Exploiting genomic data to identify proteins involved in abalone reproduction

Highlights

• A molluscan protein database was constructed from a variety of genomic resources.

• Protein identification rates were increased >60% over standard database usage.

• The gonads of sexually mature male and female abalone were profiled.

• Key proteins involved in both reproduction and energy balance were identified.

Abstract

Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography–mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis.

Biological significance

A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding > 60% more protein identifications than in a standard workflow employing public protein databases.

Introduction

Abalone are marine gastropods that can be found worldwide in temperate and tropical waters from intertidal zones and to a depth of 40 m [1]. Abalone are highly valued for their palatability in Europe, the United States and most Asian countries. In the wild, abalone resources are strictly regulated to prevent depletion of natural stocks which in turn has promoted artificial cultivation to fulfil market demands. Abalone are cultivated in many countries including Australia, New Zealand, South Africa, Japan, China, Korea, Chile, Mexico, the United States and Thailand [2]. In Australia temperate blacklip Haliotis rubra, greenlip Haliotis laevigata and their hybrid are the predominant aquaculture species farmed onshore in the coastal areas of Victoria, Tasmania, South Australia and Western Australia. In 2011–12 the value of wild-caught and farmed abalone production was estimated to be AUD$178 million [3].

Australian abalone farmers face significant challenges. Financial commitments are high as it can take up to four years to bringing temperate abalone to market. Aquatic animal health is a major concern in the abalone industry. Massive mortalities can occur due to pathogens and variations of temperature that compromise the immune systems of abalone. Another issue in temperate abalone farming is the inefficient control over spawning which hinders the application of selective breeding programmes to improve production and allow highly marketable abalone with desirable characteristics to be farmed. Selection of sexually mature abalone on farms relies upon the visual assessment of the gonad diameter and state of engorgement. Whilst this technique is not entirely accurate it allows the farmer to select potential broodstock without sacrificing the animal to evaluate its sexual maturity.

Considerable research has been performed on H. rubra and H. laevigata, however this has focused on physiological studies to improve culture conditions and on genetic studies to develop selective breeding programmes and improve animal health. There has been some progress in the use of proteomic approaches in gastropod molluscs, such as the sea slug Aplysia californica, the limpet Lottia gigantea, and pond snail Lymnaea stagnalis, where substantial genomic information is available [4], [5], [6]. In contrast, the successful application of proteomics to studies of abalone is limited by the poor availability of genomic information in public databases (e.g. UniProt, NCBI). A study employing proteomics and a NCBI Haliotis asinina database identified only 14 proteins from abalone shell matrix, two of which had previously been reported [7]. This is a low identification rate compared to similar studies in well characterised species. For example, a similar approach reported the identification of 569 proteins from limpet shell matrix [6]. Owing to the limitations of the public protein databases (34 reviewed proteins exist in UniProt-KB), characterisation of abalone proteins relies on either examination of homology to orthologous proteins in other gastropod species or predictions based on the use of genomic resources from other molluscan species. To illustrate this point, a recent study employing transcriptomics to generate a database specific to Haliotis rufescens was coupled to proteomics and facilitated the identification of 975 proteins in abalone sperm [8].

Reproduction must be efficient to ensure the continuity of any species. In broadcast spawning organisms, efficient reproduction is more complex due to the dynamic nature of their ecological niche. Abalone rely upon numerous environmental cues and chemotaxis for gametes to coincide and for fertilization to occur. Whilst reproduction is well documented in model organisms, control of synchronized spawning which is a prerequisite for efficient abalone reproduction on farms remains unsolved.

In this study, a molluscan protein database was assembled using protein sequences predicted by EST and genomic resources, which facilitated a more in-depth proteomic analysis of abalone gonad. This approach was applied to the comprehensive proteomic profiling of mature female and male H. laevigata gonads and provides the basis for ongoing studies of reproduction in farmed abalone.