Neuropeptidomes of Tenebrio molitor L. and Zophobas atratus Fab. (Coleoptera, Polyphaga: Tenebrionidae).

Neuropeptides are signaling molecules that regulate almost all physiological processes in animals. Around 50 different genes for neuropeptides have been described in insects. In Coleoptera, which is the largest insect order based on numbers of described species, knowledge about neuropeptides and protein hormones is still limited to a few species. Here, we analyze the neuropeptidomes of two closely related tenebrionid beetles: Tenebrio molitor and Zophobas atratus─both of which are model species in physiological and pharmacological research. We combined transcriptomic and mass spectrometry analyses of the central nervous system to identify neuropeptides and neuropeptide-like and protein hormones. Several precursors were identified in T. molitor and Z. atratus, of which 50 and 40, respectively, were confirmed by mass spectrometry. This study provides the basis for further functional studies of neuropeptides as well as for the design of environmentally friendly and species-specific peptidomimetics to be used as biopesticides. Furthermore, since T. molitor has become accepted by the European Food Safety Authority as a novel food, a deeper knowledge of the neuropeptidome of this species will prove useful for optimizing production programs at an industrial scale.

PaOctß2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or ß-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a ß-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an ß2-adrenergic-like octopamine receptor. The functional characterization of PaOctß2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass-Spectrometry-Imaging Protocol.

Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information from immunohistochemistry with peptidomics information from mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues; however, these studies did not detect neuropeptide complements in manners comparable to those of conventional peptidomics. The aim of our study was to improve sample preparation so that MSI could provide comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 µm spatial resolution, which was confirmed by parallel analysis of tissue extracts using electrospray-ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide-precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neuroscience-related topics, including the study of complex neuropeptide interactions within the CNS.

Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus.

One of the most thoroughly studied insect species, with respect to locomotion behavior, is the stick insect Carausius morosus. Although detailed information exists on premotor networks controlling walking, surprisingly little is known about neuropeptides, which are certainly involved in motor activity generation and modulation. So far, only few neuropeptides were identified from C. morosus or related stick insects. We performed a transcriptome analysis of the central nervous system to assemble and identify 65 neuropeptide and protein hormone precursors of C. morosus, including five novel putative neuropeptide precursors without clear homology to known neuropeptide precursors of other insects ( Carausius neuropeptide-like precursor 1, HanSolin, PK-like1, PK-like2, RFLamide). Using Q Exactive Orbitrap and MALDI-TOF mass spectrometry, 277 peptides including 153 likely bioactive mature neuropeptides were confirmed. Peptidomics yielded a complete coverage for many of the neuropeptide propeptides and confirmed a surprisingly high number of heterozygous sequences. Few neuropeptide precursors commonly occurring in insects, including those of insect kinins and sulfakinins, could neither be found in the transcriptome data nor did peptidomics support their presence. The results of our study represent one of the most comprehensive peptidomic analyses on insects and provide the necessary input for subsequent experiments revealing neuropeptide function in greater detail.

Unravelling population genetic structure with mitochondrial DNA in a notional panmictic coastal crab species: sample size makes the difference.

BACKGROUND: The extent of genetic structure of a species is determined by the amount of current gene flow and the impact of historical and demographic factors. Most marine invertebrates have planktonic larvae and consequently wide potential dispersal, so that genetic uniformity should be common. However, phylogeographic investigations reveal that panmixia is rare in the marine realm. Phylogeographic patterns commonly coincide with geographic transitions acting as barriers to gene flow. In the Mediterranean Sea and adjoining areas, the best known barriers are the Atlantic-Mediterranean transition, the Siculo-Tunisian Strait and the boundary between Aegean and Black seas. Here, we perform the so far broadest phylogeographic analysis of the crab Pachygrapsus marmoratus, common across the north-eastern Atlantic Ocean, Mediterranean and Black seas. Previous studies revealed no or weak genetic structuring at meso-geographic scale based on mtDNA, while genetic heterogeneity at local scale was recorded with microsatellites, even if without clear geographic patterns. Continuing the search for phylogeographic signal, we here enlarge the mtDNA dataset including 51 populations and covering most of the species' distribution range. RESULTS: This enlarged dataset provides new evidence of three genetically separable groups, corresponding to the Portuguese Atlantic Ocean, Mediterranean Sea plus Canary Islands, and Black Sea. Surprisingly, hierarchical AMOVA and Principal Coordinates Analysis agree that our Canary Islands population is closer to western Mediterranean populations than to mainland Portugal and Azores populations. Within the Mediterranean Sea, we record genetic homogeneity, suggesting that population connectivity is unaffected by the transition between the western and eastern Mediterranean. The Mediterranean metapopulation seems to have experienced a relatively recent expansion around 100,000 years ago. CONCLUSIONS: Our results suggest that the phylogeographic pattern of P. marmoratus is shaped by the geological history of Mediterranean and adjacent seas, restricted current gene flow among different marginal seas, and incomplete lineage sorting. However, they also caution from exclusively testing well-known biogeographic barriers, thereby neglecting other possible phylogeographic patterns. Mostly, this study provides evidence that a geographically exhaustive dataset is necessary to detect shallow phylogeographic structure within widespread marine species with larval dispersal, questioning all studies where species have been categorized as panmictic based on numerically and geographically limited datasets.

