Phylogeography of cliff racer (Platyceps rhodorachis Jan, 1865) from Punjab, Pakistan

The present study reports the existence of cliff racer, Platyceps rhodorachis from the plains of Punjab, Pakistan. A total of 10 specimens were captured during the field surveys from June to September, 2018 from different sites of Punjab. Platyceps rhodorachis was identify on the basis of morphology and confirmed through COI gene sequences. The obtained DNA sequences have shown reliable and exact species identification. Newly produced DNA sequences of Platyceps rhodorachis were submitted to GenBank and accession numbers were obtained (MK936174.1, MK941839.1 and MT790210.1). N-J tree based on COI sequences of Platyceps rhodorachis clearly separated as out-group with other members of family Colubridae based on p-distance. The intra-specific genetic variation ranges from 12% to 18%. The DNA sequences of Platyceps rhodorachis kashmirensis, Platyceps rhodorachis ladacensis, Platyceps ventromaculatus, Platyceps ventromaculatus bengalensis and Platyceps ventromaculatus indusai are not available at NCBI to validate their taxonomic positions. In our recommendations, a large scale molecular based identification of Pakistan’s herpetofauna is required to report more new or subspecies from country.

A novel molecular diagnostic method for the gut content analysis of Philaenus DNA

Philaenus spumarius is a vector of Xylella fastidiosa, one of the most dangerous plants pathogenic bacteria worldwide. There is currently no control measure against this pathogen. Thus, the development of vector control strategies, like generalist predators, such as spiders, could be essential to limit the spread of this vector-borne pathogen. In this study, a polymerase chain reaction (PCR)-based approach was developed to principally detect DNA of P. spumarius in the spider’s gut. Accordingly, 20 primer pairs, targeting the mitochondrial cytochrome oxidase I (COI) and cytochrome b (cytB) genes, were tested for specificity, sensitivity, and efficiency in detecting P. spumarius DNA. Overall, two primer sets, targeting COI gene (COI_Ph71F/COI_Ph941R) and the cytB gene (cytB_Ph85F/cytB_Ph635R), showed the highest specificity and sensitivity, being able to amplify 870 pb and 550 bp fragments, respectively, with P. spumarius DNA concentrations 100-fold lower than that of the DNA of non-target species. Among these two primer sets, the cytB_Ph85F/cytB_Ph635R was able to detect P. spumarius in the spider Xysticus acerbus, reaching 50% detection success 82 h after feeding. The feasibility of this primer set to detect predation of P. spumarius by spiders was confirmed in the field, where 20% of the collected spiders presented positive amplifications.

Genetic variability and population structure of Leucinodes orbonalis (Guenée), a severe insect pest of brinjal in India

Aim: The present study was undertaken to provide valuable insights regarding population genetic structure of Leucinodes orbonalis from diverse agro-ecologies of India. Methodology: Molecular characterization of L. orbonalis populations collected from five major agro-climatic zones of India was carried out using mitochondrial cytochrome oxidase I (COI) gene. Collected specimens were subjected to DNA extractions, partial PCR amplification and sequencing of the target gene, and multiple sequence alignments. Results: The results showed very less diversity in the nucleotide positions of the COI sequences of 79 studied specimens, with a low number of segregating sites (30), nucleotide diversity (0.00438) and overall mean genetic distance (0.004 ± 0.001). The significant negative values of neutrality tests and unimodal mismatch distribution supported the demographic expansion theory in Indian L. orbonalis. Analysis of the molecular variance revealed that 93.13% of the genetic variation was within populations, and the variation among populations was only 6.87%. The pairwise genetic differentiation was also found to be low to moderate between most of the populations. Multiple haplotypes were recorded from all the populations, and both neighbour-joining tree as well as the haplotype network showed that clustering of the haplotypes was independent of the geographical location. Interpretation: Thus, it can be inferred that Indian populations of L. orbonalis have very low genetic variation levels concerning the COI gene. There is a possible occurrence of stable inherited gene flow among populations, thereby reducing genetic variation in India.

