High CO₂ alters the hypoxia response of the Pacific whiteleg shrimp (Litopenaeus vannamei) transcriptome including known and novel hemocyanin isoforms

Abstract

Acclimation to low O2 in many organisms involves changes at the level of the transcriptome. Here we used high-throughput RNA sequencing (RNA-Seq) to explore the global transcriptomic response and specific involvement of a suite of hemocyanin (Hc) subunits to low O2 alone and in combination with high CO2, which naturally co-occurs with low O2. Hepatopancreas mRNA of juvenile L. vannamei exposed to air-saturated water, low O2, or low O2/high CO2 for 4 or 24 h was pooled, sequenced (HiSeq 2500) and assembled (Trinity: 52,190 contigs) to create a deep strand-specific reference transcriptome. Annotation of the assembly revealed sequences encoding the previously described small Hc subunit (HcS), and three full-length isoforms of the large subunit (HcL1-3). In addition to this, a previously unidentified full-length Hc subunit was discovered. Phylogenetic analysis demonstrated the subunit to be a β-type Hc subunit (denoted HcB), making this the first report of a β-type hemocyanin subunit in the Penaeoidea. RNAs of individual shrimp were sequenced; regulated genes identified from pairwise comparisons demonstrated a distinct pattern of regulation between prolonged low O2 and low O2/high CO2 treatments by GO term enrichment analysis (Roff-Bentzen, P < 0.0001), showcasing the stabilization of energetically costly translational machinery, mobilization of energy stores, and downregulation of the ubiquitin/proteasomal degradation machinery. Exposure to hypoxia for 24 h resulted in an increase in all of the full-length hemocyanin subunits (HcS, HcL1, HcL2, HcL3, and HcB). The addition of CO2 to hypoxia muted the transcriptomic response of all the Hc subunits to low O2, except for the β-type subunit.

Construction and Characterization of Two Novel Transcriptome Assemblies in the Congeneric Porcelain Crabs Petrolisthes cinctipes and P. manimaculis

“…Both of these hit ratios fell within the general range of values reported for other arthropod de novo transcriptome assemblies though there is considerable variation in hit rate reported among species ($10-50% depending on stringency criteria; Hahn et al 2009;Roeding et al 2009;Zeng et al 2011;Johnson et al 2015). The significantly higher BLAST hit rate for P. cinctipes is surprising given the close phylogenetic relationship of these two congeners.…”

“…While a sizeable microarray EST dataset does already exist for the porcelain crab Petrolisthes cinctipes ($98K ESTs; Tagmount et al 2010), the sequences generated in this study via NGS approaches nearly triple this dataset ($194K ESTs) and adds $279K sequences for the closely related species Petrolisthes manimaculis. Within just the last few years, the community of crustacean biologists have utilized NGS-powered functional genomics approaches to examine, among other topics, growth and development (lipid storage and diapause cues in Calanus finmarchicus copepods; Tarrant et al (2014); comparison of eye-development among ecotypes in the freshwater isopod, Asellus aquaticus; Stahl et al (2015)), immune and toxin responses (investigation of resistance to a delousing drug in Caligus rogercresseyi, Chávez-Mardones and Gallardo-Escárate (2015); identification of candidate genes involved in virusinduced immune response in L. vannamei; Robalino et al (2007); elucidation of differences between sexes and populations in drug resistance in the parasitic copepod L. salmonis; Poley et al (2015), regulation of molting (Y-organ molt gland profiling, Das et al (2016); global expression changes throughout the molt cycle in L. vannamei, Gao et al (2015), and responses to hypercapnia and hypoxia (characterization of a suite of hemocyanin genes involved in hypercapnic-hypoxia response in L. vannamei; Johnson et al (2015), nitrate stress (contrasting acute and chronic exposure in the river prawn Macrobrachium nipponense; Xu et al (2016), and thermal and osmotic challenges (acute and plastic heat shock responses in P. cinctipes; Teranishi and Stillman (2007); Stillman and Tagmount (2009);Ronges et al (2012); responses to acute salinity stress in L. vannamei, Wang et al (2015). Such approaches are useful in helping to develop mechanistic models of cellular processes accompanying phenotypic shifts in response to stress (phenotypic plasticity) and provide fruitful grounds for addressing hypotheses in the field of ecological physiology.…”

“…For such species however, at all sequencing depths, increasing the number of biological replicates consistently increases detection of differentially expressed genes. In light of this, we chose to utilize higher biological replication (n ¼ 5 individuals per treatment) in this study at the expense of sequencing depth ($17.7M reads/ sample and $21.9M reads/sample as compared to (Johnson et al 2015).…”

“…5) including hepatopancreas tissue in the shrimp L. vannamei (Johnson et al 2015), whole embryos of the amphipod P. hawaiensis and predicted transcripts from the sequenced genome of the water-flea D. pulex . Comparison of our gill tissue transcriptome assemblies with these assemblies revealed significant overlap in functional gene categories of annotated genes, suggesting that overall our de novo transcriptomes do not lack major functional categories.…”

Resources and Recommendations for Using Transcriptomics to Address Grand Challenges in Comparative Biology

“…Transcriptomics using NGS technology has revealed genes involved in stress response, reproduction, development, molting and growth, limb regeneration, immune response, endocrinology, and nutrition and digestion in decapod crustaceans (Sagi et al 2013; Tom et al 2013; Durica et al 2014; Ghaffari et al 2014; Hao et al 2014; Lv et al, 2014, 2015; Shen et al 2014; Song et al 2014; Tom et al 2014; Wei et al 2014a, 2014b; Abehsera et al 2015; Chandler et al 2015; Christiaens et al 2015; Huang et al 2015; Johnson et al 2015; Li et al 2015a, 2015b; Ventura et al 2014, 2015; Verbruggen et al 2015; Wang et al 2015; Das et al 2016a). It is clear from the input received at the SICB meeting and the ten symposium papers in this issue (Armstrong and Stillman 2016; Chandler et al 2016; Clark and Greenwood 2016; Das and Mykles 2016; Das et al 2016b; Havird and Santos 2016a, 2016b; Johnson et al 2016; Powell et al 2016; Tarrant et al 2016) that a greater understanding of the relationship between transcriptomic and phenotypic change in decapod crustaceans will not be achieved until more powerful and applicable “-omic” tools and resources are developed.…”