Arthropod Sequences Research Articles

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

Population Genomic Analysis of the Blue CrabCallinectes sapidusUsing Genotyping-By-Sequencing

Previous genetic studies of the blue crab Callinectes sapidus along the U.S. Atlantic and Gulf coasts have reported weak or temporally variable spatial structure, suggesting high gene flow among distant populations possibly facilitated by long-distance larval dispersal or other features of blue crab life history. The use of relatively few genetic markers, however, may have limited power to detect subtle but significant structure that could inform fisheries management. In this study, the potential for genome-scale datasets to uncover subtle patterns of population structure in the blue crab was examined using a high-throughput genotyping approach (genotyping-by-sequencing) that generated data for more than 9,600 single nucleotide polymorphisms (SNPs) in crabs from three populations: Panama City Beach, FL, Agawam River, MA, and Porto Alegre, Brazil. Principle components analyses among the three populations revealed very distinct clustering of the Brazilian samples from U.S. populations, likely reflecting restricted gene flow across the equator. Detailed analysis of population structure between the two U.S. populations revealed low but significant genetic differentiation (FST = 0.0103), with FST values ranging from -0.05 to 0.48. Previous studies have failed to detect significant genetic structure on a similar geographic scale. FST outlier analysis identified 242 loci (2.45% of total) with statistically extreme values at the false discovery rate α = 0.05 level, only 16 of which showed significant sequence homology to annotated proteins via BLASTx alignment. Top BLASTx hits were to crustacean or arthropod sequences and 8 of the 16 had high sequence similarity to transposable elements or related machinery. Finally, results of population assignment tests for the two U.S. populations showed that the full marker dataset provided good power to assign individuals back to their population of origin (∼83% and 92% success for Panama City Beach and Agawam River, respectively), which dropped significantly when using only 500 randomly selected SNPs (∼61% and 72% success). Overall, this study demonstrates the great utility of high-throughput sequencing technologies for characterizing fine-scale patterns of genetic structure in blue crabs, and this approach should substantially improve the delineation of stock structure and further advance our understanding of blue crab population connectivity and ecology.

Transcriptome analysis of the copepod Eurytemora affinis upon exposure to endocrine disruptor pesticides: Focus on reproduction and development

Copepods—which include freshwater and marine species—represent the most abundant group of aquatic invertebrates. Among them, the calanoid copepod Eurytemora affinis is widely represented in the northern hemisphere estuaries and has become a species of interest in ecotoxicology. Like other non-target organisms, E. affinis may be exposed to a wide range of chemicals such as endocrine disruptors (EDs). This study investigated the gene expression variation in E. affinis after exposure to ED pesticides—chosen as model EDs—in order to (i) improve the knowledge on their effects in crustaceans, and (ii) highlight relevant transcripts for further development of potential biomarkers of ED exposure/effect. The study focused on the reproduction function in response to ED. Copepods were exposed to sublethal concentrations of pyriproxyfen (PXF) and chlordecone (CLD) separately. After 48h, males and females (400 individuals each) were sorted for RNA extraction. Their transcriptome was pyrosequenced using the Illumina® technology. Contigs were blasted and functionally annotated using Blast2GO®. The differential expression analysis between ED- and acetone-exposed organisms was performed according to sexes and contaminants. Half of the 19,721 contigs provided by pyrosequencing were annotated, mostly (80%) from arthropod sequences. Overall, 2,566 different genes were differentially expressed after ED exposures in comparison with controls. As many genes were differentially expressed after PXF exposure as after CLD exposure. In contrast, more genes were differentially expressed in males than in females after both exposures. Ninety-seven genes overlapped in all conditions. Finally, 31 transcripts involved in reproduction, growth and development, and changed in both chemical exposures were selected as potential candidates for future development of biomarkers.

