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

BACKGROUND: Cichlid 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.
RESULTS: Utilizing 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.
CONCLUSION: We 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.

Background

Ray-finned fishes comprise ~50 % of all vertebrate species and of these, teleost fishes are the most diverse clade. They are found in most aquatic habitats and exhibit vast behavioral differences between species. Among teleost fishes, cichlids are one of the most species-rich families and the African cichlids, in particular, provide exceptional and unique opportunities for understanding speciation and behavioral adaptations in the African Great Lakes [1]. Cichlid phenotypic diversity in behavior, body shape, coloration, and ecological specialization is unparalleled. Some substrates for cichlid morphological diversity include well-conserved morphogen systems, and potentially the Hox gene clusters [2-4]. Although cichlid behavioral diversity has encouraged behavioral and neurobiological studies directed at understanding how brain evolution has been shaped by natural and sexual selection, the molecular and cellular bases of teleost/cichlid behavioral diversity are still largely unknown. This status is poised to change. Not only have analyses shown remarkable social and cognitive skills associated with cichlid group living [5], the recent sequencing of five cichlid genomes [6] and the development of cichlid transgenesis techniques [7, 8] have opened the door to greater understanding of the underlying mechanisms.

It has been speculated that the rich diversity and complexity of behaviors found in teleosts partially derives from a whole genome duplication (WGD) in the teleost ancestor after the divergence from other vertebrate lineages [9, 10]. Approximately 85 % of genes resulting from this duplication were subsequently lost, but amongst the retained duplicates, genes associated with brain function are overrepresented [9, 11]. Retained duplicated genes can be a source of novel gene function as they frequently undergo subfunctionalization, neofunctionalization, or some combination of the two [12]. In the haplochromine cichlid lineage, for example, neofunctionalization of a paralog has been linked to the morphological innovation of male fin spots involved in mating behavior [13].

Neuropeptides play a pivotal role in both ancient and recently derived examples of animal behavior. For example, the galanin peptide is functionally associated with the regulation of feeding, anxiety-related behaviors, and parental behavior in mammals [14, 15]. Galanin’s orexigenic function has been described in teleosts, but whether it serves additional neuroendocrine or behavioral functions is unknown [16]. In Astatotilapia burtoni, whole brain galanin (GAL) expression is higher in socially dominant males compared to socially subordinate males [17]. Duplication of prohormone genes such as proopiomelanocortin (POMC) that encode multiple neuropeptides may similarly be a source of behavioral innovation through change in expression and sequence. Teleosts possess duplicate POMC genes and A. burtoni, as well as several other teleost lineages, have undergone a more recent POMC1 duplication to generate POMC1A and POMC1B [18]. Many POMC peptide products, including melanocortins and β-endorphin, exert pleiotropic functions in multiple tissues, including the nervous system, reproductive system, and skin. Thus, regulation of POMC is a possible mechanistic link between behavior, physiology, and coloration within and across species [19]. Identification of which POMC versions are being expressed and which peptides are present is essential to understanding this link.

This effort to characterize the prohormone gene and novel neuropeptide complement for A. burtoni is the first comprehensive bioinformatic survey of any single ray-finned fish species. A. burtoni is a haplochromine cichlid with an advantageous phylogenetic position and a well-characterized natural history [20], and has undergone extensive physiological, neurobiological, and molecular analyses [21]. Molecular phylogenetics place this species in a sister group to the extremely large cichlid species flocks in Lakes Victoria and Malawi in East Africa. A. burtoni is hypothesized to be similar to the ancestor of these flocks because it is a trophic generalist endemic to the neighboring Lake Tanganyika and surrounding rivers [22, 23]. Thus, discoveries about the peptidome of A. burtoni are significant to the entire ‘modern haplochromine’ lineage, which represents ~7 % of all extant teleosts.

Results and discussion

We surveyed the A. burtoni genome for prohormone genes as well as the major processing enzymes used to form bioactive peptides from the prohormone proteins. While previous studies have only examined individual prohormones and specific prohormone families, our study provides a comprehensive summary of all known prohormones. As the final bioactive complement requires the prohormone and appropriate processing enzymes, we also characterized the peptides themselves within the endocrine pituitary using mass spectrometry (MS). Finally, in situ hybridization was used to localize the expression of 2 prohormones, GAL and POMC.

Our survey identified 158 A. burtoni genes, with 148 prohormone genes and 10 prohormone convertase subtilisin/kexin (PCSK) genes. All prohormone genes, including current accession numbers and genomic locations [6], are provided in Additional file 1: Table S1. All predicted sequences can be found in FASTA format in Additional file 2: Text S1, which also includes 7 genes with 2 splice variants and 1 gene with 3 splice variants. All predictions except glucagon II (GCG2), kisspeptin-2 (KISS2), neuropeptide VF precursor (NPVF), prokineticin 2 (PROK2) and parathyroid hormone A (PTHA) were supported by A. burtoni expressed sequence tag (EST) data. Predictions without A. burtoni EST data were all supported by Oreochromis niloticus (Nile tilapia) EST data. Gastrin-releasing peptide (GRP) was not identified in the assembly but was identified from A. burtoni and O. niloticus EST data.

The 148 prohormone genes consisted of 6 genes that were only identified in fish, 21 genes identified with a single copy, and 39 genes with at least 2 duplicated copies (Table 1). Compared to the average rate of gene retention following the teleost WGD of 15 % [9, 11], we found prohormone genes to be retained at 41 % (39 genes with multiple copies out of 96 unique prohormone genes). Almost 66 % of the currently identified prohormone families contained at least one gene duplication (23 out of 35 families) (Table 1). The 6 predictions lacking direct mammalian counterparts are likely due to either gene loss in tetrapods or additional duplication after divergence of cichlid and mammals.

