The Isolation of Vibrio crassostreae and V. cyclitrophicus in Lesser-Spotted Dogfish (Scyliorhinus canicula) Juveniles Reared in a Public Aquarium

The genus Vibrio currently contains 147 recognized species widely distributed, including pathogens for aquatic organisms. Vibrio infections in elasmobranchs are poorly reported, often with identifications as Vibrio sp. and without detailed diagnostic insights. The purpose of this paper is the description of the isolation and identification process of Vibrio spp. following a mortality event of Scyliorhinus canicula juvenile reared in an Italian public aquarium. Following investigations aimed at excluding the presence of different pathogens of marine fish species (parasites, bacteria, Betanodavirus), several colonies were isolated and subjected to species identification using the available diagnostic techniques (a biochemical test, MALDI-TOF MS, and biomolecular analysis). Discrepancies were observed among the methods; the limits of biochemistry as a unique tool for Vibrio species determination were detected through statistical analysis. The use of the rpoB gene, as a diagnostic tool, allowed the identification of the isolates as V. crassostreae and V. cyclotrophicus. Although the pathogenic role of these microorganisms in lesser-spotted dogfish juveniles has not been demonstrated, and the presence of further pathogens cannot be excluded, this study allowed the isolation of two Vibrio species in less-studied aquatic organisms, highlighting the weaknesses and strengths of the different diagnostic methods applied.

Orchid Identification and Collection Techniques in the Kembang Kuning Resort Area, Rinjani Mount National Park (RMNP)

The Rinjani Mount National Park (RMNP) area is one part of the tropical rain forest in the West Nusa Tenggara region. Orchid is one of the flora that has a high bioprospective in this area. The spread of natural orchids can continue to grow and there are still many that have not been identified. This study aims to train specific techniques in collecting and identifying natural orchids at The Resort of Kembang Kuning, Rinjani Mount National Park. The collection of orchids was carried out using the roaming method, while the identification of orchid species was carried out using a species determination technique through observation of morphological and anatomical descriptions. The out comes of this study are an increase in participants' appreciation, knowledge and skills in collecting and identifying species of natural orchids in the Kembang Kuning area of Rinjani ​​Mount National Park. Based on the observations obtained 9 species (7 species were identified to the species level and 2 species were identified to the genus level) from 6 genera of natural orchids in area of The Kembang Kuning Resort, The Rinjani Mount National Park (RMNP).

Technical note: Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework

The aim of this technical note is to provide an overview of methodical approaches used to develop molecular systems for species determination/DNA quantification called Ptig Qplex and individual identification called Ptig STRplex of Panthera tigris samples. Both systems will help to combat the illegal trade of endangered species and create a worldwide shared database of DNA profiles.

Raman Calibration Models for Chemical Species Determination in CO2-Loaded Aqueous MEA Solutions Using PLS and ANN Techniques

The improvement in energy efficiency is recognized as one of the significant parameters for achieving our net-zero emissions target by 2050. One exciting area for development is conventional carbon capture technologies. Current amine absorption-based systems for carbon capture operate at suboptimal conditions resulting in an efficiency loss, causing a high operational expenditure. Knowledge of qualitative and quantitative speciation of CO2-loaded alkanolamine systems and their interactions can improve the equipment design and define optimal operating conditions. This work investigates the potential of Raman spectroscopy as an in situ monitoring tool for determining chemical species concentration in the CO2-loaded aqueous monoethanolamine (MEA) solutions. Experimental information on chemical speciation and vapour-liquid equilibrium was collected at a range of process parameters. Then, partial least squares (PLS) regression and an artificial neural network (ANN) were applied separately to develop two Raman species calibration models where the Kent–Eisenberg model correlated the species concentrations. The data were paired and randomly distributed into calibration and test datasets. A quantitative analysis based on the coefficient of determination (R2) and root mean squared error (RMSE) was performed to select the optimal model parameters for the PLS and ANN approach. The R2 values of above 0.90 are observed for both cases indicating that both regression techniques can satisfactorily predict species concentration. ANN models are slightly more accurate than PLS. However, PLS (being a white box model) allows the analysis of spectral variables using a weight plot.

