Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand.

Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokae Pramual and Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity.

Epidemiology and phylogeny of Haemonchus contortus through internal transcribed spacer 2 gene in small ruminants.

Introduction: Haemonchus contortus (H. contortus) is a blood-feeding nematode causing infectious disease haemonchosis in small ruminants of tropical and subtropical regions around the world. This study aimed to explore the prevalence and phylogeny of H. contortus in small ruminants using the internal transcribed spacer-2 (ITS-2) gene. In addition, a comprehensive review of the available literature on the status of H. contortus in Pakistan was conducted. Methods: Fecal samples were collected from sheep and goats (n = 180). Microscopically positive samples were subjected to DNA extraction followed by PCR using species-specific primers. Results: The overall prevalence of H. contortus was 25.55% in small ruminants. The prevalence of H. contortus was significantly associated with months and area. The highest occurrence of haemonchosis was documented in July (38.70%), whereas the lowest occurred in December (11.11%), with significant difference. The prevalence was highest in the Ghamkol camp (29.4%) and lowest in the arid zone of the Small Ruminant Research Institute (17.5%) (p = 0.01). The results of the systematic review revealed the highest prevalence of haemonchosis (34.4%) in Khyber Pakhtunkhwa (p = 0.001). Discussion: Phylogenetic analysis revealed a close relationship between H. contortus and isolates from Asia (China, India, Iran, Bangladesh, Malaysia, and Mongolia) and European countries (Italy and the United Kingdom). It has been concluded that H. contortus is prevalent in small ruminants of Kohat district and all over Pakistan, which could be a potential threat to food-producing animals, farmers, dairy, and the meat industry. Phylogenetic analysis indicates that H. contortus isolates share close phylogenetic relationships with species from Asia and Europe.

Molecular Identification, Multigenic Characterization and Phylogenetic Analysis of Ctenocephalides Canis Fleas from Humans and Domestic Animals in Iran.

BACKGROUND: Fleas belonging to the Pulicidae are prevalent ectoparasites infesting mammals and birds in Iran. This study focused on genetically identifying and characterizing Ctenocephalides canis collected both off-host and infesting humans and various domestic animals in the country. METHODS: A total of 918 adult flea samples were collected from 10 sites in western and northwestern Iran between April 2018 and May 2019. Out of these, 71 specimens were found off-host, while the remaining fleas were collected from humans (121), sheep (126), goats (184), and dogs (416). Morphological identification at the genus level was performed on all fleas, and ten selected specimens selected based on the sampling sites and hosts were subjected to molecular detection at the species level by using partial amplification and sequencing of the internal transcribed spacer 1 and 2, as well as the cytochrome oxidase I (COXI) markers. RESULTS: The morphological identification confirmed all fleas as Ctenocephalides spp. Alignment and phylogenetic analysis of nuclear and mitochondrial partial sequences confirmed the presence of C. canis. However, molecular divergence was observed among the ten isolates based on the ITS1 and ITS2 with diversity rates estimated at 0.15% and 3.36%, respectively. Notably, the analysis of the COXI marker revealed no molecular divergence among the partial sequences representing the ten studied isolates from C. canis. CONCLUSIONS: This study explores the diversity of C. canis in the western and northwestern regions of Iran, providing insights into their molecular taxonomy and potential role as disease vectors in these areas.

Symbiodiniaceae diversity varies by host and environment across thermally distinct reefs.

