De Novo assembly and characterisation of the greentail prawn (Metapenaeus bennettae) hepatopancreas transcriptome – identification of stress response and detoxification transcripts
Introduction
Crude oil, and its post-combustion counterparts, enter marine environments, via natural seepage, and anthropogenic sources including oil spills, produced water (by-product of hydrocarbon extraction), and stormwater run-off (Farrington, 2013). Crude oil toxicity is primarily derived from polycyclic aromatic hydrocarbon (PAH) compounds which have detrimental physiological effects on marine organisms, including narcosis (Di Toro et al., 2000, Di Toro et al., 2007), reduced cell membrane integrity (Almeda et al., 2013), inhibited larval development (Mitra et al., 2012; Sørhus et al., 2015), and hepatic abnormalities (Myers et al., 1992). Research characterising the effects of crude oil on marine organisms has included identification of histological alteration (Myers et al., 1992) as well as finer scale cytochemical biochemical response (Whyte et al., 2000). Biomarkers of PAH exposure include CYP1A gene expression profiling (Balk et al., 2011), contrasts of ethoxyresorufin-O-deethlyase (EROD) and glutathione S-transferase (GST) enzyme activities (Kammann et al., 2014; Lavado et al., 2006; Whyte et al., 2000), PAH metabolite concentrations (Balk et al., 2011; Kammann et al., 2014), and comparisons of DNA adducts (Balk et al., 2011; Chatel et al., 2014). More recently, there has been a significant increase in studies using modern genomics enabled technologies, including transcriptomics, for characterising molecular mechanisms underpinning responses of marine organisms to hydrocarbon exposure, including both light unweathered (Hook et al., 2018) and heavy crude oils (Hook et al., 2010; Xu et al., 2017; Yednock et al., 2015).
Historically, the cost and logistical constraints of traditional molecular methods limited the generation of transcriptome data to model species (Riesgo et al., 2012). In contrast, modern next generation sequencing (NGS) technologies and bioinformatic tools now allow for the rapid, cost-effective sequencing and annotation of transcriptomes, for both model and non-model species (Feldmeyer et al., 2011; Lenz et al., 2014; Richardson and Sherman, 2015; Riesgo et al., 2012). Marine ecotoxicology has benefited significantly from these advances, providing new opportunities for characterising gene expression profiles associated with exposure to endocrine disrupting compounds (Benninghoff and Williams, 2008; Legrand et al., 2016), metals (Hook et al., 2014a, Hook et al., 2014b; Koskinen et al., 2004) and hydrocarbons (Hook et al., 2010, Hook et al., 2014c; Maria et al., 2005; Nakayama et al., 2008). Most transcriptomic studies relating to aquatic vertebrates tend to focus on commercially important fish species (Hook et al., 2017; Roling et al., 2004; Xu et al., 2017), whereas the invertebrate studies encompasses a broad range of taxonomic groups, including decapods (Jin et al., 2013; Sun et al., 2014; Yednock et al., 2015), amphipods (Hook et al., 2014b, Hook et al., 2014c), copepods (Legrand et al., 2016; Wang et al., 2017; Zhou et al., 2018) and bivalves (Bigot et al., 2010; Jiang et al., 2017) of commercial and non-commercial importance. Such studies have been pivotal in improving our understanding of physiological and molecular responses triggered by contaminants, and the pathways underpinning the detoxification processes in non-model species (Gomiero et al., 2006; Hook et al., 2010). However, further genetic resources are needed for non-model species such as crustaceans to increase our understanding of changes in biological, cellular and molecular regulation in response to developmental, seasonal, environmental and anthropogenic changes (Lenz et al., 2014).
Studies examining contaminant exposure effects in decapod crustaceans have been limited to date (Hui et al., 2018; Ren et al., 2014; Sun et al., 2014; Yednock et al., 2015). Decapods (including crabs, crayfish, lobsters, prawns and shrimps), occupy a range of marine, freshwater and terrestrial environments, providing critical ecosystem services as detritivores (De Silva, 1989; Hart, 1981) and represented in commercial and recreational fisheries globally (Kenny et al., 2014; Zeng et al., 2011). Many decapod species are vulnerable to contaminant exposure, particularly those occupying benthic sediments that accumulate hydrocarbons and heavy metals (Harris and Santos, 2000; Ren et al., 2014). Given the ecological and commercial importance of this group of crustaceans, ecotoxicological studies are needed to assess exposure effects and associated pathways for the detoxification and elimination of contaminants. Recent transcriptomic analyses of the benthic Argentinian freshwater prawn (Palaemonetes argentines; García et al. (2018)) and juvenile blue crab (Callinectes sapidus; Yednock et al. (2015)) have provided new insights into detoxification, oxidative stress, osmoregulation and DNA replication pathways associated with contaminant exposure in decapods. García et al. (2018) identified 27 transcripts coding for the phase I detoxification genes cytochrome (CYP) P450 superfamily, and 10 transcripts encoding GST enzymes involved in phase II detoxification mechanisms in freshwater prawns. Yednock et al. (2015) found the same genes plus an additional ~100 other genes from the gills and hepatopancreas that were differentially expressed after exposure to crude oil in the blue crab. A notable finding was the upregulation of the gene thiopurine S-methyltransferase, known to metabolise pharmaceutical compounds in humans (Woodson et al., 1983) and the gene dehydrogenase reductase SDR family member 7, involved in xenobiotic metabolic pathways in humans (Skarydova and Wsol, 2012), suggesting that upregulation of these genes in crustaceans could potentially enable metabolisation of other xenobiotic compounds including crude oil. Additionally, hundreds of other genes involved in regulating growth, reproduction, DNA replication and metabolism are significantly expressed in response to crude oil exposure, facilitating future investigation of adverse outcome pathway mechanisms responsible for exposure effects on aquatic organisms (Hook et al., 2014b; Xu et al., 2017; Yednock et al., 2015).
