Dataset Information

Detection and discovery of crustacean parasites in blue crabs (Callinectes sapidus) by using 18S rRNA gene-targeted denaturing high-performance liquid chromatography.

ABSTRACT: Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.

SUBMITTER: Troedsson C

PROVIDER: S-EPMC2493178 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Detection and discovery of crustacean parasites in blue crabs (Callinectes sapidus) by using 18S rRNA gene-targeted denaturing high-performance liquid chromatography.

Troedsson Christofer C Lee Richard F RF Walters Tina T Stokes Vivica V Brinkley Karrie K Naegele Verena V Frischer Marc E ME

Applied and environmental microbiology 20080523 14

Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected p ...[more]

PMID: 18502913

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Project description:BackgroundThe blue crab, Callinectes sapidus, is economically and ecologically important in western Atlantic and Gulf of Mexico coastal estuaries. In 2010 blue crabs in the northern Gulf of Mexico were exposed to crude oil and chemical dispersants from the Deepwater Horizon oil spill. To characterize the blue crab transcriptome and identify genes that could be regulated in response to oil exposure we sequenced transcriptomes from hepatopancreas and gill tissues of juvenile blue crabs after exposing them to a water-accommodated fraction of surrogate Macondo crude oil in the laboratory and compared them to transcriptomes from an unexposed control group.ResultsIllumina sequencing provided 42.5 million paired-end sequencing reads for the control group and 44.9 million paired-end reads for the treatment group. From these, 73,473 transcripts and 52,663 genes were assembled. Comparison of control and treatment transcriptomes revealed about 100 genes from each tissue type that were differentially expressed. However, a much larger number of transcripts, approximately 2000 from each tissue type, were differentially expressed. Several examples of alternatively spliced transcripts were verified by qPCR, some of which showed significantly different expression patterns. The combined transcriptome from all tissues and individuals was annotated to assign putative gene products to both major gene ontology categories as well as specific roles in responses to cold and heat, metabolism of xenobiotic compounds, defence, hypoxia, osmoregulation and ecdysis. Among the annotations for upregulated and alternatively-spliced genes were candidates for the metabolism of oil-derived compounds.ConclusionsPreviously, few genomic resources were available for blue crabs or related brachyuran crabs. The transcriptome sequences reported here represent a major new resource for research on the biology of blue crabs. These sequences can be used for studies of differential gene expression or as a source of genetic markers. Genes identified and annotated in this study include candidates for responses of the blue crab to xenobiotic compounds, which could serve as biomarkers for oil exposure. Changes in gene expression also suggest other physiological changes that may occur as the result of exposure to oil.

Project description:A large number of coastal ecosystems globally are subjected to concurrent hypoxic and acidified conditions that will likely intensify and expand with continued climate change. In temperate regions, the spawning of many important organisms including the Atlantic blue crab Callinectes sapidus occurs during the summer months when the severity of coastal hypoxia and acidification is the greatest. While the blue crab earliest larval stage can be exposed to co-occurring hypoxia and acidification observed in many coastal ecosystems, the effects of these concurrent stressors on larval blue crab survival is unknown. This study investigated the individual and combined consequences of low dissolved oxygen (DO) and low pH on blue crab larvae survival through a series of short-term experiments. During 14-day experiments with moderately hypoxic conditions (117-127 μM O2 or 3.74-4.06 mg L-1) and acidified conditions (pH on total scale of 7.16-7.33), low DO and low pH individually and significantly reduced larval survival by 60% and 49%, respectively, with the combination of stressors reducing survival by 87% compared to the control treatment (210-269 μM O2 or 6.72-8.61 mg L-1, 7.91-7.94 DO and pH, respectively). During 4-day experiments with lower DO levels (68-83 μM O2 or 2.18-2.62 mg L-1) and comparable pH levels of 7.29-7.39, low DO individually reduced survival by >90% compared to the control (261-267 μM O2 or 8.35-8.54 mg L-1, 7.92-7.97 DO and pH, respectively), whereas low pH had no effect and there was no interaction between stressors. Over a 4-day period, the DO threshold at which 50% of the larval blue crab population died (LC50) was 121 μM O2 (3.86 mgL-1). In 14-day experiments, the DO and pH effects were additive, yielding survival rates lower than the individual treatments, and significantly correlated with DO and pH concentrations. Collectively, these findings indicate that blue crab sensitivity to both low DO and low pH are acute within the larval stage, depend on the intensity and duration of exposure, and leads to mortality, thereby potentially contributing to the interannual variability and possible regional declines of this fishery.

Dataset Information

Detection and discovery of crustacean parasites in blue crabs (Callinectes sapidus) by using 18S rRNA gene-targeted denaturing high-performance liquid chromatography.

Publications

Detection and discovery of crustacean parasites in blue crabs (Callinectes sapidus) by using 18S rRNA gene-targeted denaturing high-performance liquid chromatography.

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