Identifying the genetic basis of interesting phenotypic variation in non-model systems is often limited by the lack of sophisticated molecular resources, such as complete genome sequences and DNA microarrys, that are available in model genetic taxa such as Drosophila [1], Anopheles [2], Caenorhabditis [3] and Apis [4]. However, the declining costs of developing genomic tools and the proliferation of accessible methods by which these tools can be generated holds promise for genomic-scale studies in organisms that offer profound insights into fundamental biological questions. Thus, there is a growing need to develop better genomic resources for these emerging systems.
The Orthoptera contain many such emerging systems. Consisting of over 25,000 species [5], the order Orthoptera is composed of two major lineages, the crickets and katydids (Ensifera) and the grasshoppers (Caelifera) [6,7] which diverged approximately 300 MYA. While well known for their economic impact on world-wide agriculture [8-13], they have been intensively studied in a wide variety of biological areas. For example, orthopterans have been used to study various aspects of neurobiology [14-17], physiology [18-21], behavior [10,22-24], development [17,25-28], sexual selection [29-35], and evolution [7,32,36-43]. However, very few genomic tools have been developed for this group of insects.
While genomic studies of many orthoptera are ongoing [44,45], large scale genomic resources have been developed for only one species in this order, Locusta migratoria (Caelifera) [45,46]. Research on Locusta has produced 12,161 unique sequences and provides a necessary counterpoint to the heavy phylogenetic bias in extant genomic resources. [47-50]. However, as described above, orthopterans are a phylogenetically diverse lineage which are being used to study a broad set of biological questions. The Gene Index presented here was developed to address three distinct but overlapping areas of orthopteran biology: neurobiology, speciation, and evolution.
For over 50 years, the Orthoptera have been used as a neurobiological model system by which the relationship between neural activity, muscular response and behavior are studied [51]. In particular, the study of orthopteran flight and song, or stridulation, have provided valuable insights into the physiological basis of behavior and the structure and function of Central Pattern Generating (CPG) circuits [52-55]. CPG circuits are responsible not only for orthopteran flight and song, but also for nearly all vital functions, such as circulation, respiration, digestion and locomotion, in both vertebrates and invertebrates. Since at least 1973, neuroethologists have called for the development of genetic tools to understand the creation, function, and diversification of the neural circuits responsible for cricket stridulation [56]. One result has been the analysis of the inheritance of species-specific songs [57,58] and a quantitative trait locus study of song (Shaw et al. in press). Yet the tools necessary to study the action and influence of individual genes remain largely absent. The EST's of this Gene Index, since they are derived from a nerve cord library, contain genes expressed in nervous system. Many of the EST's identified here may be involved in the construction of the flight and/or stridulation CPG.
Furthermore, our study organism, Laupala kohalensis, is a superb organism with which to investigate the genetic basis of CPG construction and evolution. The 38 species of Laupala have diverged within the past five million years [59]. The diversification of Laupala has been extraordinarily rapid, as Laupala contains the fastest diversifying arthropod clade recorded to date [59]. The radiation is also noteworthy for the extremely limited number of features that distinguish species. Members of this genus appear morphologically and ecologically similar and many closely related species often differ by fewer than 0.1% of nuclear gene bases [60]. However, pulse rates of male calling songs have diverged extensively in Laupala [61]. Given the diversity of pulse rate CPG's in this clade and the limited amount of genetic divergence that separates species, the release of the Laupala Gene Index will provide an extraordinary genomic tool by which CPG evolution may be studied.
In addition to providing a powerful platform for comparative studies of CPG evolution, Laupala is a well-developed model system for the study of reproductive isolation and the formation of species [33,34,38,59,60,62-66]. The 38 species within this genus are believed to have diverged in part via coordinated evolution in male song and female acoustic preference [33,34,65]. While there exists an extensive body of literature on the evolution of sexual isolation and the formation of species, identifying the specific genetic basis of either process has been limited to an extremely small number of taxa for which the appropriate genetic tools have been developed. The release of this cricket Gene Index will allow researchers to build on the genetic work of Hoy and Paul [56], which demonstrated a polygenetic basis of cricket songs, and Shaw [58,66], which supported Hoy and Paul's findings and identified several chromosomal regions associated with song, by providing the tools necessary to identify specific genes involved in cricket stridulation, sexual isolation and the formation of species. Identifying the genes involved in any of these processes would represent a significant achievement.
From a comparative perspective, the publication of the Laupala Gene Index is a significant advancement in the tools available to study molecular evolution in insects. To date, major insect genome projects have focused primarily on the Diptera (e.g., fruitflies and mosquitoes; [1,2]), Hymenoptera (e.g. honeybee; [67]), and Lepidoptera (moths and butterflies; [68-70]). All of these lineages belong to a single superorder (Endopterygota) and, thus, represent only a small portion of the phylogenetic diversity encompassed by the broader class Insecta (Figure 1 &2). While the evolution of complete metamorphosis (Holometabolous, Endopterygota) was certainly one of the most significant events in the history of insect diversification [71], the heavy phylogenetic bias of previously developed genomic resources has severely limited broader inferences about the evolutionary history of insects in general. Indeed, only recently have researchers begun to address this phylogenetic bias in studies of arthropod evolution [72,73] and the genomes of an Aphid [74] and Louse [75] soon will be available. Therefore, the compilation of a basal insect genomic resource, such as the one presented here, will facilitate genomic comparisons across 350 million years of insect diversification, and will serve as a phylogenetic link to even more distant comparisons, such as crustaceans (e.g.Daphnia) and chelicerates (e.g. tick), and beyond. For example, one of the early developmental studies of arthropod body patterning genes utilized EST sequences cloned from Schistocerca (Orthoptera: Caelifera) and Tribolium (Coleotpera) to demonstrate the homology between the Drosophila hox gene zen and its' human ortholog, HOX3 [76]. Thus, the benefits of developing sophisticated genomic resources for non-model organisms are potentially much broader than typically recognized.
The current study represents the first major initiative to develop a large genomic resource for a cricket species of the orthopteran suborder Ensifera (crickets and katydids). We present the sequences of 14,502 Expressed Sequence Tags (EST) from a Laupala kohalensis nerve cord cDNA library. We expect that the release of this Gene Index will provide much needed tools for the study of CPG construction and evolution, sexual selection and speciation, and the molecular evolution of arthropods.
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