A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum

Abstract

Insect neurohormones (biogenic amines, neuropeptides, and protein hormones) and their G protein-coupled receptors (GPCRs) play a central role in the control of behavior, reproduction, development, feeding and many other physiological processes. The recent completion of several insect genome projects has enabled us to obtain a complete inventory of neurohormone GPCRs in these insects and, by a comparative genomics approach, to analyze the evolution of these proteins. The red flour beetle Tribolium castaneum is the latest addition to the list of insects with a sequenced genome and the first coleopteran (beetle) to be sequenced. Coleoptera is the largest insect order and about 30% of all animal species living on earth are coleopterans. Some coleopterans are severe agricultural pests, which is also true for T. castaneum, a global pest for stored grain and other dried commodities for human consumption. In addition, T. castaneum is a model for insect development. Here, we have investigated the presence of neurohormone GPCRs in Tribolium and compared them with those from the fruit fly Drosophila melanogaster (Diptera) and the honey bee Apis mellifera (Hymenoptera). We found 20 biogenic amine GPCRs in Tribolium (21 in Drosophila; 19 in the honey bee), 48 neuropeptide GPCRs (45 in Drosophila; 35 in the honey bee), and 4 protein hormone GPCRs (4 in Drosophila; 2 in the honey bee). Furthermore, we identified the likely ligands for 45 of these 72 Tribolium GPCRs. A highly interesting finding in Tribolium was the occurrence of a vasopressin GPCR and a vasopressin peptide. So far, the vasopressin/GPCR couple has not been detected in any other insect with a sequenced genome (D. melanogaster and six other Drosophila species, Anopheles gambiae, Aedes aegypti, Bombyx mori, and A. mellifera). Tribolium lives in very dry environments. Vasopressin in mammals is the major neurohormone steering water reabsorption in the kidneys. Its presence in Tribolium, therefore, might be related to the animal’s need to effectively control water reabsorption. Other striking differences between Tribolium and the other two insects are the absence of the allatostatin-A, kinin, and corazonin neuropeptide/receptor couples and the duplications of other hormonal systems. Our survey of 340 million years of insect neurohormone GPCR evolution shows that neuropeptide/receptor couples can easily duplicate or disappear during insect evolution. It also shows that Drosophila is not a good representative of all insects, because several of the hormonal systems that we now find in Tribolium do not exist in Drosophila.

Introduction

Insects are the largest animal group living on earth (75% of all animal species are insects) and are extremely important for agriculture and ecology, because 70% of all flowering plants depend on insects for their pollination. Insects, however, can also be severe agricultural pests, destroying 30% of our potential annual harvest and can be the vectors for major diseases, such as malaria, which causes 1.5–2.7 million deaths each year.

Because of the importance of insects, 24 insect genome projects have been initiated during the last 8 years and several of them have been completed. These insects with a genome project are listed in Fig. 1 and are either medically or agriculturally important, or are model organisms (such as Drosophila). The most recent addition to the list of insects with a fully sequenced genome is the red flour beetle Tribolium castaneum[134]. Tribolium belongs to the insect order Coleoptera (or beetles), which is the largest insect order and which comprises about 40% of all insect species. Thus, about 30% of all animal species living on earth are coleopterans.

Insects can be subdivided into Holometabola (insects with a complete metamorphosis, from larvae to pupae to adults) and Hemimetabola (insects with an incomplete metamorphosis, where the larvae, also called nymphae, resemble the adults). So far, only the genomes from holometabolous insects have been fully sequenced (Fig. 1).

The Holometabola consist of four major insect orders, the most basal being Hymenoptera (to which honey bees, wasps and ants belong), followed by Coleoptera, Lepidoptera (butterflies and moths), and Diptera (flies) [139], [162]. The evolutionary distance between Hymenoptera and Diptera is about 340 million years [33], [139], a distance comparable to that between bony fishes and humans.

Tribolium has been selected for a genome project, because it is agriculturally important and a major global pest of stored grain, flour and many other dried and stored commodities for human consumption. Furthermore, Tribolium is a versatile genetic model for studies of eukaryotic development (http://www.hgsc.bcm.tmc.edu/projects/tribolium/TriboliumWhitePaper.pdf). The size of the Tribolium genome is about 190 Mb and it contains more than 16,400 genes [134].

The genomic data from Tribolium are highly relevant not only for our understanding of the biology of Tribolium, but also for the understanding of the other coleopterans, which comprise about 600,000 species. One would naively expect that the results from the Tribolium Genome Project would be somewhat intermediate between those from the more primitive honey bee (Hymenoptera) and the more advanced fruitfly (Diptera). However, as we can read in this review, each insect group has specialized itself and adapted to its ecological niche. As a consequence, Tribolium has obtained (or retained) characteristics that can not be found in the honey bee or in Drosophila.

G protein-coupled receptors (GPCRs)¹ are the largest superfamily of transmembrane proteins in metazoans and up to 1–2% of all genes in an animal’s genome code for GPCRs [43]. GPCRs have seven α-helical transmembrane domains and are, therefore, also called seven transmembrane, or 7TM receptors. They interact at their cytoplasmic loops with G proteins, leading to second messenger cascades [46]. GPCRs and their G proteins even occur in plants and prokaryotes, showing that these signaling systems are evolutionarily very old [50], [101].