Phylogenomics of darkling beetles (Coleoptera: Tenebrionidae) from the Atacama Desert.

Background: Tenebrionidae (Insecta: Coleoptera) are a conspicuous component of desert fauna worldwide. In these ecosystems, they are significantly responsible for nutrient cycling and show remarkable morphological and physiological adaptations. Nevertheless, Tenebrionidae colonizing individual deserts have repeatedly emerged from different lineages. The goal of our study was to gain insights into the phylogenetic relationships of the tenebrionid genera from the Atacama Desert and how these taxa are related to the globally distributed Tenebrionidae. Methods: We used newly generated transcriptome data (47 tribes, 7 of 11 subfamilies) that allowed for a comprehensive phylogenomic analysis of the tenebrionid fauna of this hyperarid desert and fills a gap in our knowledge of the highly diversified Tenebrionidae. We examined two independent data sets known to be suitable for phylogenomic reconstructions. One is based on 35 neuropeptide precursors, the other on 1,742 orthologous genes shared among Coleoptera. Results: The majority of Atacama genera are placed into three groups, two of which belong to typical South American lineages within the Pimeliinae. While the data support the monophyly of the Physogasterini, Nycteliini and Scotobiini, this does not hold for the Atacama genera of Edrotini, Epitragini, Evaniosomini, Praociini, Stenosini, Thinobatini, and Trilobocarini. A suggested very close relationship of Psammetichus with the Mediterranean Leptoderis also could not be confirmed. We also provide hints regarding the phylogenetic relationships of the Caenocrypticini, which occur both in South America and southern Africa. Apart from the focus on the Tenebrionidae from the Atacama Desert, we found a striking synapomorphy grouping Alleculinae, Blaptinae, Diaperinae, Stenochinae, and several taxa of Tenebrioninae, but not Tenebrio and Tribolium. This character, an insertion in the myosuppressin gene, defines a higher-level monophyletic group within the Tenebrionidae. Conclusion: Transcriptome data allow a comprehensive phylogenomic analysis of the tenebrionid fauna of the Atacama Desert, which represents one of the seven major endemic tribal areas in the world for Tenebrionidae. Most Atacama genera could be placed in three lineages typical of South America; monophyly is not supported for several tribes based on molecular data, suggesting that a detailed systematic revision of several groups is necessary.

Schistocerca neuropeptides - An update.

We compiled a comprehensive list of 67 precursor genes encoding neuropeptides and neuropeptide-like peptides using the Schistocerca gregaria genome and several transcriptome datasets. 11 of these 67 precursor genes have alternative transcripts, bringing the total number of S. gregaria precursors identified in this study to 81. Based on this precursor information, we used different mass spectrometry approaches to identify the putative mature, bioactive peptides processed in the nervous system of S. gregaria. The thereby generated dataset for S. gregaria confirms significant conservation of the entire neuropeptidergic gene set typical of insects and also contains precursors typical of Polyneoptera only. This is in striking contrast to the substantial losses of peptidergic systems in some holometabolous species. The neuropeptidome of S. gregaria, apart from species-specific sequences within the known range of variation, is quite similar to that of Locusta migratoria and even to that of less closely related Polyneoptera. With the S. gregaria peptidomics data presented here, we have thus generated a very useful source of information that could also be relevant for the study of other polyneopteran species.

