In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite

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Abstract

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identifieid, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown.

Graphical abstract

Highlights

► The spider mite genome was mined for neuropeptides and neurohormones. ► Fifty one genes coding for neuropeptides and neurohormones were identified. ► Sixty five genes which likely code for neuropeptide and neurohormone receptors were found.

Introduction

A large body of literature exists on the endocrine and neuroendocrine regulatory systems in insects and crustaceans, but much less is known about chelicerates. Some of the interest in insect (neuro-)endocrinology is because many species are either agricultural pests or important vectors of disease and a better understanding of their endocrine and nervous system may lead to new and better pesticides. Ticks are also important disease vectors while the scabies mite causes its own medical problems and other mites are very destructive in agriculture. With the exception of ticks, many of these species are too small for classical (neuro-)endocrine experiments, and consequently the little we know about this group comes mostly from work on blood-feeding ticks and concerns immunohistology, transcriptome and mass spectrometry data from synganglia of blood-feeding ticks. Those studies have identified neuropeptide and neuropeptide receptor EST and cDNA sequences and have also identified some peptides directly by mass spectrometry (e.g. Holmes et al., 2003, Šimo et al., 2009a, Šimo et al., 2009b, Neupert et al., 2009, Donohue et al., 2010, Bissinger et al., 2011). There are also a few publications on the horse shoe crab (e.g. Groome et al., 1990, Gaus et al., 1993).

Our understanding of insects has improved significantly with the sequencing of entire insect genomes, particulary with the one of Drosophila melanogaster, which is also a great experimental model. Although Drosophila is an arthropod, its value as a model for chelicerates is limited. To remedy this problem it has been suggested to develop the spider mite Tetranychus urticae as a general chelicerate model (Grbić et al., 2007, Khila and Grbić, 2007). Like Drosophila, this species has a very small genome, but unlike Drosophila, which rarely reaches significant pest status, this polyphagous spider mite causes very significant economic damage on a variety of crops and is furthermore a species which rapidly develops pesticide resistance. The recently sequenced genome of this species (Grbić et al., 2011) is a first step in this direction and has already greatly advanced our knowledge about this species in revealing both similarities and differences with other arthropods. The combination of a small genome size, good amount of EST sequences and extensive RNAseq data has allowed for a good and very complete Tetranychus genome which will be of great value for further work on spider mites in particular, but also for other chelicerates.

In the initial description of a genome there is no place to describe all its genes and it is here that we describe the genes coding neuropeptides, neurohormones and their G-protein coupled receptors (GPCRs). The large majority of the neuropeptide and neurohormone genes were identified by their homology to known arthropod neuropeptides and neurohormones, but a few are better known from mollusks. We found a total of 51 genes encoding neuropeptides and neurohormones and more than 60 genes encoding G-protein coupled receptors (GPCRs) that may be neuropeptide receptors. Some of the neuropeptide and neurohormone ESTs which were generated for this genome project have been previously reported (Christie et al., 2011).

Section snippets

Materials and methods

Initial searches were done on the Tetranychus genome using the BOGAS (Bioinformatics Online Genome Annotation Service) web site (http://bioinformatics.psb.ugent.be/webtools/bogas/). Details on the genome and its annotation have been described in detail elsewhere (Grbić et al., 2011). Alternatively, the genome was downloaded and analyzed on a desktop using the the BLAST package (Altschul et al., 1997) obtained from (www.ncbi.nlm.nih.gov/blast/Blast.cgi). For the identification of signal peptides

Results and discussion

We found 51 genes likely to encode neuropeptides or neurohormones and a total of 65 genes for putative neuropeptide GPCRs. Of the latter 58 belong to the A family and 7 to the B1 family. The 7 identified putative hormone receptors of the B-family all have the characteristic hormone receptor domain with its conserved cysteine residues in addition to its seven transmembrane regions. BLAST searches do not only find true homologs but also proteins that have an overall similarity to the proteins

Conclusion

A significant number of genes encoding spider mite neuropeptides and neurohormones was identified, most of them by virtue of their sequence similarity to insect orthologs. The strong resemblance between these two groups is somewhat deceiving, as homology searches reveal the neuropeptides that are conserved between insects and mites, but do not identify chelicerate or mite specific neuropeptides. One fundamental difference between insects and acari concerns juvenile hormone, ubiquitous in

Author contributions

JAV prospected the genome, analyzed the genes and wrote the paper, MG organized the sequencing of the genome and SR was responsible for the integration of the genomic and RNAseq data into a single bioinformatics platform. All authors read and approved of the final manuscript.

Acknowledgments

Prospecting a genome for the presence of specific genes is only possible after a genome has been sequenced. Analysis of the spider mite genome was immeasurably facilitated by the generation and analysis of deep RNAseq data (to aid the annotation and establish expression) by Edward J. Osborne and Richard M. Clark (University at the University of Utah, USA). We are grateful to both of them for providing these data in advance of publication. We are also grateful to those who performed the genomic

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