Table 1

Predicted A. burtoni prohormone families and duplication status

Neuropeptide Family	N Ga	N Db	Gene Symbolsc	References
Apelin	1	0	APLN	[70]
Augurin	1	0	AUGN
AVIT/Prokineticin	2	0	PROK1; PROK2
Bombesin/Neuromedin-B/Ranatensin	3	2	GRP; NMB1; NMB2	[113]
Calcitonin	8	4	ADM1A; ADM1B; ADM2A; ADM2B; ADM5; CALCA; CALCB; IAPP	[114]
CART	6	6	CARTPT1; CARTPT2; CARTPT3; CARTPT4; CARTPT5; CARTPT6	[71]
clpA/clpB family. Torsin subfamily.	1	0	TOR2A
Corticotrophin	5	2	CRH1A; CRH1B; UCN2; UCN3; UTS1	[50, 115–117]
Endothelin/Sarafotoxin	6	6	EDN1A; EDN1B; EDN2A; EDN2B; EDN3A; EDN3B	[118]
Gastrin/Cholecystokinin	3	2	CCK1; CCK2; GAST	[119]
Ghrelin/Motilin-related	2	0	GHRL; MLN	[119, 120]
Glucagon	8	4	ADCYAP1A; ADCYAP1B; GCG1A; GCG1B; GCG2; GHRH; GIP; VIP	[35, 37, 121]
Gonadotropin-releasing hormone	3	0	GNRH1; GNRH2; GNRH3 d	[28, 122–124]
Granin	9	4	CHGA; CHGB; PCSK1N; SCG2A; SCG2B; SCG3; SCG5; VGF1; VGF2	[73, 125, 126]
Hepcidin	5	5	HAMP1; HAMP2; HAMP3; HAMP4; HAMP5	[68]
Insulin	5	2	IGF1; IGF2; IGF3 d; INS1; INS2	[32, 127]
Kisspeptin/Galanin/Spexin	4	2	KISS2 d; GAL; SPX1; SPX2	[61, 62, 128–130]
Melanin-concentrating hormone	2	2	PMCH1; PMCH2	[131]
Natriuretic peptide	6	4	NPPA; NPPB; NPPC1; NPPC2; NPPC3; NPPC4	[70]
Neuropeptide B/W	2	2	NPB1; NPB2	[53]
Neurotensin	1	0	NTS
Neuromedin	2	0	NMS; NMU	[132]
Neuropeptide Y	4	4	NPY1; NPY2; PYY1; PYY2	[54, 55]
Nucleobindin	3	2	NUCB1; NUCB2A; NUCB2B
Opioid	7	5	PDYN; PENK; PNOC1; PNOC2; POMC1A; POMC1B; POMC2	[46–48, 104, 133–135]
Orexin	1	0	HCRT	[136]
Osteocrin	1	0	OSTN
Oxytocin/Vasopressin	2	0	AVP; OXT;	[137]
Parathyroid	6	4	PTH1A; PTH1B; PTH2; PTHLH1; PTHLH2; PTHLH3 d	[57, 58, 138, 139]
PDGF/VEGF growth factor	13	10	FIGF; PDGFA1; PDGFA2; PDGFB1; PDGFB2; PDGFC; PDGFD; PGF1; PGF2; VEGFA1; VEGFA2; VEGFC1; VEGFC2	[33, 34]
Relaxin	6	4	INSL3; INSL5A; INSL5B; RLN1; RLN3A; RLN3B	[33, 34, 140]
Rfamide peptide	5	2	NPFF; NPVF; QRFP; PRLH1; PRLH2	[141–143]
Somatostatin/Urotensin	8	4	SST1; SST2; SST3; SST5; URP1 d; URP2 d; UTS2A; UTS2B	[39, 40]
Tachykinin	6	6	TAC1A; TAC1B; TAC3A; TAC3B; TAC4A; TAC4B	[144]
Thyrotropin-releasing hormone	1	0	TRH	[145]

aN G: Number of identified gene sequences within family

bN D: Number of identified gene sequences within family that have been duplicated

cGene Symbol: Based on Homo sapiens gene symbols where probable duplicated genes are denoted with either a number or lower case letter. Full gene names and symbols are provided in Additional file 2

dProhormones lacking direct mammalian counterparts

Gonadotropin-releasing hormone and oxytocin/vasopressin families

The gonadotropin-releasing hormone (GNRH) and oxytocin/vasopressin families comprise the most functionally well-characterized prohormones in A. burtoni [24, 25]. Our genomic survey confirms previous findings of single copies of three GNRH genes (GNRH1, GNRH2, and GNRH3), as well as single copies of oxytocin (OXT) and arginine vasopressin (AVP). In Oryzias latipes (Japanese medaka fish), GnRH3 peptide is produced in the terminal nerve ganglion and modulates social behavior [26]. It is unknown whether this neuromodulatory role is shared across teleosts and whether this role is fulfilled by GnRH1 in tetrapods, which lack GNRH3 [27, 28]. Evidence based on the receptors suggests that GNRH, OXT, AVP and neuropeptide S (NPS) genes share a common ancestor [29, 30]. Analysis of Branchiostoma floridae (lancelet) AVP, GNRH, and NPS prohormones indicate conserved synteny of these prohormones [31]. It is theorized that the NPS system was lost in ray-finned fish after the duplication of an ancestral system that resulted in the OXT/AVP system and the NPS system [30]. Consistent with this hypothesis, there was no evidence of NPS in A. burtoni.

Insulin and relaxin families

Duplicate copies of insulin (INS1 and INS2) and single copies of insulin-like growth factors 1 (IGF1), 2 (IGF2), and 3 (IGF3) were identified. IGF3 is a teleost-specific, gonad-specific prohormone [32]. Following Wilkinson et al. [33] and Yegorov and Good [34], 2 copies of relaxin 3 (RLN3A and RLN3B) and insulin-like 5 (INSL5A and INSL5B) and single copies of relaxin 1 (RLN1) and insulin-like 3 (INSL3) were also identified. Multiple sequence alignment indicated that INS1, INS2, IGF1, and IGF2 were more similar to the mammalian counterparts than other members of the relaxin family. Similar to Yegorov and Good [34], only the 2 copies of A. burtoni RLN3 showed more similarity to the Homo sapiens (human) relaxin family counterparts than the other identified genes. The other 3 relaxin members had intermediate similarity between the H. sapiens RLN3 and the other H. sapiens relaxin family members.

Glucagon family

Searching the A. burtoni genome identified 6 of the 7 known members of the glucagon family: 2 glucagon 1 (GCG1) copies (GCG1A and GCG1B), 2 adenylate cyclase activating polypeptide 1 copies (ADCYAP1A and ADCYAP1B), and single copies of glucagon 2 (GCG2), gastric inhibitory polypeptide (GIP), growth hormone releasing hormone (GHRH), and vasoactive intestinal peptide (VIP). All identified prohormones were more similar in sequence to their respective H. sapiens and Gallus gallus (chicken) homologues than the other glucagon members. GCG2 is similar to glucagon type II found in O. latipes, G. gallus, and Xenopus (Silurana) tropicalis (western clawed frog) [35, 36]. This second glucagon has been lost in mammals since there are no detectable sequences or conserved synteny found in mammalian genomes [35]. No secretin (SCT) was identified in A. burtoni, as it is considered lost in teleosts, but the SCT receptor has been identified [37, 38].

