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.

THE ROLE AND SIGNIFICANCE OF MORPHOLOGICAL CHANGES IN INTRAORGAN VESSELS IN ANIMALS DURING PATHOGENESIS OF BABESIOSIS

The aim of this study is to investigate morphological changes in intraorgan vessels and their role in susceptible animals in the pathogenesis of babesiosis. The material of the study included the intraorgan vessels of domestic dogs with clinically and laboratory confirmed babesiosis and wild mouse-like rodents from latent foci of babesiosis in Volyn, Zhitomir, Kiev, Poltava, Sumy, Kharkov, Chernigov regions. For histological study, we used standard fixation in a 12% aqueous formalin solution in phosphate buffer (pH = 7.0-7.2). After the post-fixation and dehydration, the samples were placed into paraffin blocks; then a series of histological slices (5 μm) were prepared. The preparations were stained with hematoxylin and eosin, according to Van Gieson technique. All test animals, regardless of the species taxonomy, were divided into two groups. The control group included clinically healthy animals (∑ = 36). The group under clinical study consisted of animals with clinically and laboratory confirmed babesiosis (∑ = 91). Histological changes in intraorgan vessels in the control group corresponded to the anatomical and functional parameters in the health. The preparations taken from the group with basesiosis demonstrate that microvessels are branched, different in size, spasmodic; their walls have signs of delamination and through lesions. Delamination developed gradually, started with the detachment of a small horizontal fragment of the outer layer. Defects in the integrity of microvessels resulted in penetrations; there was an active diapedesis of erythrocytes into the periovascular space. The accumulation of erythrocytes directed to the areas of connective tissue, branching of blood vessels, and fatty fragments. Hemorrhages were local in nature, concentrated in the form of small blurred foci of irregular shape. In the pathogenesis of babesiosis, the defects in the walls of blood vessels with their further penetration and dissection, hemorrhages, microvesiculation of the endothelium, and its desquamation play a critical role. Other manifestations included the development of intravascular blood coagulation, thrombosis, stasis, microcirculation disorders followed by ischemia, destructive and degenerative changes in the walls of blood vessels.

The ground beetle tribe Platynini Bonelli, 1810 (Coleoptera, Carabidae) in the southern Levant: dichotomous and interactive identification tools, ecological traits, and distribution

The carabids of the tribe Platynini from the southern Levant (Egypt: Sinai Peninsula, Israel, Jordan) and adjacent regions of Egypt, Lebanon, Syria, Iraq, and Saudi Arabia are reviewed in terms of species taxonomy, ecological, distributional traits, and conservation biology. In addition to a classical dichotomous identification key to the 14 species of the region, identification tools are made freely available via the Xper3 knowledge database “Platynini, southern Levant”. Besides an interactive identification key, a matrix with character states for the species and single access identification keys are available. A database including all available records from the southern Levant is also provided. First faunistic records are recorded for Anchomenus dorsalis infuscatus from Sinai (Egypt), Olisthopus fuscatus from Lebanon and Iraq, and for O. glabricollis from Iraq. Threatened species are discussed, also with regard to the reasons of their decline. The majority of species lives in wetlands, especially on the shore of winter ponds and streams, which have been extremely degraded in the last decades.

Mitochondrial genome announcements need to consider existing short sequences from closely related species to prevent taxonomic errors