In this study, we present the de novo assembly and annotation of the greentail prawn (Metapenaeus bennettae) hepatopancreas transcriptome using next-generation sequencing, providing new insights into detoxification capabilities through the identification and characterisation of detoxification and stress response transcripts. Adult greentail prawns inhabits shallow, coastal marine and estuarine soft sediment habitats (0–14 m) (Kirkegaard and Walker, 1970). Distribution extends from Rockhampton, Queensland, on the east coast to the Great Australian Bight, on the southern coast of South Australia (Edgar, 2008; Kirkegaard and Walker, 1970). Greentail prawn commercial fisheries operate across the species' range, with the primary fishery located in the Brisbane River and Moreton Bay in southern Queensland (annual catch >500 t), and in New South Wales (annual catch 20–50 t) (Young et al., 2013). Greentail prawns bury themselves in sediments during the day to avoid predation, and emerge at night to feed in the water column (Kirkegaard and Walker, 1970), indicating that greentail populations are likely to be exposed to water soluble and sediment bound inorganic and organic contaminants, including PAHs (Lewtas et al., 2014; Pasquevich et al., 2013). In St Vincent Gulf, South Australia anthropogenic inputs of hydrocarbons associated with stormwater run-off and shipping activity are common and pose a significant risk to benthic organisms such as the greentail prawn (Edyvane, 1999). Chronic in situ exposure to crude oil could lead to potential bioaccumulation, detoxification and metabolism of hydrocarbons, specifically PAHs, with implications for understanding the resilience of greentail prawn populations to pollution exposure, and risks to the human population as consumers. In this study, we report the de novo assembly and annotation of the greentail prawn (Metapenaeus bennettae) transcriptome derived from Illumina short-read data. This assembly will provide a crucial resource for future targeted gene discovery and analysis of known and previously unknown adverse outcomes pathways involved in contaminant detoxification and metabolic processes, providing valuable information regarding long-term effects of crustacean health after exposure to environmentally relevant light, unweathered crude oil (Hook et al., 2018) synonymous with Australian hydrocarbon resources (Volkman et al., 1994). Furthermore, the transcriptome could contribute to the assessment of decapod health following a crude oil spill. Finally, the assembly adds to the growing resource of decapod transcriptome libraries (Northcutt et al., 2016; Tan et al., 2016; Theissinger et al., 2016).
Section snippets
Wild collections, library preparation and RNA sequencing
Thirty wild greentail prawns (Metapenaeus bennettae) were collected as part of a larger crude oil exposure trial in trawls from St Vincent Gulf, near Adelaide on board the RV Ngerin on the 9th February 2016. The prawns were transported to South Australian Research and Development Institute (SARDI) laboratories in Adelaide, where they acclimated for 2 weeks in six 2250 L flow-through seawater tanks with contaminant-free sand sediment in order for the prawns to maintain physiological and
Sequencing and assembly
Illumina sequencing of RNA libraries derived from the wild-caught prawns generated a total 65,686,395 reads, and 62,875,326 reads after quality control filtering (4.3% of total reads removed). The Trinity assemblies on filtered sequence reads produced 114,408 contigs with a N50 of 1813 bp (maximum contig length = 17,399 bp; average contig length = 890.4 bp) (Table 1). It has been shown previously that clustering assemblies can reduce redundancy by merging duplicate transcripts (95% similarity
Conclusions
This study involved the reconstruction of the first hepatopancreas transcriptome of the greentail prawn (M. bennettae), representing the 12th transcriptome for a decapod crustacean (Jin et al., 2013; Theissinger et al., 2016; Yednock et al., 2015). The assembly consisted of 86,401 contigs, with 22,252 annotated genes displaying strong homology with other crustacean and decapod species. Potential crude oil biomarkers of exposure were identified in the greentail prawn transcriptome, including
Additional information
All work with animals was conducted with the approval of the CSIRO (South Australia) Animal Ethics Committee (AEC), permit number 808.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This project was undertaken as part of the Great Australian Bight Deepwater Marine Program - a public-good research program led by CSIRO, and sponsored by Chevron Australia, with data generated to be made publically available.
Author contributions statements
SH and JM initiated and designed the current study, and coordinated RNA tissue sample collection. SS conducted RNA preparation for library formation and sequencing. EA led the transcriptome assembly and functional annotation, with additional contribution from PG, MHT, HMG and AM. EA led the interpretation of functional significance of the derived transcriptome, with additional contribution from SH and JM. EA drafted the manuscript which was subsequently reviewed by AM, JM and SH, and approved
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