Somatostatin and urotensin II families

Following Tostivint et al. [39, 40], single copies of 4 members of the somatostatin (SST) family were identified (SST1, SST2, SST3, and SST5) in A. burtoni, but no evidence of other SST versions. It is proposed that the somatostatin and urotensin II families are related by an early evolutionary event [39-43]. The urotensin II family consists of 2 members, urotensin 2 (UTS2) and urotensin 2B (UTS2B), that are widespread through many taxa including invertebrates [44]. In addition, the urotensin II family consists of two additional members, urotensin II-related peptide 1 (URP1) and urotensin II-related peptide 2 (URP2), which appear to be absent in tetrapods [40]. Both A. burtoni URP1 and URP2 contain the urotensin II domain and the dibasic cleavage site necessary to produce the urotensin 2B neuropeptide. Injection of URP1 and URP2 peptides in Oncorhynchus mykiss (rainbow trout) found that these peptides had similar, but not identical, effects on locomotor behavior and cardio-respiratory physiology to UTS2, suggesting some subfunctionalization within this family in ray-finned fishes [45].

Opioid peptide prohormone genes

Duplicates of prepronociceptin (PNOC; PNOC1 and PNOC2), and single copies of proenkephalin (PENK) and prodynorphin (PDYN), were identified in A. burtoni. Similar to the Verasper moseri (barfin flounder) and O. mykiss [46], 3 versions of POMC (POMC1A, POMC1B, and POMC2) were also identified.

It is hypothesized that these opioid genes are related through two rounds of genomic duplication [47, 48]. All 3 POMC sequences lacked the melanocyte-stimulating hormone (MSH) peptide, γ-MSH, consistent with the loss of γ-MSH in ray-finned fishes [48]. Only 2 POMC versions were similar across species, suggesting these are duplicated copies from the teleost duplication and independent duplication events led to a second paralog.

Comparisons of POMC sequences have indicated that POMC1A and POMC1B are a result of a tandem duplication in the teleost lineage near when the Pleuronectiformes (e.g., flounders) split from Perciformes (e.g., cichlids) [18]. Further, Harris et al. [18] observed a tandem duplication within the POMC2 gene that encompassed part of the N-terminal fragment, all of α-MSH, and part of the adrenocorticotropic hormone that arose before the radiation of cichlids but sometime after the radiation of Labridea (wrasses). A novel ε-MSH peptide was proposed [18], but this peptide is unlikely to occur or be bioactive in A. burtoni. While the proposed cleavage fits the RxxK motif that is cleaved as part of the known motif model [49], the sequence lacks a suitable amidation site that is present in all MSH peptides.

Corticotropin family

Duplicated copies of corticotropin-releasing hormone 1 (CRH1A and CRH1B), and single copies of urotensin 1 (UTS1), urocortin 2 (UCN2), and urocortin 3 (UCN3) genes were identified. Alignment to the mammalian versions indicated A. burtoni CRH, UTS1, and UCN3 were similar to mammalian corticotropin-releasing hormone (CRH), urocortin 1 (UCN1), and UCN3, respectively. Similar to Boorse et al. [50], UCN2 was intermediate between mammalian UCN2 and UCN3 with only the UCN2 domain shared. It is proposed that a genome duplication prior to the divergence of actinopterygian and sarcopterygian fishes gave rise to duplicated UCN3 and CRH genes [50, 51]. In the case of CRH, one gene duplicate, CRH2, was lost in teleost fishes and eutherian mammals, and A. burtoniCRH1A and CRH1B are likely duplicates of an ancestral CRH1 [51, 52].

Neuropeptide B/W family

We found duplicate versions of neuropeptide B (NPB), NPB1 and NPB2, but not neuropeptide W (NPW) in A. burtoni. Both the prohormones and neuropeptide receptors are highly related [53]. Both NPB and NPW have been identified in the genome of Monodelphis domestica (opossum; NPB: [GenBank:XM_001379652.2]; NPW: [GenBank:XM_007499923.1]) and Xenopus laevis (African clawed frog; NPB: [GenBank:XM_002937305.3]; NPW: [GenBank:XM_004918054.2]). Although NPW was not identified in the avian genomes of G. gallus and Taeniopygia guttata (zebra finch), an NPW-like sequence is identified in Pseudopodoces humilis (ground tit; [GenBank:XM_005523213.1]). Homology searching in Latimeria chalumnae (coelacanth) also indicated both NPB ([GenBank:XM_005989108.1]) and NPW (match on scaffold01390 NCBI Reference Sequence: [GenBank:NW_005820400.1]). Thus, our findings in A. burtoni support that NPW either arose after the split of the teleosts from other vertebrates or was lost in the teleost lineage.

Neuropeptide Y family

The neuropeptide Y family consists of neuropeptide Y (NPY), pancreatic prohormone (PPY), and peptide tyrosine-tyrosine (PYY) that arose by gene duplication [54, 55]. Two copies of NPY (NPY1 and NPY2) and two of PYY (PYY1 and PYY2) were identified in A. burtoni, as well as the ray-finned fish-specific pancreatic peptide Y, which has been recognized as a duplicate of PYY [54]. PPY was not identified, consistent with a duplication event after tetrapod divergence [55]. The L. chalumnae sequence ([GenBank:XM_005992227.1]) containing the partial pancreatic polypeptide sequence reported by Larhammar and Bergqvist [56] was more similar to PYY than the other neuropeptide Y family members.

Parathyroid hormone family

The parathyroid family consists of parathyroid hormone 1 (PTH1), parathyroid hormone 2 (PTH2) and parathyroid hormone-related hormone (PTHLH) [57, 58]. Duplicate versions of PTH1 (PTH1A and PTH1B) and PTHLH (PTHLH1 and PTHLH2) were identified, and a single version PTH2 were identified. In addition, a third PTHLH gene (PTHLH3) similar to the Danio rerio (zebrafish) predicted gene ([GenBank:XM_005168285.2]) was identified that had intermediate homology between the PTH and PTHLH genes and appears to have been lost in eutherian mammals [57, 58].

RFamide and kisspeptin/galanin/spexin families

The RFamide family consists of prohormones that produce C-terminal arginine and amidated phenylalanine bioactive peptide [59, 60]. It has been proposed that kisspeptin (KISS), GAL and spexin (SPXN) belong to the same family due to the sequence similarly of prohormones and receptors and that SPXN can activate the GAL receptors [61]. The relationship between these families is undergoing further revision as it has been suggested that KISS and prolactin-releasing hormone (PRLH) may not belong in the RFamide family [62].

Duplicate copies of SPXN (SXPN1 and SPXN2) and PRLH (PRLH1 and PRLH2), and single copies of neuropeptide FF-amide peptide precursor (NPFF); NPVF; pyroglutamylated RFamide peptide (QRFP), GAL and KISS2 were identified. There was no evidence for an A. burtoni galanin-like peptide (GALP) or kisspeptin-1 (KISS1).

