Genetic identification of bat species for pathogen surveillance across France

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.

Sus sp. DNA ENCODING cyt b GENE DETECTION TEST ON SOFT GEL CANDY SAMPLES USING PCR METHOD

Softgel candy is soft-textured confectionery processed by the addition of several components such as gum, pectin, starch and gelatin, to obtain a supple product and packed after aging treatment first. Gelatin is one of the main components in the manufacture of soft candy derived from the hydrolysis of collagen connective tissue and animal bone that serves as the nature of gelling agents, stabilizers or emulsifiers. However, the gelatin used in products not yet labeled halal Indonesian Council of Ulama (MUI) is particularly vulnerable to pork gelatin, since pork gelatin is cheaper than cattle. The purpose of this study was to test the contaminants of pig DNA on 17 samples of soft candles not labeled halal MUI. This research used Polymerase Chain Reaction (PCR) method. Seventeen samples were isolated by DNA, then spectrophotometry was performed, followed by PCR. The PCR product is run electrophoresis. Visualize the DNA with a UV gel documentation. Primer used is primer gene encoding cyt b DNA pork. Results showed that 17 samples were negative contaminants, while the positive control of pork showed a DNA band of 149 bp. This shows that Softgel Candy 17 samples do not contain pork gelatin.

Vanmanenia marmorata, a new species of loach (Teleostei: Gastromyzontidae) from the middle Chang-Jiang Basin in Guizhou Province, south China

The gastromyzontid genus Vanmanenia was established by Hora in 1932, based on the type species Vanmanenia stenosoma. The genus is a loach group adapted to running waters of streams from southern China, northern Vietnam and Laos. Currently, 19 valid species of the genus have been recognised. The northernmost distribution of the genus is the Yangtze River (= Chang-Jiang in Chinese) Basin and five species (V. maculata, V. intermedia, V. stenosoma, V. pseudostriata and V. gymnetrus) have been reported from the Basin. Vanmanenia marmorata, a new hillstream species of loach, is here described from the middle Chang-Jiang Basin in Guizhou Province, south China. It is distinguished from its congeners by having a combination of the following characters: three triangular-shaped rostral lobules; postdorsal saddles wider than interspaces; a more backwards-placed anus (the vent to anal distance 30.5–36.9% of the pelvic to anal distance); a larger gill opening with its upper extremity reaching the level of the middle of the orbit; anal-fin base length 5.6–6.4% of SL; caudal-peduncle length 11.6–12.9% of SL; prepelvic length 51.1–53.4% of SL. Its validity is also affirmed by its distinct cyt b gene sequence divergence with all sampled congeners and its monophyly recovered in a cyt b gene-based phylogenetic analysis.

Detection of the G143A Mutation in the Cytochrome b Gene of Corynespora cassiicola Isolates from Soybean in Tennessee

Corynespora cassiicola is the causal pathogen of target spot in soybean and cotton grown in the United States. With target spot increasing in importance, fungicides are becoming an important tool for control of this disease. Unfortunately, there are reports of C. cassiicola isolates in other crops being resistant to some fungicide classes. The objective of this study was to identify if resistance to quinone outside inhibitor (QoI) fungicides is present in Tennessee soybean and cotton isolates of C. cassiicola. Four isolates of C. cassiicola were evaluated at a range of doses for the fungicide pyraclostrobin. Isolates were also sequenced to determine if the G143A mutation was present in the cyt b gene. Two isolates previously reported to be resistant to QoIs were also used as positive checks. Two isolates of C. cassiicola from Tennessee soybean were found to have the G143A mutation. EC50 values for the two isolates ranged from 15.7 to 121 μg/ml. As a result of this study, C. cassiicola isolates have exhibited resistance to QoI fungicides in Tennessee soybean.

Haemosporidian Blood Parasites in nestling birds of prey in Mongolia

Haemosporidians are vector-transmitted intracellular parasites that happen in numerous bird species worldwide and may possibly have important effects for wild bird populations. Studies of haemosporidians most dedicated on Europe and North America, and only newly some study in the Neotropics has been done, where the occurrence and influences of the disease have been less considered and are not understood well. In this study we designed a study in the nestling birds of prey in Mongolia. We sampled blood from 72 raptors at 2 different species and evaluated avian haemosporidian infection by two nested PCR protocol and one Real time PCR protocol. Sequencing a portion of the cytochrome b (cyt b) gene of the parasite. From the sampled birds, 10 % were infected by Plasmodium. Inclusive, our findings advocate a high haemosporidian species richness in the bird community of Mongolia. In view of the frequency of local habitat loss that in this area is living, recognize how avian haemosporidians affect bird populations it is very important; in addition, more exhaustive sampling is required to fully understand the range of avian haemosporidian infection in this area.

