Diversity analyses of the eukaryotic microorganisms in the gut of marine animals is hampered by the presence of host DNA in the samples. PCR amplification of rRNA genes of eukaryotic microorganisms is inefficient with universal primers targeting 18S rRNA gene when the host DNA is dominant. In this study, we designed several blocking primers to inhibit PCR amplification of rRNA genes of the shrimp Litopenaeus vannamei, and tested their efficacy on the oyster Crassostrea hongkongensis. We first compared the intensity of PCR product bands obtained with and without the blocking primers. Then, one primer was selected for further verification using high-throughput sequencing. Our results showed that X-BP2-DPO was the most effective blocking primer in suppressing the host 18S amplification compared to nine other candidates. The inhibition rate was 99% for the amplification of shrimp rDNA, and 17% for the amplification of oyster rDNA. The concentration of the blocking primer in the PCR mixture was an important factor to be considered in the experimental design. The development of blocking primers provided a valid method to study the composition and characteristics of eukaryotic microorganisms in shrimp gut for a better understanding of its diets.

Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37–42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1–10 target DNA copies in less than 20 minutes. We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20–25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

The shell-boring Polydora websteri is described in detail in the present study for future unambiguous identification using an integrative taxonomic approach that combines morphology and molecular analysis of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Adult P. websteri exhibit a high degree of morphological plasticity in the palp pigmentation pattern, the shape of the anterior edge of the prostomium, the shape of the major spines on chaetiger 5, and the shape of the pygidium. The COI gene sequence demonstrated that the intraspecific distance of P. websteri was 0.33%, whereas the interspecific distance of P. websteri ranged from 18.88% (with P. brevipalpa) to 24.79% (with Boccardia proboscidea). The intraspecific genetic distances of polydorids examined in the present study ranged from 0.33% to 1.67%, whereas the interspecific distances ranged from 18.88% to 24.79%. Such large barcoding gaps between intra- and inter-specific distances indicate that the COI is a suitable gene marker for molecular identification of polydorid species. Our results demonstrate that not only did all COI sequences from the larvae show greater than 99% sequence identity to those from adults, but some larvae share the same haplotypes as adults. These findings clearly indicate that the larvae collected from sea waters around an oyster farm belong to P. websteri, the same species as the adult worms collected from the oyster Crassostrea hongkongensis in that locality. Two polydorid-specific primers were successfully designed, for the first time, to amplify target fragments of the COI gene. This study is the first to molecularly validate unidentified larvae from the aquatic environment through the known COI sequences of adults.