DNA barcoding reveals overlooked shark and bony fish species in landing reports of small-scale fisheries from northern Peru

Species-level identification of commercially landed fish provides pivotal information for stock assessment and fishery management. However, there is a common lack of species determination in landing records from small-scale fisheries (SSFs) worldwide. Using DNA barcoding analyses, we detected four overlooked bony fish (yellow snapper, union snook, blackspot wrasse, and steeplined drum) and one shark species (the sicklefin smooth-hound) in official landing records of SSFs from northern Peru. Of particular concern is the sicklefin smooth-hound shark Mustelus lunulatus that was found to be overlooked and could mistakenly be landed as the humpback smooth-hound M. whitneyi. Increased efforts should be made to improve species identification capacities in Peruvian fishing landings. There is an urgent need to quantify the catch levels of members of the genus Mustelus to species level. This would contribute to a better understanding of the levels of exploitation in each particular species and to improved management decisions.

Feline Demodicosis Case Report—First Molecular Characterization of Demodex Mites in Romania

Cat demodicosis is uncommon to rare, and is caused by Demodex cati, Demodex gatoi and another unnamed species. The investigated patient was a mix-breed, 10-year-old feline with no dermatological history. Alopecia, erythema, minor erosions and ulcerations and crusts, associated with pruritus and self-trauma, were observed on the head. Dark, agglutinated cerumen was also present in the external ear canal. The agent causing the skin condition in the feline patient was identified as being a Demodex genus mite, based on the specific, morphological characteristics noticed upon the microscopic examination of deep skin scrapes. Biological samples were collected from the patient with to perform a PCR assay for clear species-determination and morphological assessment. PCR amplification of DNA extracted from the Demodex mites produced a single band of ~330 bp, indicating the presence of the D. cati species. The acaricidal treatment consisted of topical treatment using a fluralaner and moxidectin-based spot-on. Upon follow-up appointments, scheduled three times at a monthly interval, the patient failed to provide a positive result upon deep skin scrapes. The negative scrapes were also accompanied by the complete resolution of the existing lesions. In conclusion, this is the first molecular study to highlight the presence of Demodex cati within the feline population of Romania, and the fluralaner-moxidectin spot-on therapy has led to a complete recovery of the feline patient affected by feline demodicosis.

Novel Tetrazoles against Acanthamoeba castellanii Belonging to the T4 Genotype

Background: Acanthamoeba castellanii is a pathogenic free-living amoeba responsible for blinding keratitis and fatal granulomatous amoebic encephalitis. However, treatments are not standardized but can involve the use of amidines, biguanides, and azoles. Objectives: The aim of this study was to synthesize a variety of synthetic tetrazole derivatives and test their activities against A. castellanii. Methods: A series of novel tetrazole compounds were synthesized by one-pot method and characterized by NMR and mass spectroscopy. These compounds were subjected to amoebicidal, and cytotoxicity assays against A. castellanii belonging to the T4 genotype and human keratinocyte skin cells respectively. Additionally, reactive oxygen species determination and electron microscopy studies were carried out. Furthermore, two of the seven compounds were conjugated with silver nanoparticles to study their antiamoebic potential. Results: A series of seven tetrazole derivatives were synthesized successfully. The selected tetrazoles showed anti-amoebic activities at 10µM concentration against A. castellanii in vitro. The compounds tested caused increased reactive oxygen species generation in A castellanii, and significant morphological damage to amoebal membranes. Moreover, conjugation of silver nanoparticles enhanced antiamoebic effects of two tetrazoles. Conclusions: The results showed that azole compounds hold promise in the development of new formulations of anti-Acanthamoebic agents.

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.