Identification and pathogenicity of Pratylenchus scribneri on tomato in Sichuan Province of People's Republic of China

Root-lesion nematodes (Pratylenchus spp.) are a group of economically important pathogens that have caused serious economic losses in many crops. In 2019, root-lesion nematodes were recovered from tomato (Solanum lycopersicum) root samples collected from Sichuan Province, People's Republic of China (PRC). Extracted nematodes were disinfected, and one individual female was cultured on a carrot disc for propagation at 25 °C by parthenogenesis and designated the SC isolate. Afterwards, the isolate was identified on the basis of morphometric and molecular markers. Both morphometric characters and molecular analysis of the internal transcribed spacer region gene (ITS) of ribosomal DNA, the D2-D3 expansion region of the 28S rDNA gene and the mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed that the species of root-lesion nematode was Pratylenchus scribneri. The Bayesian tree inferred from the ITS rDNA, 28S rDNA and mtDNA-COI gene sequences also showed that this isolate formed a highly supported clade with other P. scribneri isolates. The pathogenicity of the root-lesion nematode SC isolate on tomato was assessed, showing that tomato was a suitable host for P. scribneri. To the best of our knowledge, this is the first report of P. scribneri on tomato in Sichuan Province, PRC. These are also the first molecular data obtained from P. scribneri on tomato in the PRC, and the pathogenicity of P. scribneri to tomato was studied for the first time. This study provides scientific data for the detection, identification and control of tomato root-lesion nematode disease.

Systematic analysis of leisler’s bat Nyctalus leisleri (Kuhl, 1817) captured from FATA region, Pakistan

Extensive field surveys were carried out to explore the distribution of Leisler’s Bat Nyctalus leisleri (Kuhl, 1819) in selected area of FATA regions, Pakistan. Specimens of Leisler’s Bat Nyctalus leisleri (Kuhl, 1819) (n5) were collected from Kurram Agency (Shublan) (N33.8229788 E70.1634414) at elevation 1427m and Khyber Agency (Landi Kotel) (N34.0909899 E71.1457517) at elevation 1091m for two years survey extending from May 2013 through August 2015. The mean head and body length, hind foot length, ear length and tail length the Nyctalus leisleri specimens captured from the study area was 65.08 ± 1.58 mm, 44.06 ± 0.52 mm, 8.38 ± 0.60 mm, 13.20 ± 0.99 mm and 39.46 ± 1.46 mm, respectively. For molecular analysis the sequences of COI gene were obtained and analyzed. The mean intraspecific divergences of Nyctalus leisleri was 0.04%. The mean interspecific divergences of Nyctalus noctula and Nyctalus leisleri was 0.2%. The mean concentration of each nucleotides was A = (26.3%), T = (32.8%), G = (15.9%) and C = (25.0%). The mean A+T contents were 59.2%and C+G were 40.9%. In the phylogenetic tree Nyctalus leisleri and Nyctalus noctula clustered with significant bootstrap support value.

Molecular classification and comparative phylogeographic study of insectivorous bat species (Pipisitrellus coromandra) from Punjab, Pakistan

Molecular based identification of bat fauna in Pakistan has been relatively less explored. The current study was therefore planned to report for the first time the molecular classification of insectivorous bats (Pipistrellus coromandra) based on mitochondrion gene (COI) from Punjab, Pakistan. Specimens were collected from five different locations followed by DNA extraction with subsequent gene amplification and sequencing. All samples in the study had shown close identity matches with species (Pipistrellus coromandra) from India and (Pipistrellus tenuis) from Vietnam with percentage identity score of 96.11 and 95.58 respectively except one sequence which only revealed 86.78% identity match on Basic Local Alignment Search Tool (BLAST) and could only be assigned to genus level Pipistrellus sp. The results indicated negligible intra-population genetic distance among collected samples whereas the comparison with species from other countries had shown high intraspecific (P. coromandra) and interspecific (P. tenuis) mean genetic distances. The current study hence successfully proved the efficiency of COI gene as a molecular marker for species identification and in analyzing the patterns of genetic variation with species from other countries.

First morphometry, reproduction, and genetic data for Blennius ocellaris (Linnaeus, 1758) from the Black Sea

Two specimens of the butterfly blenny, Blennius ocellaris, were caught off the coast of Ordu (Black Sea, Turkey) in April 2021 by trammel net. The aim of this paper is to further document occurrence and distribution of the butterfly blenny for the Black Sea and for Turkish marine ichthyofauna and to provide first morphometric, reproduction, and genetic data on this species to the Black Sea fauna species. Some morphometric and meristic characters were measured and presented as the percentage of total length (TL%). All morphometric measurements except eye diameter, pre-anal length, and maximum body depth were higher in the male individual. It was observed that the head makes up almost 1/4 of the body. It was determined that the ripe eggs were in their final stage of development (Stage IV). Gonad’s weight of a female individual was 2.85 g and the number of eggs was determined as 2993. The mean egg diameter was measured as 1070.7 ±15.63 μm (from 1050.2 to 1123.1 μm). The mitochondrial DNA gene regions of 16S rRNA and COI of the specimens were sequenced and analyzed. The generated partial sequences of COI and 16S rRNA were 621 bp and 551 bp, respectively. The maximum likelihood tree generated with the COI gene sequences retrieved from the GenBank database demonstrated geographic region-based distinction and sequences of the Black Sea specimens nested with the reference specimen sequences from the Western Mediterranean Sea and the Sea of Marmara.