Endosymbiotic dinoflagellates (Symbiodiniaceae) influence coral thermal tolerance at both local and regional scales. In isolation, the effects of host genetics, environment, and thermal disturbances on symbiont communities are well understood, yet their combined effects remain poorly resolved. Here, we investigate Symbiodiniaceae across 1300 km in Australia's Coral Sea Marine Park to disentangle these interactive effects. We identified Symbiodiniaceae to species-level resolution for three coral species (Acropora cf humilis, Pocillopora verrucosa, and Pocillopora meandrina) by sequencing two genetic markers of the symbiont (ITS2 and psbAncr), paired with genotype-by-sequencing of the coral host (DArT-seq). Our samples predominantly returned sequences from the genus Cladocopium, where Acropora cf humilis affiliated with C3k, Pocillopora verrucosa with C. pacificum, and Pocillopora meandrina with C. latusorum. Multivariate analyses revealed that Acropora symbionts were driven strongly by local environment and thermal disturbances. In contrast, Pocillopora symbiont communities were both partitioned 2.5-fold more by host genetic structure than by environmental structure. Among the two Pocillopora species, the effects of environment and host genetics explained four times more variation in symbionts for P. meandrina than P. verrucosa. The concurrent bleaching event in 2020 had variable impacts on symbiont communities, consistent with patterns in P. verrucosa and A. cf humilis, but not P. meandrina. Our findings demonstrate how symbiont macroscale community structure responses to environmental gradients depend on host species and their respective population structure. Integrating host, symbiont, and environmental data will help forecast the adaptive potential of corals and their symbionts amidst a rapidly changing environment.

Comparative metabarcoding and biodiversity of gut-associated fungal assemblages of Dendroctonus species (Curculionidae: Scolytinae).

The genus Dendroctonus is a Holarctic taxon composed of 21 nominal species; some of these species are well known in the world as disturbance agents of forest ecosystems. Under the bark of the host tree, these insects are involved in complex and dynamic associations with phoretic ectosymbiotic and endosymbiotic communities. Unlike filamentous fungi and bacteria, the ecological role of yeasts in the bark beetle holobiont is poorly understood, though yeasts were the first group to be recorded as microbial symbionts of these beetles. Our aim was characterize and compare the gut fungal assemblages associated to 14 species of Dendroctonus using the internal transcribed spacer 2 (ITS2) region. A total of 615,542 sequences were recovered yielding 248 fungal amplicon sequence variants (ASVs). The fungal diversity was represented by 4 phyla, 16 classes, 34 orders, 54 families, and 71 genera with different relative abundances among Dendroctonus species. The α-diversity consisted of 32 genera of yeasts and 39 genera of filamentous fungi. An analysis of ß-diversity indicated differences in the composition of the gut fungal assemblages among bark beetle species, with differences in species and phylogenetic diversity. A common core mycobiome was recognized at the genus level, integrated mainly by Candida present in all bark beetles, Nakazawaea, Cladosporium, Ogataea, and Yamadazyma. The bipartite networks confirmed that these fungal genera showed a strong association between beetle species and dominant fungi, which are key to maintaining the structure and stability of the fungal community. The functional variation in the trophic structure was identified among libraries and species, with pathotroph-saprotroph-symbiotroph represented at the highest frequency, followed by saprotroph-symbiotroph, and saprotroph only. The overall network suggested that yeast and fungal ASVs in the gut of these beetles showed positive and negative associations among them. This study outlines a mycobiome associated with Dendroctonus nutrition and provides a starting point for future in vitro and omics approaches addressing potential ecological functions and interactions among fungal assemblages and beetle hosts.

Identification and distribution of Rhipicephalus microplus in selected high-cattle density districts in Uganda: signaling future demand for novel tick control approaches.

