

Final steps in juvenile hormone biosynthesis in the desert locust, Schistocerca gregaria.

Two genes coding for enzymes previously reported to be involved in the final steps of juvenile hormone (JH) biosynthesis in different insect species, were characterised in the desert locust, Schistocerca gregaria. Juvenile hormone acid O-methyltransferase (JHAMT) was previously described to catalyse the conversion of farnesoic acid (FA) and JH acid to their methyl esters, methyl farnesoate (MF) and JH respectively. A second gene, CYP15A1 was reported to encode a cytochrome P450 enzyme responsible for the epoxidation of MF to JH. Additionally, a third gene, FAMeT (originally reported to encode a farnesoic acid methyltransferase) was included in this study. Using q-RT-PCR, all three genes (JHAMT, CYP15A1 and FAMeT) were found to be primarily expressed in the CA of the desert locust, the main biosynthetic tissue of JH. An RNA interference approach was used to verify the orthologous function of these genes in S. gregaria. Knockdown of the three genes in adult animals followed by the radiochemical assay (RCA) for JH biosynthesis and release showed that SgJHAMT and SgCYP15A1 are responsible for synthesis of MF and JH respectively. Our experiments did not show any involvement of SgFAMeT in JH biosynthesis in the desert locust. Effective and selective inhibitors of SgJHAMT and SgCYP15A1 would likely represent selective biorational locust control agents.