Our research group is especially interested in neurohormone GPCRs and their ligands (biogenic amines, neuropeptides and protein hormones), because these molecules occupy a central position in the physiology of animals and steer important processes, such as behavior, reproduction, and development. Neurohormone GPCRs are an important, but only smaller subset of the large GPCR superfamily. These GPCRs belong to two subfamilies, family-A (family-1, or rhodopsin-like) GPCRs, and family-B (family-2, or calcitonin-like) GPCRs [46]. The biogenic amine GPCRs belong to family-A, whereas the neuropeptide and protein hormone GPCRs cover both families. Because the Drosophila genome was the first insect genome to be sequenced [1], most information on insect neurohormone GPCRs is available from Drosophila[56], [57].

In general, after an insect genome has been sequenced and assembled, software programs are applied to predict all the genes present in the genome and, by comparison with genes already identified from other genomes, to propose a function for these genes (annotations). These automated annotations have, of course, to be verified by manual curation and subsequent experimental approaches, such as PCR and cloning. Shortly after the publication of the Drosophila genome [1], Hewes and Taghert [59] annotated 21 genes as coding for biogenic amine GPCRs and 44 genes for neuropeptides and protein hormone GPCRs. During the last few years, we have found 5 additional neuropeptide GPCRs, bringing the total number of neurohormone GPCRs in Drosophila first to 69 [56], [57], and now to 70 (present paper, Fig. 2, Fig. 3A, Fig. 3B, Fig. 4). Already before the publication of the Drosophila genomic sequence [1], a few neuropeptide and biogenic amine GPCRs had been cloned and their ligands determined. The publication of the Drosophila genome, however, has greatly boosted this line of research and currently the ligands for 17 biogenic amine GPCRs and 31 neuropeptide and protein hormone GPCRs have experimentally been identified [57]. Thus, for 48 or nearly 70% of all Drosophila neurohormone GPCRs, the ligands are known (we say that these Drosophila GPCRs have been deorphanized). Although the remaining 30% of these Drosophila GPCRs are certainly more difficult to deorphanize, we are confident that this will happen and that we will have a rather complete picture of Drosophila GPCRs and their ligands within the next few years.

To deorphanize a neurohormone GPCR, its cDNA has to be cloned and its coding region expressed in cultured mammalian cells or Xenopus oocytes [25], [26], [57]. The transformed cells are then exposed to peptides or other ligands from a chemical library or to a tissue extract containing the ligand. When a second messenger response occurs (or an ion current in the case of oocytes), we have a bioassay enabling the ligands to be purified and identified. There are many ways to measure the various second messenger responses or to manipulate the host cells in such a way that they are optimally reacting to GPCR activation by its ligand. Our research group has especially applied a technique, originally developed by Stables et al. [146], where Chinese hamster ovary (CHO) cells are transfected with both DNA coding for a “universal” (or “promiscuous”) G protein α-subunit, Gα-16, and DNA for the insect GPCR. Activation of Gα-16 in these cells leads to an increase in intracellular Ca²⁺ concentration, which is measured as a strong (up to 400× over background) bioluminescence response [57]. More than half of the above-mentioned 48 identified Drosophila receptors has been deorphanized with this technique by us and other groups.

Our increased knowledge of Drosophila neurohormone GPCRs and their ligands strongly contributes, of course, to our understanding of Drosophila. This understanding of Drosophila’s neurobiology and endocrinology will be further improved by the application of genetic tools such as the knocking-out of GPCR and neuropeptide genes. Since a few years, there has existed a Drosophila Gene Disruption Project, where, using P-element or piggyBac insertions, more than half of all Drosophila genes have been disrupted (http://flypush.imgen.bcm.tmc.edu/pscreen/). The current collection of mutants also contains flies with disrupted neurohormone GPCR and neuropeptide genes and it can be expected that the phenotypic characterization of these Drosophila mutants will teach us much about the functioning of the neurohormone/GPCR couples. This will certainly challenge many of the existing paradigms in insect neuroendocrinology.

One can expect that Drosophila neurohormone GPCRs will become a reference for our understanding and interpretation of other insect neurohormone GPCRs. For example, in a previous review on honey bee neurohormone GPCRs, we have used the Drosophila GPCRs as a “gold standard” to annotate the honey bee orthologues [57]. On the other hand, Drosophila can not be regarded as a perfect representative of all holometabolous insects and there are many biological processes absent in Drosophila that exist in other insects and vice versa. To obtain a better insight into the biology of insects, therefore, it is crucial also to investigate representatives of other insect orders and to study them with the same rigorousness as in Drosophila. The first step in this direction is the sequencing of their genomes (Fig. 1), followed by proper annotation and analysis of their genes.

In our current review, we have annotated the neurohormone GPCR genes in the first beetle with a sequenced genome, the red flour beetle Tribolium, using mainly Drosophila GPCR genes as a reference. Furthermore, we have compared these results with the neurohormone GPCR genes present in the honey bee and fruitfly. Tribolium is living in very dry surroundings and it can be expected that its endocrine system is organized in a different way from that of Drosophila. Therefore, we have not only used Drosophila GPCRs for our TBLASTN searches, but also GPCRs from several other animals, including mammals.

In a recent paper, we have determined the occurrence of neuropeptides and protein hormone genes in the genome of Tribolium and we have also experimentally confirmed the presence of many of these neurohormones, using mass spectrometry [100]. A summary of these neurohormone findings is given in Table 1. We found that our current neurohormone GPCR annotations perfectly fit to the independently annotated neurohormones. The outcome from the two studies is highly relevant for our understanding of the neurobiology of Tribolium. They also give us excellent insights into the evolution of neurohormone GPCRs in insects and their co-evolution with the ligands.