BACKGROUND: Rhipicephalus (Boophilus) microplus (Canestrini, 1888), the Asian blue tick, is a highly invasive and adaptable ectoparasite. This tick species has successfully established itself in most regions of the world, with movement of cattle being a major driver for its spread. In the recent past, R. microplus ticks have been reported in three districts of Uganda. Information on its spread and distribution are vital in deepening our understanding of the ecological scenarios that lead to tick persistence and in the formulation of control strategies. This is especially important in the cattle-dense districts. METHODS: We randomly collected tick specimens from 1,461cattle spread across seven cattle dense districts located in the Central, Karamoja and West Nile regions of Uganda from January to September 2020. The ticks were identified using standard morpho-taxonomic keys and the R. microplus tick species identities were confirmed by sequencing of the ITS2 region, 12S rRNA and 16S rRNA genes and phylogenetic analyses. RESULTS: Adult ticks (n = 13,019) were collected from 1,461 cattle. Seventeen tick species were identified based on morpho-taxonomic keys and the majority (47.4%; n=6184) of these were R. appendiculatus. In total, 257 R. microplus ticks were found infesting cattle in 18 study sites in the districts of Amudat, Kaabong, Napak (Karamoja region) and Arua (West Nile region). The identity of R. microplus was confirmed using molecular technics. No R. microplus tick was recorded in the districts of Lyantonde and Nakaseke (Central region). Arua district accounted for 82.1% (n=211) of the R. microplus ticks recorded followed by Napak district at 16.3% (n=42), while Amudat and Kaabong districts accounted for 1.5% (n=4). Rhipicephalus microplus and R. decoloratus co-existed in 6 of the 13 study sites in Arua district, while in another 6 study sites, no R. decoloratus was recorded. In the Karamoja region districts R. decoloratus co-existed with R.microplus. Of the total 618 ticks belonging to four species of the subgenus Boophilus recorded in this study, R. decoloratus accounted for 50.04% (n=334), followed by R. microplus at 41.58% (n=257), R. geigyi at 2.75% (n=17) and R. annulatus at 1.61% (n=10). In the districts of Amudat, Kaabong and Napak, R. decoloratus was more dominant (76.1%; n=179) of the three Rhipicephalus (Boophilus) tick species recorded, followed by R. microplus (19.5%; n=46) and R. geigyi (4.2%; n=10). Contrariwise, R. microplus was more dominant (84%; n=211) in Arua district followed by R. decoloratus (10.7%; n=27), R. annulatus (3.9%; n=10) and R. geigyi (1.1%; n=3). Phylogenetic analyses of the ITS2 region, 12S rRNA and 16S rRNA genes revealed subgrouping of the obtained sequences with the previously published R. microplus sequences from other parts of the world. CONCLUSION: Rhipicephalus microplus ticks were found infesting cattle in four districts of Uganda. The inability to find R. decoloratus, an indigenous tick, from six sites in the district of Arua is suggestive of its replacement by R. microplus. Rhipicephalus microplus negatively affects livestock production, and therefore, there is a need to determine its distribution and to deepen the understanding of the ecological factors that lead to its spread and persistence in an area.

Comparison of the first time detected Oesophagostomum asperum with Oesophagostomum columbianum in sheep and goats in Bangladesh based on the trinity: Morphology, morphometry and genetic diversity.

Oesophagostomum spp. (Family: Chabertiidae) is keeping a low profile in terms of severity in Bangladesh while maintaining economic loss through disguise within sheep and goats. The study was performed to identify prevalence, confirmation of species through morphology and morphometry followed by phylogeny using ITS2 and COX1 genes. In total 384 slaughterhouse-sourced small and large intestines were pooled from Mymensingh, Kishoreganj, Netrokona, Sherpur and Tangail districts of Mymensingh division. Followed by isolation, O. columbianum and O. asperum were identified following their key morphological features. Notably, O. asperum was first time detected in Bangladesh. The overall prevalence of Oesophagostomum spp. was found 60.93%. The prevalence of O. columbianum (64.95%) was almost double than that of O. asperum (35.04%). Among several characters, only the distance between anus to tail tip showed a significant morphological disparity in female. The Neighbor-joining (NJ) phylogenic trees based on ITS2 and COX1 genes confirmed the study species. The first time identified O. asperum along with morphometry and phylogeny will add value to the fact that nematodes are invisibly present with high prevalence in this country. This study will help to draw specific attention to command a practical control strategy for intervening in economic loss.

Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

Genetic Diversity of Leishmania major Isolated from Different Dermal Lesions Using ITS2 Region.

BACKGROUND AND OBJECTIVE: Cutaneous leishmaniasis (CL) is still considered to be an uncontrolled endemic disease that spreads in many countries. The current study aimed to determine intra-species relationships of L. major using ITS2 sequencing. METHODS: The study was conducted from the beginning of March to the end of November 2022. All medical information regarding CL was collected from patients of Thi-Qar province who attended the Dermatology Department of Al-Hussein Teaching Hospital in Nasiriyah city. Seventy-three samples were selected for the molecular identification after confirming microscopy with Giemsa stain. In this study, the primers were designed using NCBI GenBank sequence database and Primer 3 plus primer design online software. RESULTS: The results recorded 21 (28.77%) positive samples of L. major using the internal transcribed spacer 2 region (ITS2) in ribosomal RNA gene. The local L. major IQN.1-IQN.10 were submitted to NCBI GenBank database with accession numbers OM069357.1-OM069366.1, respectively. The phylogenetic analysis revealed that local isolates of L. major showed a close relationship with NCBI-BLAST L. major Iran isolate (KU680848.1). CONCLUSION: ITS2-PCR is suitable for identifying Leishmania spp. and determining genetic diversity. A phylogenetic data analysis may provide an idea on the genetic homogeneity of local isolates and knowing the genetic origin of the dermal lesion. However, the local isolates showed genetic proximity to the KU680848.1 isolate. This signifies the possibility of infection prevalence from Iranian areas. In general, genetic variation of L. major isolates may give several clinical manifestations of the cutaneous lesion. Therefore, determination of the heterogeneity is important for detecting the infection origin, epidemiology, therapy, and control strategies.