Sex-dependent transcriptome analysis and single nucleotide polymorphism (SNP) discovery in the brine shrimp Artemia franciscana

In order to enhance genomic resources for the brine shrimp Artemia franciscana, RNA-Seq analysis was conducted for adult females and males. Through de novo assembly, 36,896 high quality contigs were obtained, of which 13,749 sequences were annotated with arthropod sequences. Just 4.5% matched against previously reported sequences for Artemia spp. Additionally, different transcriptional patterns between males and females were found, evidencing sex-related transcriptional responses. Furthermore, 221 and 534 putative SNPs were identified exclusively in males and females, respectively. These results will build the foundation for further genomic studies in A. franciscana.

Bioinformatic analyses of the publicly accessible crustacean expressed sequence tags (ESTs) reveal numerous novel neuropeptide-encoding precursor proteins, including ones from members of several little studied taxa

ESTs have been generated for many crustacean species, providing an invaluable resource for peptide discovery in members of this arthropod subphylum. Here, these data were mined for novel peptide-encoding transcripts, with the mature peptides encoded by them predicted using a combination of online peptide prediction programs and homology to known arthropod sequences. In total, 70 mature full-length/partial peptides representing members of 16 families/subfamilies were predicted, the vast majority being novel; the species from which the peptides were identified included members of the Branchiopoda ( Daphnia carinata and Triops cancriformis), Maxillopoda ( Caligus clemensi, Caligus rogercresseyi, Lepeophtheirus salmonis and Lernaeocera branchialis) and Malacostraca ( Euphausia superba, Marsupenaeus japonicus, Penaeus monodon, Homarus americanus, Petrolisthes cinctipes, Callinectes sapidus and Portunus trituberculatus). Of particular note were the identifications of an intermediate between the insect adipokinetic hormones and crustacean red pigment concentrating hormone and a modified crustacean cardioactive peptide from the daphnid D. carinata; Arg 7-corazonin was also deduced from this species, the first identification of a corazonin from a non-decapod crustacean. Our data also include the first reports of members of the calcitonin-like diuretic hormone, FMRFamide-related peptide (neuropeptide F subfamily) and orcokinin families from members of the Copepoda. Moreover, the prediction of a bursicon α from the euphausid E. superba represents the first peptide identified from any member of the basal eucaridean order Euphausiacea. In addition, large collections of insect eclosion hormone- and neuroparsin-like peptides were identified from a variety of species, greatly expanding the number of known members of these families in crustaceans.

The complete mitochondrial genome of the cockroach Eupolyphaga sinensis (Blattaria: Polyphagidae) and the phylogenetic relationships within the Dictyoptera

We present the complete mitochondrial DNA sequence of Eupolyphaga sinensis. This closed circular molecule is 15553 bp long and consists of 37 genes that encode for 13 inner membrane proteins, 2 ribosomal RNAs and 22 transfer RNAs. The genome shares the gene order and orientation with previously known Blattaria mitochondrial genomes. All tRNAs could be folded into the typical cloverleaf secondary structure, but the tRNASer (AGN) appears to be missing the DHU arm. The A + T-rich region is 857 bp long and longer than other cockroaches. Based on the concatenated amino acid sequences of all protein coding genes of E. sinensis in conjunction with those 23 other arthropod sequences, we reconstruct the phylogenetic tree. Phylogenetic analyses shows that Blataria (including Isoptera) and the Mantodea are sister groups. Furthermore the relationship of the three basal clades of winged insects are different from the three previous hypotheses ((Ephemeroptera + Odonata) +Neoptera, Ephemeroptera + (Odonata + Neoptera), Odonata + (Ephemeroptera +Neoptera)). The Ephemeroptera (Parafronurus youi) clusters with the Plecoptera (Pteronarcys princes).

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

Can comprehensive background knowledge be incorporated into substitution models to improve phylogenetic analyses? A case study on major arthropod relationships.

