Intensive Commercialization of Endangered Sharks and Rays (Elasmobranchii) Along the Coastal Amazon as Revealed by DNA Barcode

Elasmobranchs represent a well-defined group, composed of about 1,150 species inhabiting diverse aquatic environments. Currently, several of these species have been classified as threaten due to overexploitation. Therefore, we used DNA barcode to identify traded species of sharks and stingrays in the municipality of Bragança (Amazon coastal region), a major fishery landing site in northern Brazil. We collected a total of 127 samples labeled into 24 commercial nomenclatures over 1-year period. Twenty species were discriminated and 13 of them are recognized under some threatening status. In relation to sharks, Carcharhinidae showed the highest number of species, half of them classified as endangered, followed by hammerhead sharks (Sphyrnidae), with four species also regarded as threatened with extinction. The Rhinopteridae and Dasyatidae rays were the most abundant groups, with trade records of the following threatened species: Rhinoptera brasiliensis, Rhinoptera bonasus, Hypanus berthalutzae, and Fontitrygon geijskesi. It is noteworthy that threatened species of elasmobranchs have been frequently and regularly exploited because of inefficient fishery management policies. Therefore, effective inspection practices need to be incorporated in fisheries, including the use of DNA barcode to enable a reliable method of species authentication and to assure the proper commercialization.

Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn

Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen’s DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, “Bar-cas12a” for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM’s gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.

Bar-cas12a, a Novel and Rapid Method for Plant Species Authentication: A Case of Phyllanthus Species

The rapid and accurate species diagnosis accelerates the performance to investigate various biology fields and its relevant, perhaps but morphology-based species taxonomy/identification hamper. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen’s DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-hyphenated with cas12a assay, “Bar-cas12a” for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM’s gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication and is likely to implement point-of-care testing.