Integrative taxonomy of Anaporrhutum mundae sp. nov. (Trematoda: Gorgoderidae), a parasite of the Munda round ray Urotrygon munda (Urotrygonidae) in Costa Rica.

A new species of trematode of anaporrhutine gorgoderid, from the gill chambers of the Munda round ray Urotrygon munda in Costa Rica is described, based on an integrative taxonomic approach that includes the use of light and scanning electron microscopy, ITS2 and 28S rDNA sequencing, and phylogenetic analysis. Anaporrhutum mundae sp. nov. can be distinguished from congeneric species by a combination of morphological traits and particularly by having the genital pore opening at the level of the intestinal bifurcation. The new species also can be distinguished from all other species of Anaporrhutum, except A. euzeti Curran, Blend & Overstreet, 2003, by having fewer testicular follicles per testis. Anaporrhutum mundae sp. nov. also differs from A. euzeti in its forebody shape and by having different morphology and location of the vitellaria. The study of the tegumental surface of A. mundae sp. nov., as revealed by scanning electron microscopy, allowed detection of new morphological characters for a member of Anaporrhutinae that may be of taxonomic value. These are: a stylet cavity dorsal to the oral sucker with a large penetration gland opening on each side of the cavity and small penetration gland openings located ventral to the stylet cavity, arranged in a circle around the mouth. This represents the first record of an Anaporrhutum species from Costa Rica. Further, A. mundae sp. nov. represents the first parasite described or reported in this host.

Genotypic diversity and epidemiology of Trichomonas gallinae in Columbidae: Insights from a comprehensive analysis.

Trichomonas gallinae is a protozoa that parasitizes the upper gastrointestinal and respiratory tracts of various animals and birds, including Columbidae, Passeriformes, and Falconiformes. Polymerase chain reaction-based T. gallinae ITS1/5.8S/ITS2 gene typing yields inconsistent results owing to methodological differences. To standardize the statistical analysis of T. gallinae genotype distributions, this study employed MEGA-X software with the Tamamura 3-parameter (T92) + G model in the neighbor-joining method, with 2,000 bootstrap replicates, to calculate a systematic evolutionary tree. The resulting tree comprised 12 branches, ITS-OBT-Tg-1 to ITS-OBT-Tgl, with similar phylogenetic relationships. Relevant literature review yielded T. gallinae prevalence data in Columbidae. Statistical analysis was conducted from two perspectives: non-biological and biological factors, using chi-square tests and ordered logistic regression analysis. T. gallinae positivity rates differed significantly across diverse regions (χ2 = 4,609.9, P = 0.000, df = 4) and at various times (χ2 = 2,810.8, P = 0.000, df = 3). However, temperature and precipitation did not significantly affect T. gallinae positivity rates. Additionally, T. gallinae positivity rates differed significantly among diverse hosts (χ2 = 2,958.6, P = 0.000, df = 14) and by host age (χ2 = 478.5, P = 0.000, df = 2) and sex (χ2 = 96.00, P = 0.000, df = 1). This comprehensive analysis aimed to control T. gallinae transmission, reduce economic and species resource losses, and provide a foundation for future related research.

Using capillary electrophoresis to identify Anopheline species in routine sampling sites.