New records of the occurrence of Megaleporinus macrocephalus () (Characiformes, Anostomidae) from the basins of the Itapecuru and Mearim rivers in Maranhão, Northeastern Brazil

The “piaussu”, Megaleporinus macrocephalus is an anostomatid fish species native to the basin of the Paraguay River, in the Pantanal biome of western Brazil. However, this species has now been recorded in a number of other drainages, including those of the upper Paraná, Uruguay, Jacuí, Doce, Mucuri, and Paraíba do Sulrivers. This study presents two new records of the occurrence of M. macrocephalus, in the basins of the Itapecuru and Mearim rivers in the state of Maranhão, in the Brazilian Northeast. The piaussu is a large-bodied fish of commercial interest that is widely raised on fish farms, and its occurrence in the Itapecuru and Mearim rivers is likely the result of individuals escaping from fish tanks when they overflow during the rainy season. Morphological analyses and sequences of the Cytochrome Oxidase Subunit I (COI) gene confirmed the taxonomic identification of the specimens as M. macrocephalus. The COI sequences were 99.66% similar to those of M. macrocephalus deposited in the BOLDSystems database. These records extend the known distribution of M. macrocephalus to the basins of the Itapecuru and Mearim rivers in the Brazilian Northeast, highlighting a new case of introduction of exotic fish species into Brazilian river basins.

Genetic variation in the small bivalve Nuculana inaequisculpta along a retreating glacier fjord, King George Island, Antarctica

Climate change is strongly influencing regions of Antarctica but the consequences on microevolutionary processes have been little studied. Patterns of population genetic diversity were analysed in the Antarctic bivalve Nuculana inaequisculpta (Protobranchia: Nuculanidae) from a fjord with 70 years of documented climate-forced glacier retreat. Thirty-nine individuals from five sites at different distances from the glacier terminus were collected, and the COI gene was sequenced from each individual. No statistically significant genetic differentiation was found between sites nor a significant correlation between the proximity of glaciers and genetic diversity, suggesting a high dispersal capability and therefore, a planktonic larval stage for this species. Nevertheless, we encourage increasing the sample size and number of loci in future studies to confirm our findings.

Three new species of Byrsopteryx Flint microcaddisflies from Peru (Insecta: Trichoptera) including DNA-based larval associations

In this paper, we have described and illustrated three new species of Byrsopteryx from Peru: Byrsopteryx inti, sp. nov. Byrsopteryx mamaocllo sp. nov., and Byrsopteryx mancocapac sp. nov. Larvae of the latter two were also associated to male specimens based on comparison of a fragment of COI gene and pharate male identification. Byrsopteryx inti sp. nov. and Byrsopteryx mamaocllo sp. nov. share a unique feature: a semi-dome process formed by a thickened area on male forewings. The three species can be easily identified by wing coloration and male genitalia. Furthermore, Byrsopteryx inti sp. nov. can be recognized by its sternum VIII with a median digitate process on posterior margin, slightly capitate; and by long dorsolateral processes from segment VIII, which cross each other apically in dorsal view. Byrsopteryx mamaocllo sp. nov. can be distinguished by sternum VIII bearing a pair of short, posterior, spinelike processes, which are curved inwards and bordered by a rounded, membranous structure, and by a pair of short, heavily sclerotized, dorsolateral processes. Byrsopteryx mancocapac sp. nov. can be distinguished by strong spine-like processes arising dorsally from subgenital plate and by sternum VIII with posterior margin divided into two plate-like lobes. Larvae of B. mamaocllo sp. nov. and B. mancocapac sp. nov. are similar to other Byrsopteryx larvae known. They can be distinguished from each other by the shape of the operculum formed by terga VIII and IX, and number of setae on the second abdominal pleurite. Maximum likelihood analyses of 20 COI sequences, including nine Byrsopteryx species, placed B. inti sp. nov. and B. mamaocllo sp. nov. as sister species and related to a clade including B. gomezi, B. tapanti, and B. esparta, while B. mancocapac sp. nov. was found as sister to B. abrelata. Despite the close phylogenetic relationship found between B. inti sp. nov. and B. mamaocllo sp. nov., they are separated by 14.9% minimum K2P divergence of COI. The highest intraspecific distance observed was 1.4% for B. mancocapac sp. nov. individuals. Although the Peruvian caddisfly fauna has around 320 known species and almost a third of them are microcaddisflies, in this paper we present the first descriptions of Byrsopteryx species for the country.