Research paper
Gonadulins, the fourth type of insulin-related peptides in decapods

https://doi.org/10.1016/j.ygcen.2020.113528Get rights and content

Abstract

Insulin and related peptides play important roles in the regulation of growth and reproduction. Until recently three different types of insulin-related peptides had been identified from decapod crustaceans. The identification of two novel insulin-related peptides from Sagmariasus verreauxi and Cherax quadricarinatus suggested that there might a fourth type. Publicly available short read archives show that orthologs of these peptides are commonly present in these animals. Most decapods have two genes coding such peptides, but Penaeus species have likely only one and some palaemonids have three. Interestingly, expression levels can vary more than thousand-fold in the gonads of Portunus trituberculatus, where gonadulin 1 is expressed by the testis and gonadulin 2 by the ovary. Although these peptides are also expressed in other tissues, the occasionally very high expression in the gonads led to them being called gonadulins.

Introduction

Neuropeptides and neurohormones originated early during evolution and are well conserved between decapods and insects (e.g. Veenstra, 2016). In several cases their functions are also the same or at least similar, this is e.g. the case of crustacean cardioactive peptide (CCAP) that plays crucial roles during molting in both decapods and insects (Webster et al., 2013). Other peptides on the other hand likely have very different functions. EFLamide for example is very abundantly expressed and effects many different tissues in decapods (Dickinson et al., 2019), but is present in only some insect species and when present expressed in very few neurons (Veenstra and Šimo, 2020, Kotwica-Rolinska et al., 2020). Allatotropin on the other hand is commonly present in insects but absent from decapods (Veenstra, 2016).

Insulin and related peptides seem to be present in all animals and have been extensively studied in insects where they are important growth hormones. They have been particularly well investigated in Drosophila (see e.g. Brogiolo et al., 2001, Nässel and Vanden Broeck, 2016), but also in other species such as the silk worm Bombyx mori (Mizoguchi and Okamoto, 2013). The best known crustacean insulin-related peptide is the one secreted by the androgenic gland. This gland has been found in all Malacostraca and secretes a hormone that is able to change the sex of animals from female to male as first demonstrated in an amphipod by transplanting those glands (Charniaux-Cotton, 1954). After the initial identification of the hormone made by these glands from an isopod as an insulin-related peptide (Martin et al., 1999) it has also been identified from several decapods (e.g. Ventura et al., 2009, Ventura et al., 2011, Chung et al., 2011). However the physiological relevance of the other insulin-related peptides in crustaceans remains unclear.

Both growth and sex determination are essential for the survival of a species and hence one would expect that the way these processes are regulated is under significant evolutionary pressure and might be conserved between insects and crustaceans. The insect insulin-like growth hormones and the decapod Insulin-like Androgenic Gland hormone, that will be called androgenin from here on, are evolutionarily related and I previously suggested that perhaps the androgenins might be orthologs of Drosophila insulin-related peptide 8 (dilp 8), one of the Drosophila insulin-related peptides (Veenstra, 2016). Unlike most insulin-related peptides that stimulate a receptor tyrosine kinase (RTK) dilp 8 acts through a leucine-rich repeat G-protein coupled receptor (LGR; Vallejo et al., 2015, Gontijo and Garelli, 2018). Dilp8 is made by the imaginal disks, that are only present in holometabolous insects, as well as the ovary and testis. The specific LGR (Drosophila LGR3) that is the dilp 8 receptor has orthologs in many other arthropods, including decapods (Veenstra, 2016), meaning that dilp 8 orthologs must be common. Nevertheless, the primary amino acid sequences of dilp 8 and the decapod androgenins are very different. The sequence of dilp 8 is also very poorly conserved within insects. When only three different types of insulin-like peptides had been identified in decapods it seemed an attractive hypothesis that dilp 8 might be an ortholog of androgenin (Veenstra, 2016). After all, the other two are clear orthologs of dilp 7, a relaxin-like peptide, and the insect insulin-related peptides that stimulate growth (Brogiolo et al., 2001, Nässel and Vanden Broeck, 2016). However, soon afterwards two novel decapod insulin-like peptide precursors were identified from Sagmariasus verreauxi and Cherax quadricarinatus that look more similar to dilp 8 than androgenin (Chandler et al., 2017). This suggests that decapods do not have three but at least four different types of insulin-like peptides and that dilp 8 is probably not an ortholog of androgenin. This provided stimulation to exploit the large amount of genomic and especially trancriptomic sequences available for decapods for additional clues as to the presence and distribution of insulin-related peptides in decapods. I here report that the peptides initially described from S. verreauxi and C. quadricarinatus belong to a novel type of decapod insulin-like peptides that in many species is produced by two and in some species even three paralogous genes, one or two of which seem to be rarely expressed. However, under particular conditions, that remain to be identified, the transcripts for these peptides are occasionally made in very large numbers by the gonads, at least in the crab Portunus trituberculatus. As the gonads seem to be an important site of expression of these peptides, they have been baptized gonadulins.

Section snippets

Materials and methods

I analyzed transcriptome and genome SRAs using the tools from the SRAtoolkit (https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software), Trinity (Grabherr et al., 2011) and Artemis (Rutherford et al., 2000) on the publicly available decapod genomes and draft genomes, as well as a large number of transcriptome and genome SRAs (Table S1). Details of the methods that were used have been described in more detail elsewhere (Veenstra and Khammassi, 2017, Veenstra, 2019). A list of all SRAs

Results

This manuscript describes the gonadulins and their putative receptors, however other decapod insulin-like peptides were also identified in an attempt to establish where all the different insulin-related peptides and their putative receptors are expressed. Although gonadulins were identified in many decapod species, identification of their putative receptors and the expression of these genes was limited to eight species. These are Penaeus vannamei, P. monodon, Macrobrachium nipponense, Cherax

Discussion

Three different types of insulin-related peptides have previously been shown to be commonly present in decapod crustaceans, the relatively well known androgenin, arthropod relaxin, and a third peptide, insulin, that shares characteristics with its mammalian homonyme. The similarity of these peptides with their putative orthologs from insects suggested that androgenin might be an ortholog of dilp 8 (Veenstra, 2016). However, this hypothesis was challenged by the description of yet another type

Acknowledgements

I thank three anonymous reviewers for their constructive criticism and help for improving this manuscript. As always for this type of research I am analyzing data I did not collect myself and using methods developed by others. Without either these tools or data, I would not be able to do this and I express my sincere gratitude to all of those who made this possible. I apologize to all those whose SRA data I used without citing the papers that described them; there are simply too many.

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