The presence of KISS2 and the lack of KISS1 in A. burtoni is consistent with KISS gene evolution [63]. Interestingly, some ray-finned fish and Ornithorhynchus anatinus (platypus) have both KISS1 and KISS2, whereas eutherian mammals have maintained only KISS1. There is some indication of a KISS2-like gene in apes [64] that may be a pseudogene because it is a single exon compared to the 2 exons in other species. The presence of a single GAL gene appears to be consistent with other teleost species, with the exception of Cypriniformes (e.g., D.rerio and Carassius auratus (goldfish)) that appear to have a duplication of GAL (GAL1 and GAL2) [16, 65]. Two GAL isoforms were determined from EST evidence. The longer of the A. burtoni splice isoforms introduces an in-frame insertion of 72 bp due to alternative splicing of exon 3 and 4. A similarly generated splice isoform has also been identified in other teleosts and avian species [65, 66].

The lack of evidence of GALP is consistent with our previous studies suggesting it may only be present in some eutherian mammals [67]. The partial L. chalumnae GALP, predicted by Kim et al. [61], appears to be a duplicated GAL gene. Homology indicated that it is more similar to the X. laevis GAL2 ([GenBank:XM_004916293.1]) than the X. laevis GAL1 ([Genbank:XM_002941642.3]). It is likely that this is a duplicated GAL gene rather than GALP because the X. laevis sequence contains the galanin message associated peptide (GMAP), which is not present in mammalian GALP, and because X. laevis and L. chalumnae GAL2 genes lack synteny with H. sapiens GALP [61].

Hepcidin antimicrobial peptide

Five HAMP sequences were identified in A. burtoni (HAMP1, HAMP2, HAMP3, HAMP4 and HAMP5), however 2 sequences (HAMP2 and HAMP4) were virtually identical. These 2 sequences were located on different contigs and the location of one sequence at the end of a contig indicates these may be the result of an assembly error. The different HAMP versions are a consequence of WGD and additional tandem gene duplication, possibly related to host-pathogen interaction or changes in oxygen availability [68, 69].

Natriuretic peptides

Single copies of natriuretic peptide A (NPPA) and natriuretic peptide B (NPPB) were identified as well as the expected 4 copies of natriuretic peptide C (NPPC1, NPPC2, NPPC3, and NPPC4) [70]. Multiple chromosomal duplications resulted in 4 versions of the ancestral NPPC gene with the subsequent loss of the NPPC1, NPPC2 and NPPC3 versions in tetrapods. Prior to the NPPC3 loss in tetrapods, tandem duplication of the NPPC3 gene gave rise to NPPA and NPPB [70], which is evident by less than 6,000 bps between these genes in the A. burtoni assembly.

CART prepropeptide

Similar to O. latipes, 6 different CART prepropeptide (CARTPT) prohormones were identified in A. burtoni. CARTPT1 was most similar to mammalian CARTPT than the other CARTPT prohormones identified. The relationship between different prohormones is unclear because all versions are located on different scaffolds in the current A. burtoni genome assembly. Multiple sequence alignment suggests greater sequence similarity between two pairs of CARTPT prohomones: CARTPT1 with CARTPT2, and CARTPT3 with CARTPT4. O. latipes also has 6 CARTPT copies [71] and D. rerio has 4 CARTPT copies [72].

The granin family

A. burtoni orthologs were identified for the mammalian granin family members chromogranin A (CHGA), chromogranin B (CHGB), secretogranin II (SCG2), secretogranin III (SCG3), secretogranin V (SCG5), proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N), and vascular endothelial growth factor (VGF) [73]. In addition, two copies of SCG2 (SCG2A and SCG2B) and VGF (VGF1 and VGF2) were found. Although a match for GNAS complex locus was found, there was no homology to the neuroendocrine secretory protein-55 isoform. Similar to Kudo et al. [74], there was limited similarity between mammalian and the identified versions of PCSK1N genes; the A. burtoni version contained PEN-like and little-LEN peptides but lacked the SAAS peptides. This suggests that the gain of PCSK1N functionality is only in mammals.

Salusin peptides and torsin family 2, member A

Unlike most prohormones, the TOR2A gene undergoes alternative splicing where one isoform is cleaved into the salusin peptides [75]. While the A. burtoni TOR2A gene was identified, there was no predicted isoform that could produce the salusin peptides. Subsequent searches for the salusin peptides in other species indicate that this isoform may have only arisen within eutherian mammals.

Prohormone convertases family

The bioactive peptides produced from the prohormones described above depend on the specific set of prohormone convertases (as well as other enzymes responsible for post-translational modifications) present [76]. A search of the A. burtoni genome identified PCSK type 1 (PCSK1), 2 (PCSK2), 5 (PCSK5), 7 (PCSK7), and 9 (PCSK9) genes. There were no matches to mammalian PCSK type 4 and 6 genes. Generally these PCSK genes showed higher similarity to their mammalian counterparts than to each other. Two copies of PCSK5 (PCSK5A and PCSK5B) and FURIN (FURIN1 and FURIN2) were identified and single copies of PCSK1, PCSK2, PCSK7, PCSK9, and membrane-bound transcription factor peptidase, site 1 (MBTPS1) were identified. Only the PCSK5B gene was similar to the mammalian version, with PCSK5A appearing to be a paralog of the mammalian gene. An identified PCSK-like gene may be an incomplete gene or an assembly error because the prediction lacks the FU domains that are present in other PCSK genes.

Tandem mass spectrometry peptide identification in the pituitary

Peptides resulting from the previously detected prohormones were identified using MS-based peptidomics [77-79]. We first assessed the feasibility of peptidomic experiments by testing peptide content in freshly frozen individual pituitaries using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS. Mass fingerprinting of peptides measured by direct tissue MALDI-TOF MS detected and putatively identified 46 peptides from 12 proteins, including 29 peptides from POMC1A (Fig. 1), and all peptides were subsequently confirmed by tandem MS (Additional file 3: Table S2).

Fig. 1

Peptide mass fingerprinting of POMC1A in male Astatotilapia burtoni pituitary by direct tissue MALDI-TOF MS. Masses matching the POMC1A peptides subsequently confirmed by tandem MS in the pituitary extracts are labeled with asterisks; masses matching oxytocin and arginine vasopressin are labeled by gene symbols OXT and AVP, respectively. List of all mass matches to sequenced peptides is given in Additional file 3: Table S2

Tandem MS detected a total of 680 peptides from 620 unique peptide sequences after accounting for different modifications on the same sequence (Additional file 4: Table S3). There were 22 peptides detected with a single amino acid substitution compared to the predicted sequence from the genome, which may be due to genetic differences between the A. burtoni populations used for tandem MS and genome sequencing. The unique peptide sequences were matched to 125 unique proteins. Proteins identified by more than 9 unique characterized peptides each are listed in Table 2.