In the Anopheles genus, various mosquito species are able to transmit the Plasmodium parasites responsible for malaria, while others are non-vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected in the field is an essential but problematic task. Morphological identification requires expertise and cannot be checked after processing samples in a destructive treatment, while sequencing of numerous samples is costly. Here, we introduce a method of Species identification via Simple Observation Coupled with Capillary Electrophoresis Technology (SOCCET). This molecular technique of species identification is based on precise determination of ITS2 length combined with a simple visual observation, the colour of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analysed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus colour, which allows discrimination of the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of ITS2 amplicons. Using the SOCCET method, we characterised the composition of Anopheles populations in five locations of French Guiana, where we detected a total of nine species. Anopheles braziliensis and Anopheles darlingi were detected in four locations each and represented 13 and 67% of our samples, respectively. The SOCCET method can be particularly useful when working with routine sampling sites with a moderate species diversity, that is, when the number of local species is too high to define species-specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may be further implemented for other mosquito genera.

Variations of the fungal microbiome in Corydalis Rhizoma with different collection areas, processing methods, and storage conditions.

Corydalis Rhizoma (CR, Yanhusuo in Chinese) has been widely used as an analgesic in herbal medicine and functional food. Cases of fungal and mycotoxin contamination in CR have been reported. In this study, the composition and diversity of fungal microbiome in CR samples from four herbal markets and two processing methods were investigated by DNA metabarcoding. Variations of the fungal microbiome in CR during cold and conventional storage were monitored. Results showed that Aspergillus was the dominant genus and saprotroph was the dominant trophic mode. Six potential toxigenic fungi, namely, Aspergillus fumigatus, Aspergillus ostianus, Aspergillus terreus, Penicillium citrinum, Penicillium oxalicum, and Trichothecium roseum, were detected. Differences in fungal composition and diversity among various groups based on collection areas and processing methods were also observed. Moreover, the relative abundance of dominant genera in CR samples stored at different temperatures was significantly different and changed with storage time. This study is the first to reveal the influence of collection areas, processing methods, and storage conditions on the fungal microbiome in CR, which was expected to provide a basis for control strategies of fungal contamination in the industrial chain of CR.

Chicory (Cichorium intybus) reduces cyathostomin egg excretion and larval development in grazing horses.

Cyathostomins are the most prevalent parasitic nematodes of grazing horses. They are responsible for colic and diarrhea in their hosts. After several decades of exposure to synthetic anthelmintics, they have evolved to become resistant to most compounds. In addition, the drug-associated environmental side-effects question their use in the field. Alternative control strategies, like bioactive forages, are needed to face these challenges. Among these, chicory (Cichorium intybus, Puna II cultivar (cv.)) is known to convey anthelmintic compounds and may control cyathostomins in grazing horses. To challenge this hypothesis, we measured fecal egg counts and the rate of larval development in 20 naturally infected young saddle horses (2-year-old) grazing either (i) a pasture sown with chicory (n = 10) or (ii) a mesophile grassland (n = 10) at the same stocking rate (2.4 livestock unit (LU)/ha). The grazing period lasted 45 days to prevent horse reinfection. Horses in the chicory group mostly grazed chicory (89% of the bites), while those of the control group grazed mainly grasses (73%). Cyathostomins egg excretion decreased in both groups throughout the experiment. Accounting for this trajectory, the fecal egg count reduction (FECR) measured in individuals grazing chicory relative to control individuals increased from 72.9% at day 16 to 85.5% at the end of the study. In addition, larval development in feces from horses grazed on chicory was reduced by more than 60% from d31 compared to control individuals. Using a metabarcoding approach, we also evidenced a significant decrease in cyathostomin species abundance in horses grazing chicory. Chicory extract enriched in sesquiterpenes lactones was tested on two cyathostomins isolates. The estimated IC50 was high (1 and 3.4 mg/ml) and varied according to the pyrantel sensitivity status of the worm isolate. We conclude that the grazing of chicory (cv. Puna II) by horses is a promising strategy for reducing cyathostomin egg excretion and larval development that may contribute to lower the reliance on synthetic anthelmintics. The underpinning modes of action remain to be explored further.

At a crossroads: Genetic lineages and dispersal routes of Morimusasper (Sulzer, 1776) s.l. (Coleoptera, Cerambycidae) in Bulgaria.

