Marked changes in neuropeptide expression accompany broadcast spawnings in the gastropod Haliotis asinina

Abstract

Introduction: A huge diversity of marine species reproduce by synchronously spawning their gametes into the water column. Although this species-specific event typically occurs in a particular season, the precise time and day of spawning often can not be predicted. There is little understanding of how the environment (e.g. water temperature, day length, tidal and lunar cycle) regulates a population's reproductive physiology to synchronise a spawning event. The Indo-Pacific tropical abalone, Haliotis asinina, has a highly predictable spawning cycle, where individuals release gametes on the evenings of spring high tides on new and full moons during the warmer half of the year. These calculable spawning events uniquely allow for the analysis of the molecular and cellular processes underlying reproduction. Here we characterise neuropeptides produced in H. asinina ganglia that are known in egg-laying molluscs to control vital aspects of reproduction.

Results: We demonstrate that genes encoding APGWamide, myomodulin, the putative proctolin homologue whitnin, FMRFamide, a schistosomin-like peptide (SLP), a molluscan insulin-related peptide (MIP) and a haliotid growth-associated peptide (HGAP) all are differentially expressed in the anterior ganglia during the two week spawning cycle in both male and female abalone. Each gene has a unique and sex-specific expression profile. Despite these differences, expression levels in most of the genes peak at or within 12 h of the spawning event. In contrast, lowest levels of transcript abundance typically occurs 36 h before and 24 h after spawning, with differences in peak and low expression levels being most pronounced in genes orthologous to known molluscan reproduction neuromodulators.

Conclusions: Exploiting the predictable semi-lunar spawning cycle of the gastropod H. asinina, we have identified a suite of evolutionarily-conserved, mollusc-specific and rapidly-evolving neuropeptides that appear to contribute to the regulation of spawning. Dramatic increases and decreases in ganglionic neuropeptide expression levels from 36 h before to 24 h after the broadcast spawning event are consistent with these peptides having a regulatory role in translating environmental signals experienced by a population into a synchronous physiological output, in this case, the release of gametes.

Figure 1

Characterisation of Has-APGWamide . (A) Nucleotide and predicted amino acid sequence of Has-APGWamide (nucleotide and amino acids are numbered on left). A predicted signal sequence is underlined, and predicted dibasic and tribasic cleavage site residues are boxed. The Has-APGWamide qPCR primers correspond to the nucleotides 750–771 and 830–853, identified here in bold. (B) Multiple sequence alignment of molluscan APGWamide prepropeptide sequences. Shading to four levels shows conservation as per Nicholas et al. (1997) [33]. The start of each H. asinina APGWamide is indicated with an asterisk. HaliotisH. asinina [GenBank:JN606061]; LottiaLottia gigantea[34]; LymnaeaLymnaea stagnalis ([GenBank:1811269A], [35]); AplysiaA. californica ([GenBank: NP_001191561], [30]). (C) Schematic representation of H. asinina anterior ganglia showing regions analysed by MALDI-TOF-MS. Representative mass peaks were identified that match APGWamide (m/z 428.9) and the acetylated CP3 peptide Ac-TLDILEDYT (m/z 1124.8) (right). Signal, signal sequence; LG1, left cerebral ganglia region 1; LG2, left cerebral ganglia 2; RG1, right cerebral ganglia 1; RG2, right cerebral ganglia 2; CC, cerebral commissure; PGM, pleuropedal ganglia middle; Ac, acetylation; amide, amidation; m/z, mass to charge ratio.

Figure 2

Characterisation of Has-Myomodulin . (A) Nucleotide and predicted amino acid sequence of Has-Myomodulin (nucleotide and amino acids are numbered on left). A predicted signal sequence is underlined and predicted monobasic and dibasic cleavage sites are boxed. Predicted myomodulins cleaved from the precursor are shaded. Has-Myomodulin qPCR primers described in Table 1 correspond to nucleotides 1034–1057 and 1122–1144, here shown in bold. (B) Multiple sequence alignment of molluscan myomodulin prepropeptide sequences. Shading is to 4 levels, and indicates conservation as described in Nicholas et al. (1997) [33]. Asterisks indicate the start of H. asinina myomodulins. HaliotisH. asinina [GenBank:JN606062]; LottiaLottia gigantea (Joint Genome Institute, Protein ID 159404); LymnaeaLymnaea stagnalis ([GenBank:X96933]; [39]); AplysiaA. californica ([GenBank:S64300], [38]). The C-terminal Q has been removed from the Aplysia sequence. (C) Schematic representation of H. asinina anterior ganglia showing regions analysed by MALDI-TOF-MS. Identification by MALDI-TOF-MS of the most commonly encoded myomodulin PMNMLRLamide in regions of H. asinina anterior ganglia. Peak shown is representative of PMNMLRLamide (m/z 872.9) (right). Signal, signal sequence; LG1, left cerebral ganglia region 1; LG2, left cerebral ganglia 2; RG1, right cerebral ganglia 1; RG2, right cerebral ganglia 2; CC, cerebral commissure; PGM, pleuropedal ganglia middle; amide, amidation; m/z, mass to charge ratio.

