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 (dN/dS), 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 dS 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

The Regulation of Juvenile Hormone in Dictyoptera: A Functional and Evolutionary Study of USP/RXR and Allatostatin

The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.MAS

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

Abstract 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: β-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

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

Characterization of the Juvenile Hormone Pathway in the Viviparous Cockroach, <i>Diploptera punctata</i>

<div><p>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 <i>Diploptera punctata</i>. 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 <i>Vg</i> and ovarian vitellin content significantly decreased. In addition, silencing <i>HMGR</i> and <i>JHAMT</i> resulted in a decrease in the transcript levels of other genes in the pathway.</p></div

q-RT-PCR primer sequences and reaction efficiencies and correlation coefficients in the q-RT-PCR assay.

<p>q-RT-PCR primer sequences and reaction efficiencies and correlation coefficients in the q-RT-PCR assay.</p

The effect of JH precursors on JH biosynthesis by CA from mated female <i>D</i>. <i>punctata</i>.

<p>JH biosynthesis was determined in CA that were first incubated in medium TC199 (control), and then in medium with JH precursor (treatment). (A) JH precursors stimulate JH biosynthesis by CA from day 7 mated female cockroach, <i>D</i>. <i>punctata</i>. 100μM of JH precursor was added to the medium during the second incubation. (B) The sensitivity of CA to JH precursors during the first gonadotrophic cycle. 40μM farnesol was added during the second incubation. Values represent mean ± SEM (n≥10). Significant differences are indicated ***p < 0.001.</p

Tissue specific expression of genes encoding JH biosynthetic enzymes.

<p>All tissues were dissected from day 4 mated adult females, except accessory glands (AG) and testes (Test) were from day 4 adult male. Abbreviations on the X-axis: Brain (Br), corpora allata (CA), nerve cord (NC), midgut (Mg), Malpighian tubules (MT), fat body (Fb), and ovary (Ov). Bars represent the mean of three biologically independent pools of ten animals run in triplicate and normalized to <i>Tubulin</i> and <i>EF1α</i>. Vertical error bars indicate SEM.</p

Primers for dsRNA construction.

<p>Primers for dsRNA construction.</p

Developmental expression of genes encoding JH biosynthetic enzymes during the first gonadotrophic cycle of <i>D</i>. <i>punctata</i>.

<p>Measurements were taken every day during the cycle (day 0 to day 7 after the final molt). Bars represent the mean of three biologically independent pools of ten animals run in triplicate and normalized to <i>Armadillo</i> and <i>EF1α</i>. Vertical error bars indicate SEM. Inset at the right bottom shows JH biosynthesis per individual CA (n = 12) and the transcript level of <i>DpVg</i> in fat body during the first gonadotrophic cycle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117291#pone.0117291.ref027" target="_blank">27</a>]. Vertical error bars indicate SEM.</p