The present study fills a knowledge gap in the distribution and genetic variation of Morimus populations in the Balkans, by studiyng the representatives of the genus in Bulgaria - M.asperfunereus Mulsant, 1862, M.verecundusbulgaricus Danilevsky, 2016 and M.orientalis Reitter, 1894. Additional information is provided for Albania and northern Greece. The mitochondrial cytochrome C oxidase subunit I (COI) marker and the nuclear internal transcribed spacer 2 (ITS2) were used for the genetic analyses. Three of the previously-defined mitochondrial lineages (Lb/HgA, L2 and L3) were detected in Bulgaria, as well as a new lineage (Str) from the Strandzha Mountains (south-eastern Bulgaria). A total of 24 distinct haplotypes, 20 of them in Bulgaria, were found. Bulgarian populations of Morimus demonstrated relatively high nucleotide diversity. The L3 COI lineage was confirmed as the most diverse and frequent in the Balkans. The L3 lineage is dominant in most of Bulgaria, but was not identified in the easternmost parts near the Black Sea coast, where the L2 and Str lineages were found. New data highlighted two dispersal routes of the L2 mitochondrial lineage on the Balkan Peninsula: 1) northwards along the Black Sea coast and 2) westwards, across the Balkans where only disjunct populations remain. North-western Bulgaria seems to be the eastern limit of the basal lineage Lb/HgA distribution. Our results show high levels of genetic exchange between most of the mitochondrially defined lineages, yet some of the easternmost populations probably remained isolated for comparatively longer periods.

Patterns of Equine Small Strongyle Species Infection after Ivermectin Intervention in Thailand: Egg Reappearance Period and Nemabiome Metabarcoding Approach.

The indiscriminate use of anthelmintics to control parasitic nematodes in horses has led to the emergence of anthelmintic resistance worldwide. However, there are no data available on using ivermectin for treating strongyle infections within domesticated horses in Thailand. Therefore, this study aimed to use the fecal egg count reduction (FECR) test to determine the strongylid egg reappearance period (ERP). Additionally, the nemabiome metabarcoding approach is incorporated to study patterns of strongyle species infection following ivermectin treatment. The study results indicate that, although ivermectin effectively eliminated adult strongyle parasites within two weeks post-treatment, the ERP was shortened to 6 weeks post-treatment with a mean FECR of 70.4% (95% CI 46.1-84.0). This potentially indicates a recent change in drug performance. In addition, nemabiome metabarcoding revealed that strongyle species have different levels of susceptibility in response to anthelmintic drugs. The reduction in ERP was associated with the early reappearance of specific species, dominated by Cylicostephanus longibursatus and Cylicocyclus nassatus, indicating the lower susceptibility of these species. In contrast, Poteriostomum imparidentatum, Triodontophorus nipponicus, and Triodontophorus serratus were not found post-treatment, indicating the high level of susceptibility of these species. This information is vital for comprehending the factors contributing to the emergence of resistance and for devising strategies to manage and control strongyle infections in horses.

How reliable is metabarcoding for pollen identification? An evaluation of different taxonomic assignment strategies by cross-validation.

Metabarcoding is a powerful tool, increasingly used in many disciplines of environmental sciences. However, to assign a taxon to a DNA sequence, bioinformaticians need to choose between different strategies or parameter values and these choices sometimes seem rather arbitrary. In this work, we present a case study on ITS2 and rbcL databases used to identify pollen collected by bees in Belgium. We blasted a random sample of sequences from the reference database against the remainder of the database using different strategies and compared the known taxonomy with the predicted one. This in silico cross-validation (CV) approach proved to be an easy yet powerful way to (1) assess the relative accuracy of taxonomic predictions, (2) define rules to discard dubious taxonomic assignments and (3) provide a more objective basis to choose the best strategy. We obtained the best results with the best blast hit (best bit score) rather than by selecting the majority taxon from the top 10 hits. The predictions were further improved by favouring the most frequent taxon among those with tied best bit scores. We obtained better results with databases containing the full sequences available on NCBI rather than restricting the sequences to the region amplified by the primers chosen in our study. Leaked CV showed that when the true sequence is present in the database, blast might still struggle to match the right taxon at the species level, particularly with rbcL. Classical 10-fold CV-where the true sequence is removed from the database-offers a different yet more realistic view of the true error rates. Taxonomic predictions with this approach worked well up to the genus level, particularly for ITS2 (5-7% of errors). Using a database containing only the local flora of Belgium did not improve the predictions up to the genus level for local species and made them worse for foreign species. At the species level, using a database containing exclusively local species improved the predictions for local species by ∼12% but the error rate remained rather high: 25% for ITS2 and 42% for rbcL. Foreign species performed worse even when using a world database (59-79% of errors). We used classification trees and GLMs to model the % of errors vs. identity and consensus scores and determine appropriate thresholds below which the taxonomic assignment should be discarded. This resulted in a significant reduction in prediction errors, but at the cost of a much higher proportion of unassigned sequences. Despite this stringent filtering, at least 1/5 sequences deemed suitable for species-level identification ultimately proved to be misidentified. An examination of the variability in prediction accuracy between plant families showed that rbcL outperformed ITS2 for only two of the 27 families examined, and that the % correct species-level assignments were much better for some families (e.g. 95% for Sapindaceae) than for others (e.g. 35% for Salicaceae).