Prevalence, species, and antimicrobial resistance of Acinetobacter in surgical practice and laboratory dog husbandry room environments

Acinetobacter is a bacteria found in the environment and clinical specimens, causing nosocomial infection and antimicrobial resistance (AMR) threats. This study examined the prevalence, species, and AMR characteristics of Acinetobacter isolated from surgical practice and the laboratory dog husbandry room environments (n = 235) at Rajamangala University of Technology Tawan-ok veterinary hospital during 2018-2019. The prevalence of Acinetobacter in the laboratory dog husbandry room and veterinary belongings were 2.55% and 0.43%, respectively. Species determination was Acinetobacter hemolyticus (2.1%) and Acinetobacter baumannii (0.4%) from environments in the laboratory dog husbandry room, and Acinetobacter junii (0.4%) from the shoes used in the surgical practice room. AMR was observed in both study environments and the specimens sent to the Veterinary Diagnostic Center. These isolates had a high resistant percentage to amoxicillin-clavulanic acid (84.62%), sulfamethoxazole-trimethoprim (61.54%), and cephalexin (53.85%) but were susceptible to imipenem. Compared to the isolates recovered from the clinical specimens, most isolates derived from environments exhibited multidrug resistance and shared correlated resistance patterns. These results highlight the need for sanitization in the dog husbandry room. Furthermore, the AMR results can be used as a preliminary baseline for studying AMR Acinetobacter contamination in animals and their environments.

Machine Learning for Species Identification: The HebelomaProject from database to website

Attempts to use machine learning (ML) for species identification of macrofungi have usually involved the use of image recognition to deduce the species from photographs, sometimes combining this with collection metadata. Our approach is different: we use a set of quantified morphological characters (for example, the average length of the spores) and locality (GPS coordinates). Using this data alone, the machine can learn to differentiate between species. Our case study is the genus Hebeloma, fungi within the order Agaricales, where species determination is renowned as a difficult problem. Whether it is as a result of recent speciation, the plasticity of the species, hybridization or stasis is a difficult question to answer. What is sure is that this has led to difficulties with species delimitation and consequently a controversial taxonomy. The Hebeloma Project—our attempt to solve this problem by rigorously understanding the genus—has been evolving for over 20 years. We began organizing collections in a database in 2003. The database now has over 10,000 collections, from around the world, with not only metadata but also morphological descriptions and photographs, both macroscopic and microscopic, as well as molecular data including at least an internal transcribed spacer (ITS) sequence (generally, but not universally, accepted as a DNA barcode marker for fungi (Schoch et al. 2012)), and in many cases sequences of several loci. Included within this set of collections are almost all type specimens worldwide. The collections on the database have been analysed and compared. The analysis uses both the morphological and molecular data as well as information about habitat and location. In this way, almost all collections are assigned to a species. This development has been enabled and assisted by citizen scientists from around the globe, collecting and recording information about their finds as well as preserving material. From this database, we have built a website, which updates as the database updates. The website (hebeloma.org) is currently undergoing beta testing prior to a public launch. It includes up-to-date species descriptions, which are generated by amalgamating the data from the collections of each species in the database. Additional tools allow the user to explore those species with similar habitat preferences, or those from a particular biogeographic area. The user is also able to compare a range of characters of different species via an interactive plotter. The ML-based species identifier is featured on the website. The standardised storage of the collection data on the database forms the backbone for the identifier. A portion of the collections on the database are (almost) randomly selected as a training set for the learning phase of the algorithm. The learning is “supervised” in the sense that collections in the training set have been pre-assigned to a species by expert analysis. With the learning phase complete, the remainder of the database collections may then be used for testing. To use the species identifier on the website, a user inputs the same small number of morphological characters used to train the tool and it promptly returns the most likely species represented, ranked in order of probability. As well as describing the neural network behind the species identifier tool, we will demonstrate it in action on the website, present the successful results it has had in testing to date and discuss its current limitations and possible generalizations.