Table 2

Pituitary proteins confirmed by the highest number of peptides using tandem MS

Type	Symbol	N. peptides	Gene locus	Accession No.	Gene name
Prohormone	AVP	16	LOC102310610	NP_001273257.1	arginine vasopressin
Prohormone	CHGB	9	LOC102304831	XP_005917770.1	chromogranin B
Prohormone	GNRH1	9	LOC102291227	NP_001273225.1	gonadotropin-releasing hormone 1
Prohormone	NUCB2B	9	LOC102313482	XP_005931689.1	nucleobindin 2B
Prohormone	OXT	26	LOC102312886	XP_005919106.1	oxytocin
Prohormone	PCSK1N	11	LOC102305204	XP_005931188.1	proprotein convertase subtilisin/kexin type 1 inhibitor
Prohormone	PMCH1	24	LOC102310003	XP_005921781.1	pro-melanin-concentrating hormone 1
Prohormone	PMCH2	14	LOC102314388	XP_005934290.1	pro-melanin-concentrating hormone 2
Prohormone	POMC1A	164	LOC102296817	NP_001273262.1	proopiomelanocortin 1A
Prohormone	SCG2A	13	LOC102306907	XP_005948590.1	secretogranin IIa
Prohormone	SCG2B	12	LOC102295815	XP_005938347.1	secretogranin IIb
Prohormone	SCG3	22	LOC102313744	XP_005949052.1	secretogranin III
Prohormone	TAC1B	10	none	none	tachykinin precursor 1B
Other	ACTB	13	LOC102293972	XP_005952745.1	actin, cytoplasmic 1-like isoform X1
Other	CALR	12	LOC102296215	XP_005936722.1	calreticulin-like
Other	HBA1	21	LOC102301295	XP_005920277.1	hemoglobin subunit alpha-A-like
Other	HBB	13	LOC102301591	XP_005920278.1	hemoglobin subunit beta-A-like
Other	HSP90B1	15	LOC102308602	XP_005929149.1	endoplasmin-like
Other	PDIA3	20	LOC102293196	XP_005931703.1	protein disulfide-isomerase A3-like
Other	PDIA4	9	LOC102309651	XP_005912150.1	protein disulfide-isomerase A4-like
Other	PPIB	9	LOC102303154	XP_005931742.1	peptidyl-prolyl cis-trans isomerase B-like

Most of the peptides detected (164) were derived from POMC prohormones. There were 42 peptides with at least one post-translational modification. No α-MSH was detected but an N-terminal peptide of POMC1A was detected, which was expected from the sequence [48]. Subsequent alignment of all POMC peptides indicated that most of these were derived only from POMC1A (120), 39 peptides were derived from an identical peptide region in POMC1A and POMC1B, and 7 peptides with an identical sequence in POMC1A, POMC1B, and POMC2. There were no peptides uniquely identified to POMC1B and POMC2, indicating that POMC1B and POMC2 prohormones were not present at detectable levels.

Both pro-melanin-concentrating hormone 1 (PMCH1) and pro-melanin-concentrating hormone 2 (PMCH2) were detected with 24 and 14 peptides, respectively. In addition, a single peptide was detected that shared a sequence between 2 copies of melanin-concentrating hormone (MCH), MCH1 and MCH2. This peptide corresponds to the MCH encoding sequence at the C-terminal of both MCH prohormones. Peptides from PMCH1 corresponded to the same region as mammalian neuropeptide-glycine-glutamic acid. The predicted PMCH2 sequence could not form this neuropeptide-glycine-glutamic acid peptide, and, unlike the mammalian prohormone sequence, this PMCH1 peptide is surrounded by dibasic amino acids. Neither PMCH1 nor PMCH2 prediction included a peptide corresponding to neuropeptide-glutamic acid-isoleucine.

Both arginine vasopressin and oxytocin neuropeptides were detected as well as other peptides from their respective prohormones. Overall, OXT and AVP provided 26 and 16 unique peptides, respectively. Oxytocin and arginine vasopressin peptides were detected, as well as C-terminal peptides corresponding with copeptin from both OXT and AVP. The remaining peptides detected were OXT fragments from the neurophysin 1 peptide. The detection of these peptides and the similarity of prohormone sequences indicate that mammalian OXT has undergone greater divergence than AVP since the tetrapod divergence.

The other prohormone with multiple peptides was SCG3, in which the majority of the peptides were near the signal peptide or near the C-terminus. The peptides near the signal peptide likely correspond to polypeptides resulting from cleavage at the arginine pair following removal of the signal peptide. Peptides from this region have also been detected in mammals (e.g., Fricker et al. [80]). However, since neither region had obvious NeuroPred-predicted [81] cleavage sites, it is unclear whether these are post-processing degradation products of the large SCG3 peptide, or resulted from post-translational enzymatic cleavage.

Examination of the peptides from V-set and transmembrane domain-containing protein 2-like protein-like (VSTM2A) indicates a novel C-terminal peptide (Fig. 2). Evaluation of the predicted protein sequence identified an immunoglobulin domain and dibasic sites commonly cleaved by prohormone convertases, and a signal peptide was predicted using SignalP v4.1 [82]. This protein is longer than the mammalian VSTM2A sequence. The observed peptides corresponded to regions between cleavage sites predicted by NeuroPred [81], with both vertebrate and invertebrate models. This predicted gene was found with similar length and dibasic sites in some ray-finned fish species (e.g., O. latipes: [GenBank:XM_004070260.2]; Poecilia reticulata (Guppy): [GenBank:XM_008413143.1]; Cynoglossus semilaevis (tongue sole): [GenBank:XM_008318238.1]; Xiphophorus maculatus (southern platyfish): [GenBank:XM_005807649.1]). However, some species, such as Takifugu rubripes (Japanese puffer) and Lepisosteus oculatus (spotted gar), appear to lack one or more of the dibasic sites, and others such as L. chalumnae and D. rerio lack any match to the region containing these peptides.

