Among the many Callinectes spp. across the western Atlantic, the blue crab C. sapidus has the broadest latitudinal distribution, encompassing both tropical and temperate climates. Its life history varies latitudinally, from extended overwintering at high latitudes to year-round activity in tropical locations. Callinectes sapidus reovirus 1 (CsRV1) is a pathogenic virus first described in North Atlantic C. sapidus and has recently been detected in southern Brazil. Little information exists about CsRV1 prevalence at intervening latitudes or in overwintering blue crabs. Using a quantitative reverse transcription PCR (RT-qPCR) method, this study investigated CsRV1 prevalence in C. sapidus across latitudinal differences in temperature and crab life history, as well as in additional Callinectes spp. and within overwintering C. sapidus . CsRV1 prevalence in C. sapidus was significantly correlated with high water temperature and blue crab winter dormancy. Prevalence of CsRV1 in C. sapidus on the mid-Atlantic coast was significantly lower in winter than in summer. CsRV1 infections were not detected in other Callinectes spp. These findings revealed that CsRV1 is present in C. sapidus across their range, but not in other Callinectes species, with prevalence associated with temperature and host life history. Such information helps us to better understand the underlying mechanisms that drive marine virus dynamics under changing environmental conditions.
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Rapid Genetic Identification of the Blue Crab Callinectes sapidus and Other Callinectes spp. Using Restriction Enzyme Digestion and High Resolution Melt (HRM) Assays
The blue crab Callinectes sapidus is one of the most widely studied marine crustaceans due to its high economic value and ecological significance. Despite extensive research on the blue crab in North America, many questions remain about the distribution and abundance of the species in the subtropics and tropics. In many places, C. sapidus is sympatric with morphologically similar Callinectes spp., which has implications for seafood mislabeling. To enable rapid identification of the species, we designed and tested two PCR-based assays targeting the 12S rRNA mitochondrial gene. The first assay discriminates C. sapidus from other Callinectes spp. via post-PCR restriction digestion (PCR-RFLP) and the second assay discriminates among multiple Callinectes spp. through High Resolution Melting (HRM) analysis and supervised machine learning analyses. A total of 58 DNA samples from five Callinectes spp. (validated via 12S gene sequencing) were used for assay testing. The PCR RFLP assay was 100% accurate identifying C. sapidus from other Callinectes spp. HRM analysis of amplicons showed good discrimination among species, with distinct clusters formed between species with higher sequence homology. Linear discriminant analysis (LDA) classification of HRM curves was quite successful given the small dataset available, producing ∼90–91% mean accuracy in classification over all species with 100-fold cross validation. Much of the error came from misclassifications between C. similis and C. danae, which are ∼99% similar in sequence for the amplicon; collapsing them into a single class increased overall classification success to 94%. Error also arose from C. bocourti classifications, which had a reference set containing only three samples. Classification accuracy of C. sapidus alone via HRM was 97.5%. Overall, these assays show great promise as rapid and inexpensive methods to identify Callinectes spp. and have application for both ecological research and seafood identification or labeling.
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- NSF-PAR ID:
- 10186598
- Date Published:
- Journal Name:
- Frontiers in marine science
- Volume:
- 7
- ISSN:
- 2296-7745
- Page Range / eLocation ID:
- 663
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)The blue crab, Callinectes sapidus (Rathbun, 1896) is an economically, culturally, and ecologically important species found across the temperate and tropical North and South American Atlantic coast. A reference genome will enable research for this high-value species. Initial assembly combined 200× coverage Illumina paired-end reads, a 60× 8 kb mate-paired library, and 50× PacBio data using the MaSuRCA assembler resulting in a 985 Mb assembly with a scaffold N50 of 153 kb. Dovetail Chicago and HiC sequencing with the 3d DNA assembler and Juicebox assembly tools were then used for chromosome scaffolding. The 50 largest scaffolds span 810 Mb are 1.5–37 Mb long and have a repeat content of 36%. The 190 Mb unplaced sequence is in 3921 sequences over 10 kb with a repeat content of 68%. The final assembly N50 is 18.9 Mb for scaffolds and 9317 bases for contigs. Of arthropod BUSCO, ∼88% (888/1013) were complete and single copies. Using 309 million RNAseq read pairs from 12 different tissues and developmental stages, 25,249 protein-coding genes were predicted. Between C. sapidus and Portunus trituberculatus genomes, 41 of 50 large scaffolds had high nucleotide identity and protein-coding synteny, but 9 scaffolds in both assemblies were not clear matches. The protein-coding genes included 9423 one-to-one putative orthologs, of which 7165 were syntenic between the two crab species. Overall, the two crab genome assemblies show strong similarities at the nucleotide, protein, and chromosome level and verify the blue crab genome as an excellent reference for this important seafood species.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmars.2020.00633
