Exploiting genomic data to identify proteins involved in abalone reproduction
Graphical abstract
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.
Section snippets
Protein preparation and digestion using Filter Aided Sample Preparation (FASP)
Sexually mature H. laevigata (100–130 mm; 36 months of age) with a visual gonad index (VGI) of 3 were selected from production tanks at the Kangaroo Island Abalone (KIAB) farm. The VGI is a non-destructive technique for the assessment gonad development during spawning season [9] and consists of four categories (0–3) that relate to changes in the size and shape of the gonad wherein VGI 3 refers to animals with a swollen gonad with rounded tip and are classified as sexually mature. Gonads were
Results and discussion
A total of 162 and 110 proteins were identified in female and male gonads respectively using a proteomic approach employing high resolution MS and database searching utilising both protein and DNA sequences housed in public resources. The benefit of using genomic resources in poorly characterised species is highlighted by the application of the custom-built database described here. Additional gains of 60% (female) and 72% (male) in the identification rates (from only 101 and 64 proteins
Conclusions
This study has revealed the identity of 232 proteins, the majority of which are reported for the first time in H. laevigata. Despite the high economic importance for many aquaculture focused countries, abalone is one of the least studied commercial molluscs. The most significant aspect in this study is the identification of 47 proteins related to sexual maturity, fertilization and reproduction. Inconsistent spawning in the Australian abalone aquaculture industry has hindered the application of
Competing interests
All authors declare no competing interests.
Author's contributions
OMP participated in sample collection, sample processing, data acquisition, data interpretation, and manuscript preparation. NAB, MTC and JOH assisted in aspects of aquaculture and abalone reproduction including experimental design, execution and sampling. SMM produced the customised molluscan protein database. GW contributed with expertise in biology, database development and manuscript preparation. MLC provided expertise in mass spectrometry, bioinformatics and manuscript preparation.
Transparency document
Acknowledgements
OMP acknowledges and thanks the following people and organisations: the staff of Kangaroo Island Abalone and manager Mr. David Connell for the kind donation of abalone samples; Mr. Harry Goswami from CSIRO for assistance with mass spectrometry; CSIRO internal reviewers Brian Shiell and Harry King for their valuable contributions; the Mexican Council of Science and Technology (CONACyT) for supporting OMP through the provision of a PhD scholarship (number 308662) and the Australian Seafood CRC
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2023, Developments in Aquaculture and Fisheries ScienceLong-read RNA sequencing of Pacific abalone Haliotis discus hannai reveals innate immune system responses to environmental stress
2022, Fish and Shellfish ImmunologyCitation Excerpt :Nevertheless, several processes concerning the immune system in H. discus hannai remain unclear, such as the linkages and cross-talks between distinct signaling pathways, as well as the immunological regulation of the immune system by diverse stressors. The use of a genomics platform was successfully adopted in H. discus hannai, and many investigations are now ongoing to better understand the growth, development, molecular adaptability, pathologies, or innate immune defense mechanisms in the Haliotis genus in response to environmental challenges [45–54]. Furthermore, transcriptome research of Haliotis species and many other aquaculture mollusks have been conducted and reported in recent years utilizing high-throughput sequencing technology [46,55–72].
Use of a gene encoding zona pellucida 4 as a female-specific marker for early stage sexual differentiation and size dimorphism in the pacific abalone Haliotis discus hannai
2021, Animal Reproduction ScienceCitation Excerpt :Zona pellucida domain proteins have also been identified in the outer membranes of abalone, amphibians, and fish (Kubo et al., 1997; Wang and Gong, 1999). The gene encoding zp12 is expressed exclusively in female gonads in H. discus discus (Yu et al., 2018), and in a proteomic study there was identification of several vitelline envelope zona pellucida domain proteins (vezp) that were expressed in female gonads of H. laevigata (Mendoza-Porras et al., 2014). There were more than 30 genes encoding the vezp domain identified in H. rufescens that are markedly different in structure than those for the mammalian homologues (Aagaard et al., 2010).
Characterization and expression analysis of a GnRH-like peptide in the Pacific abalone, Haliotis discus hannai
2020, Agri GeneCitation Excerpt :H. discus hannai is deemed to be high valued seafood due to its content in health beneficial bioactive molecules, in addition to its basic nutritional value (Suleria et al., 2015). Many research groups have relied on the genomic and proteomic approaches, linkage map development, construction of bacterial artificial chromosome (BAC) libraries, and whole genome sequence to unravel the mechanisms underlying the growth, development, reproduction, and molecular adaptation of the Haliotis genus (Palmer et al., 2013; Mendoza-Porras et al., 2014; Jiang et al., 2016; Nam et al., 2017). Several studies have reported on isolation and expression of GnRH in various tissues of protostomian and ascidian species (Zhang et al., 2000; Di Cristo et al., 2002; Iwakoshi-Ukena et al., 2004; Tsai et al., 2003; Di Fiore et al., 2000).