BackgroundWhenever different data sets arrive at conflicting phylogenetic hypotheses, only testable causal explanations of sources of errors in at least one of the data sets allow us to critically choose among the conflicting hypotheses of relationships. The large (28S) and small (18S) subunit rRNAs are among the most popular markers for studies of deep phylogenies. However, some nodes supported by this data are suspected of being artifacts caused by peculiarities of the evolution of these molecules. Arthropod phylogeny is an especially controversial subject dotted with conflicting hypotheses which are dependent on data set and method of reconstruction. We assume that phylogenetic analyses based on these genes can be improved further i) by enlarging the taxon sample and ii) employing more realistic models of sequence evolution incorporating non-stationary substitution processes and iii) considering covariation and pairing of sites in rRNA-genes.ResultsWe analyzed a large set of arthropod sequences, applied new tools for quality control of data prior to tree reconstruction, and increased the biological realism of substitution models. Although the split-decomposition network indicated a high noise content in the data set, our measures were able to both improve the analyses and give causal explanations for some incongruities mentioned from analyses of rRNA sequences. However, misleading effects did not completely disappear.ConclusionAnalyses of data sets that result in ambiguous phylogenetic hypotheses demand for methods, which do not only filter stochastic noise, but likewise allow to differentiate phylogenetic signal from systematic biases. Such methods can only rely on our findings regarding the evolution of the analyzed data. Analyses on independent data sets then are crucial to test the plausibility of the results. Our approach can easily be extended to genomic data, as well, whereby layers of quality assessment are set up applicable to phylogenetic reconstructions in general.

In silico analyses of peptide paracrines/hormones in Aphidoidea

The Aphidoidea is an insect superfamily comprising most of the known aphid species. While small in size, these animals are of considerable economic importance as many members of this taxon are serious agricultural pests, inflicting physical damage upon crop plants and serving as vectors in the transmission of viral plant diseases. In terms of identifying the paracrines/hormones used to modulate behavior, particularly peptides, members of the Aphidoidea have largely been ignored, as it is not tractable to isolate the large pools of tissue needed for standard biochemical investigations. Here, a bioinformatics approach to peptide discovery has been used to overcome this limitation of scale. Specifically, in silico searches of publicly accessible aphidoidean ESTs were conducted to identify transcripts encoding putative peptides precursors, with the mature peptides contained within them deduced using peptide processing software and homology to known arthropod sequences. In total, 39 ESTs encoding putative peptides precursors were identified from four aphid species: Acyrthosiphon pisum (14 ESTs), Aphis gossypii (four ESTs), Myzus persicae (20 ESTs) and Toxoptera citricida (one EST). These precursors included ones predicted to encode isoforms of B-type allatostatin, crustacean cardioactive peptide, FMRFamide-related peptide (both myosuppressin and short neuropeptide F subfamilies), insect kinin, orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide and tachykinin-related peptide. In total, 83 peptides were characterized from the identified precursors, most novel, including two B-type allatostatins possessing the variant −W X 7Wamide motif, two N-terminally extended proctolin isoforms and an N-terminally truncated and substituted SIFamide. Collectively, these results expand greatly the number of known/predicted aphid peptide paracrines/hormones, and provide a strong foundation for future molecular and physiological investigations of peptidergic control in this insect group.

Neuropeptide discovery in Ixodoidea: An in silico investigation using publicly accessible expressed sequence tags

The Ixodoidea (ticks) are important vectors in the transmission of many human diseases; for example, the blacklegged tick Ixodes scapularis is the major vector in the transmission of Lyme disease, the most frequently reported vector-borne illness in the United States. The development of expressed sequence tags (ESTs) for ixodoidean cDNA libraries, and their public deposition, has generated a rich resource for protein discovery in members of this taxon, thereby providing an opportunity for better understanding the physiology and behavior of these disease vectors. Here, in silico searches of publicly accessible ESTs were conducted to identify transcripts encoding putative ixodoidean neuropeptide precursors, with the mature peptides contained within them predicted using online peptide processing programs and homology to known arthropod sequences. In total, 37 putative neuropeptide-encoding ESTs were identified from three ixodoidean species: I. scapularis (29 ESTs), Rhipicephalus microplus (seven ESTs) and Amblyomma americanum (one EST). Among those identified from I. scapularis were ones predicted to encode isoforms of corazonin, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone (both calcitonin- and corticotropin-releasing factor-like), FMRFamide-related peptide (both short neuropeptide F and sulfakinin subfamilies) orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide, and tachykinin-related peptide. Collectively, 80 distinct ixodoidean neuropeptides were characterized from the identified precursors. These results not only expand greatly the number of known/predicted ixodoidean neuropeptides, but also provide a strong foundation for future molecular and physiological investigations of peptidergic control in this important group of disease-transmitting arthropods.