Fig. 2

Identification of novel V-set and transmembrane domain-containing protein 2-like protein-like (VSTM2A) peptides. a Multiple sequence alignment VSTM2A proteins. C-terminal peptides identified with tandem MS are underlined and dibasic cleavage residues marked with black circles. Sequence accession numbers are Mus musculus (NP_941029.2), Danio rerio (XP_009300365.1), Takifugu rubripes (XP_011620087.1), and Oryzias latipes (XP_004070308.1). Fragment ion series for the 2 C-terminal peptides are shown in b and c

Tandem MS identified peptides from alternatively spliced forms of CHGB and GAL. The CHGB isoforms are identical except that the longer CHGB isoform retains an intron that contains an MS-identified peptide. The galanin peptide detected by MS is encoded by the shorter A. burtoni splice isoform, and this peptide is distinguished by two aspects. First, the N-terminal 14 amino acids of galanin are highly conserved across vertebrates and are important for receptor binding [14]. Previously, the only known exception was in Thunnus albacares (yellowfin tuna), which has a serine to alanine substitution at position 6 [83]. A. burtoni galanin is also an exception, with a leucine to methionine substitution at position 4 (Fig. 3a). Second, the A. burtoni peptide is 32 amino acids long, making it the longest sequenced galanin peptide (Fig. 3b). The length of galanin is highly conserved across vertebrate evolution at 29 amino acids and previously only H. sapiens galanin was known to exceed this length [84]. Our understanding of galanin receptor function in teleosts is only beginning to emerge [85], therefore, the significance of this substitution is unknown. If A. burtoni galanin receptors are similar to D. rerio galanin receptors, and display affinity for galanin and spexin peptides, then the unusual form of A. burtoni galanin may suggest that there has been pronounced ligand-receptor co-evolution in the GAL/SPXN system in cichlids.

Fig. 3

Characterization of galanin mature peptide and GAL prohormone expression. a Alignment of vertebrate galanin peptides. Astatotilapia burtoni sequences have a bolded species label. Sequences supported by peptide sequencing are marked with asterisks (*). Alignment made with the ClustalW program. b The A. burtoni galanin peptide fragmented and sequenced via tandem MS. c–g Representative transverse sections showing GAL-expressing neurons in the brain and pituitary. GAL-expressing cells were found in the preoptic area c, nucleus of the lateral tuberalis (NLT) d, medial region of the nucleus of the lateral recess (NRL) and in the region dorsal to the nucleus of the posterior recess (NRP) e, region of the periventricular nucleus of the posterior tuberculum f, and in the hindbrain along the lateral border of the vagal lobe (VL) e. CC, cerebellar crest; ON, optic nerve; pit, pituitary. Scale bars = 100 μm c; 25 μm d–g

Detection of numerous peptides from non-secreted proteins such as hemoglobin subunit-α-like and β-like, endoplasmin-like, actin, and cytoplasmic 1-like protein, among others in the pituitary, is not surprising due to their ubiquitous nature. High protein sequence coverage was obtained for some of these proteins due to multiple detected peptides (Additional file 3: Table S2). In fact, enzymatic processing of cytosolic proteins can generate non-classical peptides that may have biological activity [86, 87]. In particular, hemoglobin-derived peptides, such as hemorphins and hemopressins, have diverse functions in various tissues and are expressed by neurons [87, 88]. Therefore, A. burtoni peptides from hemoglobin subunit-α-like (20 unique peptide sequences) and hemoglobin subunit-β-like (8 peptides) may in part represent bioactive peptides. The disulfide-isomerase proteins, protein disulfide-isomerase A3-like (18 unique peptide sequences), and protein disulfide-isomerase A4-like (9 peptides), regulate folding and redox state of proteins via formation, reduction, or isomerization of disulfide bonds [89]. Endoplasmin (15 unique peptides) is a molecular chaperone involved with the processing and transport of secreted proteins [90]. Actin, cytoplasmic 1-like (12 peptides), possibly reflects the role of actin in vesicle transport [91]. Calreticulin (12 peptides) is involved with maintaining adequate calcium levels in the system, and functions as a chaperone in the folding of other proteins [92].

Localization of galanin prohormone expression

Galanin is one of the better characterized peptides within ray-finned fishes; having identified the mature A. burtoni galanin peptide, we next sought to identify GAL-expressing cells in the A. burtoni brain using in situ hybridization (Fig. 3c–g). The distribution of galanin in many ray-finned fish species has been investigated primarily through detection of galanin-like immunoreactivity using anti-porcine galanin antibodies [16]. These studies have shown that in ray-finned fish, pituitary galanin is exclusively neural in origin, rather than both neural and pituitary-derived, as seen in mammals [14]. The Xiphophorus and Anableps genera may be exceptions [93, 94]. In situ hybridization of the A. burtoni pituitary supports all pituitary galanin in this species being neural-derived (Fig. 3d).

Neuroanatomical locations of GAL cells in the A. burtoni brain were determined according to brain atlases from Burmeister et al. [95] and Cerdá-Reverter et al. [96]. The most anterior cell population identified was in the anterior preoptic area (POA) (Fig. 3c). Along the dorsal-ventral axis, this population spanned from the anterior commissure and approached the ventral edge of the brain. This group of cells displayed the most intense signal of all populations, as well as the greatest diversity in cell soma diameter (10–30 μm). Moving posteriorly, a small, sparse set of cells was present in the nucleus of the lateral tuberalis (NLT) (Fig. 3d). A few, faintly-stained cells were also observed along the midline, in the periventricular nucleus of the posterior tuberculum (Fig. 3e). In the caudal hypothalamus, GAL-expressing cells were distributed in the anterior portion of the nucleus of the lateral recess (NRL), and directly dorsal to the nucleus of the posterior recess (NRP) (Fig. 3f). The most posterior group was found in the hindbrain, bordering the vagal lobe (Fig. 3g). Hindbrain galanin cells have been previously described in the locus coeruleus of cyprinodonts [97].

The presence of galanin-expressing cells in the POA and NLT is conserved across ray-finned fishes. In contrast, the presence and locations of more posterior populations exhibits greater diversity across ray-finned fishes. The NRL and NRP are considered components of the fish homolog to the nonmammalian vertebrate paraventricular organ, which contains galanin-immunoreactive cells in amphibians [98, 99]. An NRL population has been described in C. auratus [100], and both NRP and NRL populations described in Anguilla anguilla (eel) [101], Apteronotus leptorhynchus (brown ghost knifefish) [102], and O. mykiss [103]. Only the POA and NLT populations were identified in the non-haplochromine cichlid Alcolapia grahami (Lake Magadi tilapia) [97]. The lack of more caudal populations in A. grahami could be due to differences in specificity between techniques, diversification of the galanin system within cichlids, or a combination of the two.

Subfunctionalization of POMC prohormones

We show here the first exploration of three POMC prohormones in the same species on the cellular level in the brain and pituitary using in situ hybridization (Table 3). Expression of all three POMC prohormones was found in both the corticotrope (rostral pars distalis)- and melanotrope (pars intermedia)-containing regions of the A. burtoni pituitary (Fig. 4). POMC1B showed the most intense signal in the pituitary, particularly in the pars intermedia, which is consistent with reverse transcription polymerase chain reaction (RT-PCR) results [18]. Similarly, expression of three POMC prohormones of V. moseri was detected in whole pituitary samples [104].