Sensitivity of C. fructicola and C. siamense of peach in China to multiple classes of fungicides and characterization of pyraclostrobin-resistant isolates

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including C. fructicola and C. siamense, is a devastating disease of peach. The chemical control has been widely used for years and management failures have increased towards commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance and 26 % were low resistant, all of the C. siamense isolates were low resistant. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin, even they are members of the same quinone outside inhibitor (QoI) fungicide group, and between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and low-resistant isolates of C. fructicola. Molecular characterization of Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the current resistance status of C. fructicola and C. siamense to different fungicides and their future perspectives. Demethylation inhibitor (DMI) fungicides are the best option among different chemicals to control peach anthracnose in China.

Molecular Phylogeny and Genetic Diversity of Domestic Yaks (Bos grunniens) in Pakistan based on Mitochondrial and Microsatellite Markers

The complete Cytchrome b gene and partial mtDNA control region were sequenced for the Pakistani domestic yak (Bos grunniens) within the Bovidae family. A total of 300 samples were genotyped using 27 bovine microsatellite markers from the Gilgit-Baltistan and Skardu regions of Pakistan. We identified a total of 35 mutations and 9 haplotypes based on D-loop sequences, with a haplotype and nucleotide diversity of 0.9640±0.051 and 0.02172±0.00224, respectively. For the Cyt b gene, a total of 23 variable sites and six different haplotypes were observed with 0.885±0.067 haplotype and 0.00989±0.003 nucleotide diversity. Phylogenetic analysis of D-loop and Cyt b gene suggested that domestic yak sequences cluster into two highly divergent maternal lineages (lineages I and II), while three haplogroups A, C, and D were identified of the six previously known haplogroups. Haplogroups A and C were dominant and widely distributed among all investigated yak samples. All microsatellites were polymorphic and a total of 138 alleles were observed, with average polymorphic information content (PIC) of 0.56 indicating their effectiveness. The average heterozygosity was observed at 0.6071 with allele diversity of 5.1111 and gene diversity of 0.4830. The implications of these findings can be applied for yak conservation.

Re-description of the loach species Leptobotia citrauratea (Teleostei, Botiidae), with the description of L. brachycephala from southern Zhejiang Province, China

Leptobotia citrauratea (Nichols, 1925), a loach species, originally described from Dongting Lake, was recently rehabilitated, based on the examination of the holotype and non-topotypical specimens. Several field surveys conducted from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, P.R. China, yielded many specimens of Leptobotia which were initially identified as L. citrauratea. Molecular and morphological analyses of these specimens demonstrated that two distinct species are involved. One was identified as L. citrauratea, represented by specimens from both the Poyang and Dongting Lake (type locality) systems in Jiangxi and Hunan Provinces, and the other species is described as L. brachycephala, represented by specimens from the Ou-Jiang and Qu-Jiang, two coastal rivers of Zhejiang Province, China. Leptobotia brachycephala resembles L. citrauratea and L. micra in having a row of orange dots or an orange stripe along the dorsal mid-line of the body, extending from the nape to the caudal-fin base – a unique character in Leptobotia. Leptobotia brachycephala differs from L. citrauratea and L. micra Bohlen & Šlechtová, 2017, in caudal-fin shape and pelvic-fin insertion and proportional measurements including caudal-fin length, head length, predorsal length and anal-fin length. Its species status was further corroborated by position in a molecular phylogenetic analysis, based on the mitochondrial cyt b gene and its minimum uncorrected p-distance (2.9%) from congeneric species.

Population diversity of striped snakehead, Channa striata (Bloch, 1793) from Bekasi, West Java and Barito Kuala, South Kalimantan using Cytochrome B gene

Channa striata or striped snakehead is one of species from family Channidae that widely distributed from India, Southern China to Southeast Asia including Indonesia. It is a commercially important freshwater fish because of its taste and health benefits. High demand of this species trigger many efforts to increase its production, one of them is genetic monitoring. This study used complete Cytochrome b gene sequence of mtDNA for determining genetic variation in wild population of C. striata. C. striata samples (n=31) from two different locations in Indonesia were amplified and analyzed using MEGA ver 7.0. Sequences of 1140 bp complete cyt b gene revealed the presence of 2 haplotypes with 1137 bp conserved sites and 3 bp variable sites (0,26%). Overlapping haplotype was observed in samples from Bekasi, however there were only one haplotype in samples from South Borneo. Interspecies genetic were analysed with species from Genebank and showed that C. striata from Indonesia has close genetic relationships with C. striata from Borneo-Indonesia (MN057164.1) with genetic distance 0%. This study also revealed that C. striata from Indonesia were phylogenetically distinct with C. striata from China with 9,2%K2P genetic distance. Complete cyt b gene has been proven for assessing phylogenetic relationships and population diversity of C. striata in Indonesia.