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

DNA metabarcoding analysis of fungal community on surface of four root herbs.

Objective: Angelicae Sinensis Radix (ASR, Danggui in Chinese), Cistanches Herba (CH, Roucongrong in Chinese), Ginseng Radix et Rhizoma (PG, Renshen in Chinese), and Panacis Quinquefolii Radix (PQ, Xiyangshen in Chinese), widely used as medicine and dietary supplement around the world, are susceptible to fungal and mycotoxin contamination. In this study, we aim to analyze their fungal community by DNA metabarcoding. Methods: A total of 12 root samples were collected from three main production areas in China. The samples were divided into four groups based on herb species, including ASR, CH, PG, and PQ groups. The fungal community on the surface of four root groups was investigated through DNA metabarcoding via targeting the internal transcribed spacer 2 region (ITS2). Results: All the 12 samples were detected with fungal contamination. Rhizopus (13.04%-74.03%), Aspergillus (1.76%-23.92%), and Fusarium (0.26%-15.27%) were the predominant genera. Ten important fungi were identified at the species level, including two potential toxigenic fungi (Penicillium citrinum and P. oxalicum) and eight human pathogenic fungi (Alternaria infectoria, Candida sake, Hyphopichia burtonii, Malassezia globosa, M. restricta, Rhizopus arrhizus, Rhodotorula mucilaginosa, and Ochroconis tshawytschae). Fungal community in ASR and CH groups was significantly different from other groups, while fungal community in PG and PQ groups was relatively similar. Conclusion: DNA metabarcoding revealed the fungal community in four important root herbs. This study provided an important reference for preventing root herbs against fungal and mycotoxin contamination.

Secondary Structure Analysis of Fasciola from Semi-wild Ruminants of Northeast India.

PURPOSE: The objective of this study is to study the secondary structure analysis of Fasciola flukes from a rare mithun host from Manipur. Fascioliasis, a neglected tropical trematodiasis, is poorly studied in India and is widely believed to be predominantly caused by F. gigantica. Through this study, we want to assess the flukes from the rare semi-wild ruminants of Northeast India. This study is important as the mithun population is semi-wild and its population is declining in Manipur. METHODS: Sample collected from the difficult and challenging terrain of Northeast India. The sample was collected from mithun and observed under the microscope. DNA was isolated, sequenced, and analyzed using various bioinformatics tools. The secondary structure analysis of the Internal Transcribed Spacer 2 (ITS2) region was also performed. RESULTS: The secondary structure species tree corroborated the Bayesian inference and, hence, strengthened the phylogeny reconstructed. The annotated ITS2 sequence and RNA secondary of the Manipur isolate displayed the typical four-helix or four-domain model. Helix III reveals the presence of the UGGU motif with other deviations like UGG and GGU. CONCLUSION: This is an in-depth analysis of the secondary structure of Fasciola species. The present study has demonstrated the usefulness of ITS2 and its secondary structures for characterizing parasites. The information on fascioliasis in the mithun's population presents itself useful with regards to their conservation strategy as their populations in both Manipur and Nagaland are dwindling.