Table 3

Localization of the proopiomelanocortin (POMC) gene family in the brain and pituitary of Astatotilapia burton i

Brain Regiona	POMC1A	POMC1B	POMC2
Telencephalon
Dm	-b	-	+
Dld	-	-	+
Dlg	-	-	+
Dlv	-	-	+
Diencephalon
POA	-	-	+
NLT	+	+	+
ATn	-	-	+
Pituitary
RPD	+	+	+
PI	+	+	+
Mesencephalon
T	-	-	+
Metencephalon	-	-	-
Rhombencephalon
NCC	-	-	+

a Brain Region: ATn anterior tuberal nucleus, Dm, medial part of the dorsal telencephalon; Dld, Dlg, Dlv, dorsal, granular, and ventral zones of the lateral part of the dorsal telencephalon; NCC commissural nucleus of Cajal; NLT nucleus of the lateral tuberalis, POA preoptic area, PI pars intermedia, RPD rostral pars distalis, T tectum

b +, detected; -, not detected

Fig. 4

Distribution of POMC1A, POMC1B, and POMC2 in the hypothalamus and pituitary gland of Astatotilapia burtoni. In situ hybridization shows POMC1A and POMC1B staining in NLTv and rostral ATn, and POMC2 in ATn. All three POMCs were found in the RPD and PI of the pituitary. Brain is 20 μm transverse sections and pituitary is 20 μm sagittal sections. ATn, anterior tuberal nucleus; hc, horizontal commissure; NH, neurohypophysis; NLT, nucleus of the lateral tuberalis; NLTv, ventral part of NLT; PI, pars intermedia; PPD, proximal pars distalis; RPD, rostral pars distalis;. Scale bars = 100 μm

Localization of POMC prohormones within the brain showed that POMC1A and POMC1B occurred in the same locations, while POMC2 was more widely expressed. POMC1A and POMC1B expression were restricted to two hypothalamic nuclei, the NLT and rostral anterior tuberal nucleus (ATn) (Fig. 4). There were numerous small (4–10 μm diameter) POMC1A and POMC1B cells in the ventral part of the NLT (NLTv) above the infundibular recess and pituitary, and a smaller population of larger neurons (10–15 μm diameter) located along the midline in the most rostral part of the ATn near the horizontal commissure. This expression pattern matches that of the single POMC1 gene in Tetraodon nigroviridis (green spotted puffer) [105]. The range of POMC2 expression in the A. burtoni brain and pituitary encompassed that of the POMC1 genes but within the NLT, POMC2 expression was more predominant in the medial and inferior parts of the NLT, while POMC1s were localized to the NLTv. POMC2 expression also extended to the dorsolateral telencephalon, POA, tectum, and commissural nucleus of Cajal in the hindbrain (Additional file 5: Figure S1). This expression pattern is broader than that in T. nigroviridis, in which POMC2 is restricted to the POA [105].

Although all three POMC genes are expressed in the hypothalamus and the pituitary, the tandem MS results suggest that POMC1B and POMC2 peptide products are either not present, are at an undetectable level, or are expressed at different times than POMC1A in pituitary (Additional file 4: Table S3). Whether POMC1B and POMC2-derived peptides are present in other tissues or developmental stages remains to be explored. Increased brain POMC1A expression is associated with dominant status in adult A. burtoni [17], but it is unknown which brain regions contribute to this increase and whether POMC2 exhibits similar social status-dependent expression.

It remains to be determined what function the extrahypothalamic POMC-expression serves, as well as whether POMC1A or POMC2 are generally more varied in expression pattern across teleosts. For example, β-endorphin-like immunoreactive cells have been described in the thalamus and cerebellum of other teleosts [106, 107]. Since the beta-endorphin region of POMC2 has degenerated in many teleost lineages, it is unclear which prohormones are involved.

Conclusions

Our systematic survey of prohormone genes in the A. burtoni genome identified 167 sequences from 141 prohormone, 7 prohormone-related and 10 PCSK genes, with experimental evidence for numerous peptides derived from proteins encoded by many of these genes. In addition, tandem MS identified a possible novel fish-only neuropeptide from VSTM2A. Identification of peptides across fish species will facilitate functional testing of prohormone families, and whether there are any synergistic or collective mechanisms contributing to fish behavioral diversity. Two thirds of prohormone families contain duplicate genes, most deriving from the teleost WGD, indicating that this gene group has retained duplicates nearly three times the genome-wide average. These duplicates may serve as substrates for behavioral and physiological diversification within fishes and may have contributed to the remarkable speciation in the African cichlid species. In the case of POMC, we show that all three A. burtoni POMC genes are expressed in the hypothalamus and pituitary, but MALDI-TOF and tandem MS analysis of the pituitary suggest only one gene yields peptide products. Whether duplicates described in A. burtoni have undergone functional changes to give rise to different roles can be pursued in the wider context of the remarkably diverse African cichlids. The elucidation of A. burtoni’s prohormone complement comes at an exciting time in cichlid research, and follows recent developments in genome assembly and transgenic technologies.

Methods

Prohormone identification in silico

Detection of teleost prohormones requires a two-phase approach to address the impacts of WGD, tandem duplication, reciprocal gene loss, and ligand–receptor coevolution. In the first phase, orthologs of known prohormones and any paralogs are identified. Subsequently, the second phase searches for any previously unidentified prohormone paralogs across different genomes.

In the first phase, 109 candidate genes, including known gene duplications and possible pseudogenes, were derived from prior mammalian and avian studies [67, 108, 109]. Each sequence was searched in the genome using our previously documented approach [67]. The protein sequence of each candidate gene was matched to the A. burtoni genome assembly using TBLASTN with the default settings (E-value < 10 and BLOSUM62 scoring matrix) and filtering disabled on the cichlid data site (http://cichlid.umd.edu/cichlidlabs/kocherlab/bouillabase.html). All scaffold position matches with E-values < 1 were evaluated as possible prohormone genes to account for WGD, tandem duplication, and ligand–receptor coevolution. Partial matches were also used to query the A. burtoni EST database in order to provide a more accurate match as well as any alternative splicing. When there was no suitable BLAST match to a candidate gene, the other cichlid resources were used to confirm any missing candidate gene or provide a more suitable candidate. The resulting matches were classified into similar matches based on E-value and percentage identity to separate duplicated genes from genes from the same prohormone family. Prohormone protein sequences were predicted using the gene parsing tool Wise2 [110]. The final predictions were then bioinformatically screened for alignments to related genes in the same neuropeptide family across other species to ensure the accuracy of prediction.

Compared to tetrapods, each teleost prohormone was expected to have two paralogous copies due to the third tetraploidization. In the second phase, candidate genes with only a single match were further investigated for reciprocal gene loss. Initially the searches were conducted using the genomic resources of the other sequenced cichlids, primarily O. niloticus. Unsuccessful searches were then conducted in other published fish genomes, notably Takifugu species, T. nigroviridis, O. latipes, Gasterosteus aculeatus (three-spined stickleback), and D. rerio, to determine if a more closely related version of the candidate gene could be found. Finally, a literature search was conducted to find evidence that the candidate gene is duplicated in any fish species. Any potential sequence was further screened using the previous tools and databases to confirm the presence of a duplicated prohormone.