Figure 3

Characterisation of Has-Whitnin . (A) Nucleotide and predicted amino acid sequence of Has-Whitnin (nucleotide and amino acids are numbered on left). A predicted signal sequence is underlined. Predicted monobasic and dibasic basic cleavage sites are boxed. Predicted peptides cleaved from the precursor are shaded. Putative disulphide bonded cysteines within the predicted ERYM peptide are circled. Nucleotides 246–269 and 399–423, in bold, correspond to the Has-Whitnin qPCR primers. (B) Multiple sequence alignment of H. asinina Whitnin prepropeptide with molluscan homologues. The 4-level shading indicates conservation, as detailed in Nicholas et al. (1997) [33]. HaliotisH. asinina GenBank:JN606063; LottiaLottia gigantea[34]; LymnaeaLymnaea stagnalis ([GenBank:AAF36485]; [42]); AplysiaA. californica ([GenBank:AAV84472]; [41]). (C) Schematic representation of H. asinina anterior ganglia showing regions analysed by MALDI-TOF-MS. MALDI-TOF-MS detection of predicted Has-Whitnin gene products in regions of H. asinina anterior ganglia. Asterisks indicate that masses consistent with both disulphide bonded and non-disulphide bonded versions of the predicted ERYM peptide were found in LG1 and RG2. Peaks shown represent LPADEamide (m/z 541.7), SPTR (m/z 1656.6), and ERYM with disulphide bonded cysteines (m/z 2699.9) (right). Signal, signal sequence; LG1, left cerebral ganglia region 1; LG2, left cerebral ganglia 2; RG1, right cerebral ganglia 1; RG2, right cerebral ganglia 2; CC, cerebral commissure; PGM, pleuropedal ganglia middle; amide, amidation; m/z, mass to charge ratio.

Figure 4

Quantitative PCR expression profiles of neuromodulators during the H. asinina reproductive cycle. (A-D) Relative gene expression in anterior ganglia during the reproductive cycle of male and female H. asinina. N = 4 anterior ganglia/data points for male or female as relevant. Error bars represent standard error of the mean. Asterisks indicate a significant difference in expression between genders for the indicated timepoint. Notable events during the spawning cycle are indicated: D, dissociation of Cohort I oocytes from trabeculae; G, germinal vesicle breakdown at onset of oocyte maturation; S, time of spawn; V, vitelline envelope appears around the developing, almost full size, Cohort I oocytes. Heat maps (right) indicate level of significance (P < 0.05) for comparisons of gene expression between gender/timepoint groups by Tukey’s HSD test. Background blue cells indicate male, green cells indicate female, aqua cells indicate corresponding time points between males and females. Significant differences in expression are indicated: yellow cells P < 0.05; orange cells P < 0.01; red cells P < 0.001. Day of spawn is highlighted in grey.

Figure 5

Quantitative PCR expression profiles of growth-related neuropeptides during the reproductive cycle. (A-C) Relative expression of indicated genes in H. asinina male and female anterior ganglia over the reproductive cycle. N = 4 anterior ganglia/data point for male or female as relevant. Error bars display standard error of the mean. Indicated notable spawning cycle events are as per Figure 4. Heat maps (right) indicate significant differences in gene expression between gender/timepoint groups, as per Figure 4.

Figure 6

Comparison of quantitative PCR expression profiles surrounding the spawning event. Male expression shown in blue (N = 4 anterior ganglia/data point), female expression shown in red (N = 4 anterior ganglia/data point). Error bars display standard error of the mean. Asterisks indicate a significant difference in expression between genders for the indicated timepoint. Tidal cycle is shown; all timepoints were taken at high tide.