The reconstruction of complete mitochondrial genomes (mitogenomes) has considerable potential to clarify species relationships in cases where morphological analysis and DNA sequencing of individual genes are inconclusive. However, the trend to use only mitogenomes for the phylogenies presented in mitogenome announcements carries the inherent risk that the study species’ taxonomy is incorrect because no mitogenomes have yet been reconstructed for its sister species. Here, I illustrate this problem using the mitogenomes of two seahorses, Hippocampus capensis and H. queenslandicus. Both specimens used for mitogenome reconstruction originated from traditional Chinese medicine markets rather than native habitats. Although mitogenome phylogenies placed these specimens correctly among the seahorses from which mitogenomes were available at the time, incorporating single-marker sequence from closely related species into the phylogenies revealed that both mitogenomes are problematic. The mitogenome of the endemic South African H. capensis did not cluster among single-marker DNA sequences of seahorses from the species’ native habitat, but among sequences submitted under the names H. casscsio, H. fuscus and H. kuda that originated from all over the Indo-Pacific, including China. Phylogenetic placement of the mitogenome of H. queenslandicus within a cluster of seahorses that also included H. spinosissimus confirms an earlier finding that H. queenslandicus is a synonym of H. spinosissimus, a widespread Indo-Pacific species that also occurs in China. It is recommended that mitogenome announcements incorporate available single-marker sequences of closely related species, not only mitogenomes. The reconstruction of mitogenomes can exacerbate taxonomic confusion if existing information is ignored.

A Current Update on the Distribution, Morphological Features, and Genetic Identity of the Southeast Asian Mahseers, Tor Species

The king of rivers or mahseer comprises three genera: Tor, Neolissochilus, and Naziritor, under the Cyprinidae family. The Tor genus has been classified as the true mahseer due to the presence of a median lobe among the three genera. The Tor species are widely distributed across Southeast (SE) Asia, and 13 Tor species have been reported previously: Tor ater, Tor dongnaiensis, Tor douronensis, Tor laterivittatus, Tor mosal, Tor mekongensis, Tor putitora, Tor sinensis, Tor soro, Tor tambra, Tor tambroides, Tor tor and Tor yingjiangensis. However, the exact number of valid Tor species remains debatable. Different and unstandardized approaches of applying genetic markers in taxonomic identification and morphology variation within the same species have further widened the gap and ameliorated the instability of Tor species taxonomy. Therefore, synchronized and strategized research among Tor species researchers is urgently required to improve and fill the knowledge gap. This review is a current update of SE Asia’s Tor species, outlining their distribution, morphology, and genetic identification. In addition, the present review proposes that there are ten valid Tor species in the SE Asian region. This list will serve as a template and standard to improve the taxonomy of the SE Asian Tor species, which could serve as a basis to open new directions in Tor research.

Research on Echinoderms in Latin America V

It is a pleasure for us to present this compendium that represents the sixth product of publications of the Iberoamerican Echinoderm Network (RIE), corresponding to 40 scientific papers presented at the Fourth Latin American Conference of Echinoderms, held from November 10th to 15th 2019 in the city of La Paz, Baja California Sur, Mexico. These works encompass aspects of echinoderms such as descriptions of new species, taxonomy, lists of species, new distribution records, paleontology, ecology, reproduction, physiology and aquaculture.

Genomes reveal the cohesiveness of bacterial species taxa and provide a path towards describing all of bacterial diversity.

This book chapter argues that bacterial systematists of the mid-20th century fortuitously created a species-level systematics that actually fits an important universal theory of speciation by discussing taxonomy would allow us to infer the important characteristics of any unknown organism once we classify it to species. It turns out, unexpectedly, that bacterial species taxa share a species-like property with the species taxa of zoology and botany. While recombination within species taxa of all these groups fails to prevent diversification within species, recombination nevertheless appears to act universally as a force of cohesion within species taxa. That is, recurrent recombination within species limits neutral sequence divergence within species taxa of plants, animals, and bacteria; recombination also allows a sharing of generally adaptive genes across a species range. The 95% ANI criterion that demarcates the traditionally defined species taxa of bacteria fortuitously also yields groups of bacteria that are subject to the species-like property of cohesion, where recombination prevents neutral sequence divergence among ecotypes within a species. Use of the ANI criterion, then, not only provides an easily used algorithm for demarcating bacterial species; it also places bacterial demarcation on the same theory-based foundation as the species taxonomy of animals and plants.