The neuropeptidome of Carabus (Coleoptera, Adephaga: Carabidae).

Neuropeptides are signaling molecules involved in the regulation of virtually all physiological functions of Metazoa. In insects, more than 50 neuropeptide genes can be present in a single species, and thus neuropeptidergic systems are attractive targets for the development of environmentally friendly pesticides. Such approaches require not only knowledge of the neuropeptidomes of pests, but also detailed knowledge of the corresponding systems in beneficial insects. In Coleoptera, there is no profound knowledge of the neuropeptides in the adephagan lineage, which contains many of the ecologically important predators of caterpillars. We analyzed by transcriptomics, mass spectrometry and immunohistochemistry the neuropeptidomes of the two Carabus species C. violaceus and C. problematicus. This information, which contains detailed data on the differential processing of CAPA peptides, allows for the recognition of features typical only of the polyphagan lineage with its many pests. The neuropeptidomics data, which also confirmed the processing of a number of protein hormones, represent the highest number of neuropeptides that have been identified so far from Coleoptera. The sequences of the mature neuropeptides of the two Carabus species, whose ancestors separated about 13 Mya, are highly similar and no sequence substitutions were found in single-copy neuropeptides.

Phylogenetic and morphometric differentiation reveal geographic radiation and pseudo-cryptic speciation in a mangrove crab from the Indo-West Pacific.

The presence of boundaries to dispersal has been recently documented for many Indo-West Pacific (IWP) species with planktonic propagules and a widespread distribution. We studied the phylogeography of the mangrove crab Neosarmatium meinerti (Brachyura: Sesarmidae) and the phylogenetic relationship to its presumed sister species N. fourmanoiri in the IWP in order to compare intraspecific with interspecific diversity. Portions of the mitochondrial genes 16S and CoxI were sequenced for 23 specimens of N. meinerti and 5 N. fourmanoiri, while a fragment of the 28S was obtained for a subset of specimens. Genetic data are supplemented by morphometric and based on 37 adult males of N. meinerti and 9 males of N. fourmanoiri. The conserved nuclear 28S reveals the existence of a genetic break between the Indian and Pacific oceans. Otherwise, mitochondrial genes as well as morphometry clearly support the presence of a species complex within N. meinerti composed by four well structured and geographically defined lineages: East African coast; western Indian Ocean islands; South East Asia; and Australia.

Coordinated RNA-Seq and peptidomics identify neuropeptides and G-protein coupled receptors (GPCRs) in the large pine weevil Hylobius abietis, a major forestry pest.

Hylobius abietis (Linnaeus), or large pine weevil (Coleoptera, Curculionidae), is a pest of European coniferous forests. In order to gain understanding of the functional physiology of this species, we have assembled a de novo transcriptome of H. abietis, from sequence data obtained by Next Generation Sequencing. In particular, we have identified genes encoding neuropeptides, peptide hormones and their putative G-protein coupled receptors (GPCRs) to gain insights into neuropeptide-modulated processes. The transcriptome was assembled de novo from pooled paired-end, sequence reads obtained from RNA from whole adults, gut and central nervous system tissue samples. Data analysis was performed on the transcripts obtained from the assembly including, annotation, gene ontology and functional assignment as well as transcriptome completeness assessment and KEGG pathway analysis. Pipelines were created using Bioinformatics tools and techniques for prediction and identification of neuropeptides and neuropeptide receptors. Peptidomic analysis was also carried out using a combination of MALDI-TOF as well as Q-Exactive Orbitrap mass spectrometry to confirm the identified neuropeptide. 41 putative neuropeptide families were identified in H. abietis, including Adipokinetic hormone (AKH), CAPA and DH31. Neuropeptide F, which has not been yet identified in the model beetle T. castaneum, was identified. Additionally, 24 putative neuropeptide and 9 leucine-rich repeat containing G protein coupled receptor-encoding transcripts were determined using both alignment as well as non-alignment methods. This information, submitted to the NCBI sequence read archive repository (SRA accession: SRP133355), can now be used to inform understanding of neuropeptide-modulated physiology and behaviour in H. abietis; and to develop specific neuropeptide-based tools for H. abietis control.

Correction: Demographic History and Reproductive Output Correlates with Intraspecific Genetic Variation in Seven Species of Indo-Pacific Mangrove Crabs.