Animals

Laboratory bred A. burtoni adults between 6 and 8.5 cm in standard length were housed in mixed sex communities in 60 l aquaria under conditions mimicking natural habitat conditions (26.5 °C; pH 8.5; 12 h dark: 12 h light with full spectrum illumination) [111]. Animals were fed daily with cichlid pellets and flakes (AquaDine, CA, USA). Animals were euthanized by rapid cervical transection prior to pituitary and/or brain dissection.

MALDI-TOF MS of pituitary tissue

Freshly frozen individual pituitaries (three dominant males and three non-brooding females) were used for MALDI-TOF MS. Each pituitary was transferred onto a MALDI sample plate, divided into 10–15 pieces using electrolytically sharpened tungsten needles, each tissue piece was transferred onto a new sample spot and mixed with 0.5 μl of MALDI matrix (2,5-dihydroxybenzoic acid, 50 mg/ml of 50 % acetone). Spectra were manually acquired on a Bruker ultrafleXtreme mass spectrometer equipped with a smartbeam-II™ laser (Bruker Daltonics, MD, USA) operated at 1 kHz speed in reflectron mode. External calibration was performed using Bruker Peptide Mix II standards in identical matrix.

Pituitary peptide extraction

Pituitaries were rapidly dissected from a second cohort of 5 adult animals (1 male, 4 females) and homogenized in 0.25 M acetic acid (Sigma-Aldrich, CA, USA) using a Dounce homogenizer. Homogenates were pooled and then centrifuged for 30 min at 4 °C and 15,000 x g. The supernatant pH was adjusted to ~4 using 1 M NaOH (Fisher, PA, USA) and desalted using Pierce C18 Spin Columns (Pierce, IL, USA) according to manufacturer’s instructions. Column eluate was then dried (Savant SpeedVac, Thermo Scientific, Waltham, MA, USA) and reconstituted in 0.1 % formic acid (Sigma-Aldrich).

Pituitary peptide analysis by LC-MS/MS

Samples were first acidified and purified on stage tips and eluted in 60 % acetonitrile/40 % H2O, fractions were then dried (SpeedVac, Thermo Scientific) and reconstituted in 2 % acetonitrile/97.8 % H2O/0.2 % formic acid and injected onto a self-packed 15 cm C18 analytical column with a flow rate of 300 nL/min directly infused into the mass spectrometer. Ultra performance liquid chromatography (UPLC) was performed using a Waters Acquity system (Waters, Milford, MA). The electrospray ionization (ESI) ion trap (IT) mass spectrometer (LTQ Orbitrap Velos; Thermo Scientific) was set in data-dependent mode to fragment the top 8 most intense, multiply charged ions using higher-energy collisional dissociation (HCD). The survey scan mass resolution was set to 60 K and the HCD fragment ion resolution to 7.5 K.

Bioinformatic peptide identification from the tandem MS data

We performed the peptide identification on the tandem MS data exported as an mzXML file using PEAKS Studio software versions 5.3 and 7.0 (Bioinformatics Solutions, Waterloo, Canada). The PEAKS workflow included creation of de novo sequence tags that were then queried against a database of predicted A. burtoni prohormones and a database of NCBI-predicted proteins from the AstBur1.0 assembly using both standard (PEAKS DB) and homology (SPIDER) searches [112]. Standard search identified the peptides whose sequences matched those in a database, while homology search revealed peptides with slightly different sequences, which could be due to polymorphism or database error. Search parameters included 20 ppm mass error tolerance for monoisotopic precursor ions and 0.1 Da for fragment ions, precursor charge state 1–5, no enzyme cleavage, and a maximum of three variable modifications (pyroglutamate from E and Q, acetylation of N-terminus or lysine, disulfide bond, oxidation and amidation). The search results were filtered with -10lgP of 20; all tandem MS spectra with scores lower than 30 were manually inspected and false positives removed.

Tissue preparation for in situ hybridization

Brains and pituitaries from 4 adult A. burtoni (2 males, 2 females) were prepared for GAL in situ hybridization and brains and pituitaries from an additional group of 5 adults (2 males, 3 females) for all POMCs. These animals were separate from those used for MS experiments. Tissues were fixed in 4 % paraformaldehyde overnight at 4 °C, rinsed in PBS, and then cryoprotected with 30 % sucrose overnight at 4 °C. Tissues were then embedded in Tissue Tek OCT media (Sakura Finetek, MA, USA) in vinyl specimen molds (Sakura Finetek) and frozen on dry ice. Tissues were sectioned at 20 micron thickness. All GAL tissues were sectioned in the transverse plane. POMC tissues were sectioned in either the transverse (1 male, 2 female) or sagittal plane (1 male, 1 female). Sections were thaw-mounted onto three replicate slide sets (Superfrost White, VWR, PA, USA) and dried at room temperature for two nights. Slides were stored at –80 °C until processed for in situ hybridization.

In situ hybridization

To localize GAL, POMC1A, POMC1B, and POMC2-expressing cells in brain and pituitary tissue, chromogenic in situ hybridization of brain and pituitary tissue was performed as previously described [51]. RT-PCR was used to amplify target sequences from A. burtoni whole brain cDNA and introduce T3 RNA promoter sequences to the template. For antisense probe generation, the T3 RNA promoter sequence was introduced in the reverse primer. For sense probe generation, the T3 RNA promoter sequence was introduced in the forward primer. We identified a target region of low sequence identity (~40 %) between the 3′UTRs of POMC1A ([GenBank : KC464872.1]) and POMC1B (Broad A. burtoni brain transcriptome [6], comp114_c0_seq1_indC_brain) mRNAs by sequence alignment using ClustalW (Geneious 8.0.5, Biomatters Inc.). The following primers were used to generate a template for cRNA antisense probe synthesis: GAL forward primer 5′-CTA GAT GGA CTA CAT GGA CAC AC-3′, GAL reverse primer 5′-AAT TAA CCC TCA CTA AAC GGA TTG GCC AGT-3′; POMC1A forward primer 5′- GAG AAA AGA GGG AGG GAT GGA G-3′, POMC1A reverse primer 5′-TGC AGT TGT GAA TA-3′; POMC1B forward primer 5′-AGA CGA GAA GAA GAT GAG GCA-3′, POMC1B reverse primer 5′-GTC TAA TTG CCT TG-3′; POMC2 forward primer 5′-GAC CTC TTA CTC AGC GTT ATT C-3′, POMC2 reverse primer 5′-AGA TAG CAA CGA GTT TGT GTA A-3′.