Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning

Abstract

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.

Keywords: Haliotis laevigata; abalone; genome; maturation; spawning.

Figure 1

Phylogeny of Haliotis laevigata (greenlip abalone). (a) Maximum likelihood tree showing the phylogenetic relationship between ten molluscan species (one brachipod and two annelids were used as outgroups). This tree was based on 748 orthologous genes (127,598 amino acids) present in thirteen species. The robustness of the relationship was evaluated using the bootstrap re-sampling procedure, and the values based on 100 pseudo-replicates are given on bifurcating nodes. (b) A linearized time tree based on the in-silico proteomes of eleven Lophotrochozoan species and two annelids. The divergence times were estimated using a Bayesian Markov chain Monte Carlo method. The estimations were based on the maximum likelihood tree, which was calibrated using four well-defined fossil ages.

Figure 2

Neuropeptide precursor clustering across different molluscan classes. Included are: class Gastropoda (greenlip abalone Haliotis laevigata, Mediterranean white land snail Theba pisana, California sea slug Aplysia californica, owl limpet Lottia gigantea, Giant triton snail Charonia tritonis, marsh snail Biomphalaria glabrata), class Bivalvia (Pacific oyster Crassostrea gigas, Sydney rock oyster Saccostrea glomerata) and class Cephalopoda (California two-spot octopus Octopus bimaculoides). The total number of neuropeptides is 64 (for the list of NPPs and abbreviations, refer to File S2).

Figure 3

Tissue-specific expression of 43 neuropeptide precursor genes in Haliotis laevigata. Heatmap shows hierarchical clustering and normalized expression abundance across eleven tissues of neuropeptide precursor genes.

Figure 4

Neuropeptide identification in Haliotis laevigata central nervous system. (a) Neuropeptides detected and normalized abundance of each peptide in the central nervous system, in years 1 and 3 of reproductive maturation. The stages of gonad maturation are represented by the visual gonad indices (0-2). F, represents female and M, represents male. (b) Schematics of neuropeptide precursors showing the position of mass spectrometry peptides.

Figure 5

Identification and comparative analysis of egg laying hormone (ELH). (a) Schematics showing ELH precursor organization. BCP, bag cell peptide; CDCP, caudodorsal cell peptide. (b) Multiple sequence alignment of molluscan ELH. Shading in alignment shows levels of conservation. Sequence logo (above alignment) also provides an indication of amino acid conservation.