Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni

Abstract

BackgroundCichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade.ResultsUtilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages.ConclusionWe characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2914-9) contains supplementary material, which is available to authorized users.

Distribution and female reproductive state differences in orexigenic and anorexigenic neurons in the brain of the mouth brooding African cichlid fish, Astatotilapia burtoni

“…For npy, while mRNA was primarily localized to all of the same regions labeled with the NPY antibody ( Figure 2c,d) (with the exception of weak to no label in DT and gTN neurons with ISH, but strong antibody label), there were also numerous additional cells labeled throughout the brain by ISH that were not detected by the antibody (e.g., ICL, Vs, PGZ, IO, SGn, SVn). Duplicate npy genes (npya, npyb) are identified in A. burtoni (Hu et al, 2016), and our npy ISH results likely represent the distribution of npya. We chose to examine this form rather than the teleost-specific duplicate, npyb, because in the related Nile tilapia O. niloticus, npya is expressed at much higher levels in the brain compared to npyb, responds to changes in feeding conditions that are not evident in npyb, and intracranial injections stimulate food intake (Yan et al, 2017).…”

Plasticity for colour adaptation in vertebrates explained by the evolution of the genes pomc, pmch and pmchl

“…The alignment of the various PMCHL shows an Mgrp putative sequence only in Group 8 species, where Nile tilapia sits (Figure ), supporting the idea that taxonomic grouping of fish reflects evolutionary lineage. Of note, mass spectrometry shows Mgrp is present in the pituitary of Group 8 medaka ( Oryzias latipes ) (Suehiro et al, ), but not in flounders ( Verasper moseri and Astatotilapia burtoni ), although PMCH, PMCHL, MCH/L peptides are all present (Hu et al, ; Takahashi et al, ).…”

Expression of tachykinin3 and related reproductive markers in the brain of the African cichlid fish Astatotilapia burtoni

“…Despite tac3 expression in the pituitary of tilapia (Biran et al, ), zebrafish (Biran et al, ; Ogawa et al, ), goldfish (Qi et al, ), grass carp (Hu, He, Ko, Lin, & Wong, ), and orange spotted grouper (Chen et al, ), we did not detect tac3a mRNA in the A. burtoni pituitary via PCR or ISH. Similarly, a recent study of pituitary peptides detected tachykinin1 (substance P) and tachykinin4 (hemokinin) but no tac3 (NKB) product by mass spectrometry of A. burtoni pituitaries (Hu et al, ), further suggesting that tac3/NKB is absent from the A. burtoni pituitary despite its presence in the pituitary of the closely related Nile tilapia (Biran et al, ). It is possible that expression levels were too low for detection, however other studies have found pituitary levels of tac3 to be similar to that found in the hypothalamus (Biran et al, ; Qi et al, ).…”

“…The tac3a gene closely resembled tac3 genes previously identified in other African cichlids (Biran et al, ). In addition, tac3b was identified based on the scaffold location produced from a bioinformatics study of A. burtoni pituitary prohormones (Hu et al, ). The location identified in that study yielded a sequence that partially resembled the tac3b sequence of other fishes.…”

“…The gene encoding NKB, tachykinin3 , has multiple forms in some fishes, like zebrafish and goldfish (Biran et al, ; Ogawa et al, ; Qi et al, ), but more recently evolved fishes lost the tac3b gene (Biran et al, ; H. Chen et al, ). A bioinformatics study of the genomes of several African cichlids failed to identify a tac3b gene (Biran et al, ); however, a recent study of Astatotilapia burtoni prohormones identified a potential tac3b gene (Hu et al, ), which has yet to be further investigated. Since studies have been performed in three cyprinid fishes with known tac3a and tac3b genes, it is important to also examine whether tac3b exists in percomorph fishes.…”