[This corrects the article DOI: 10.1371/journal.pone.0158582.].

Identification of mature peptides from pban and capa genes of the moths Heliothis peltigera and Spodoptera littoralis.

By transcriptome analysis, we identified PBAN and CAPA precursors in the moths Spodoptera littoralis and Heliothis peltigera which are among the most damaging pests of agriculture in tropical and subtropical Africa as well as in Mediterranean countries. A combination of mass spectrometry and immunocytochemistry was used to identify mature peptides processed from these precursors and to reveal their spatial distribution in the CNS. We found that the sites of expression of pban genes, the structure of PBAN precursors and the processed neuropeptides are very similar in noctuid moths. The sequence of the diapause hormone (DH; tryptopyrokinin following the signal peptide), however, contains two N-terminal amino acids more than expected from comparison with already published sequences of related species. Capa genes of S. littoralis and H. peltigera encode, in addition to periviscerokinins, a tryptopyrokinin showing sequence similarity with DH, which is the tryptopyrokinin of the pban gene. CAPA peptides, which were not known from any noctuid moth so far, are produced in cells of abdominal ganglia. The shape of the release sites of these hormones in H. peltigera represents an exceptionally derived trait state and does not resemble the well-structured abdominal perisympathetic organs which are known from many other insects. Instead, axons of CAPA cells extensively ramify within the ventral diaphragm. The novel information regarding the sequences of all mature peptides derived from pban and capa genes of H. peltigera and S. littoralis now enables a detailed analysis of the bioactivity and species-specificity of the native peptides, especially those from the hitherto unknown capa genes, and to explore their interactions with PBAN/DH receptors.

Demographic History and Reproductive Output Correlates with Intraspecific Genetic Variation in Seven Species of Indo-Pacific Mangrove Crabs.

The spatial distribution and the amount of intraspecific genetic variation of marine organisms are strongly influenced by many biotic and abiotic factors. Comparing biological and genetic data characterizing species living in the same habitat can help to elucidate the processes driving these variation patterns. Here, we present a comparative multispecies population genetic study on seven mangrove crabs co-occurring in the West Indian Ocean characterized by planktotrophic larvae with similar pelagic larval duration. Our main aim was to investigate whether a suite of biological, behavioural and ecological traits could affect genetic diversities of the study species in combination with historical demographic parameters. As possible current explanatory factors, we used the intertidal micro-habitat colonised by adult populations, various parameters of individual and population fecundity, and the timing of larval release. As the genetic marker, we used partial sequences of cytochrome oxidase subunit I gene. Genetic and ecological data were collected by the authors and/or gathered from primary literature. Permutational multiple regression models and ANOVA tests showed that species density and their reproductive output in combination with historical demographic parameters could explain the intraspecific genetic variation indexes across the seven species. In particular, species producing consistently less eggs per spawning event showed higher values of haplotype diversity. Moreover, Tajima's D parameters well explained the recorded values for haplotype diversity and average γst. We concluded that current intraspecific gene diversities in crabs inhabiting mangrove forests were affected by population fecundity as well as past demographic history. The results were also discussed in terms of management and conservation of fauna in the Western Indian Ocean mangroves.

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies.

Heterologous protein production is a key technology for biotechnological, health sciences and many other research fields. Various approaches have been developed for its optimization, but the research emphasis has been on optimization of protein yield rather than protein quality. In this study, we have established a workflow for synthetic gene optimization for heterologous protein expression that combines bioinformatics, laboratory experiments, mass spectrometry and statistical analysis. Two gene primary structure analysis platforms, Anaconda and EuGene, and multivariate optimization methods were employed to re-design the Plasmodium falciparum lysyl-tRNA synthetase gene for optimal expression in Escherichia coli. Synthetic genes were expressed from common vectors, and amino acid mis-incorporations in the expressed proteins were detected and quantified using mass spectrometry. The association between the identified amino acid mis-incorporations and 23 gene variables was then analysed. The synthetic genes yielded significantly higher levels of protein relative to the wild-type gene, but 71 amino acid mis-incorporation sites were observed along the whole protein and across the synthetic genes that were statistically associated with specific codons and protein secondary structures. The optimization method that led to production of the most accurate protein was based on a multivariate approach that combined variables that are known to influence mRNA translation.