Super paramagnetic clustering of DNA sequences.

An unsupervised clustering of 4541 DNA sequences containing active promoter regions from vertebrate and arthropod classes (including their viral genes) was performed. All necessary information was solely gathered a priori from the DNA sequences by measuring frequencies of tri-nucleotides and tetra-nucleotides. We employed Super Paramagnetic Clustering, a novel clustering algorithm based on physical properties of an inhomogeneous granular ferromagnet. This method utilizes Swendsen-Wang cluster Monte Carlo simulations to distinguish clusters by measuring pairs of correlation functions from different resolutions. We identified two strongly separated clusters of human viral genes corresponding to the Epstein-Barr virus and the Herpes Simplex virus type 1. In addition, vertebrate and arthropod sequences were successfully separated into two different classes with merely 9.25% of arthropod sequences being misclassified. From a functional perspective, these sequences have high gene function correlations with sequences from the vertebrate cluster. By tuning a clustering parameter, Super Paramagnetic Clustering was able to classify vertebrate class further into two major clusters, from where a large number of housekeeping genes and tissue-specific genes were found respectively. The indications came from observation of gene expression function and consensus transcription factors which were found grouped together in specific positions of the DNA sequences.

The structure of the USP/RXR of Xenos pecki indicates that Strepsiptera are not closely related to Diptera

The receptor for the insect molting hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.

Genome-scale evidence of the nematode-arthropod clade

The most extensive phylogenetic analysis carried out to date, including 11 complete genomes, is shown to support the Ecdysozoa hypothesis in the open-ended debate of the Coelomata-Ecdysozoa evolutionary problem.

THE SCUTOCOXIFERA TAX. NOV. AND THE INFORMATION CONTENT OF NUCLEAR SSU RDNA SEQUENCES FOR RECONSTRUCTION OF ISOPOD PHYLOGENY (PERACARIDA: ISOPODA)

The nuclear ssu rRNA gene of several isopods (Crustacea, Peracarida) was sequenced to study its phylogenetic information content. Several areas had to be cut out of the alignment of 31 isopod sequences and selected outgroup arthropod sequences due to the lack of alignable patterns. The final alignment had 2,533 positions and 43 sequences. The length of the isopod nuclear ssu rRNA genes varies between 2,098 and 3,402 bp. In some clades the gene length increases; in others like the cymothoids and bopyrids, long deletions occur. Some insertions are specific for major groups (e.g., amphipods, isopods). Most elongation areas evolve rapidly and are not alignable among higher taxa. Information content is visualized with spectra of supporting positions. Only a few groups are unambiguously supported with a signal distinctly higher than background noise. The results of maximum parsimony analyses are congruent with major aspects of earlier hypotheses on isopod phylogeny. Some contradictions are discussed. The latter are mainly based on a lack of reliable information. A major monophyletic group found in the molecular phylogenies and also supported by distinct morphological characters is named Scutocoxifera tax. nov., composed of the Oniscidea, Valvifera, Sphaeromatidea, Anthuridea, and Cymothoida. SEM photographs are presented to document the apomorphic state of the coxa in the Scutocoxifera.

Expression of Arginine Kinase Enzymatic Activity and mRNA in Gills of The Euryhaline Crabs Carcinus Maenas and Callinectes Sapidus

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.