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Ceresini treehoppers are present in northern California vineyard ecosystems, including the closely related Spissistilus and Tortistilus (Hemiptera: Membracidae). These membracids are not direct pests of wine grapes, but S. festinus is a vector of grapevine red blotch virus (GRBV). No information is available on the ability of Tortistilus spp. to transmit GRBV. In this study, Tortistilus were collected on yellow panel cards across 102 vineyard sites and surrounding areas in Napa Valley, California, USA in 2021-2023. Specimens were morphotyped, sexed and tested for GRBV ingestion and acquisition by multiplex PCR or qPCR. Phylogenetic analysis of the partial sequence of mt-COI and ITS gene fragments of a subset of 40 Tortistilus specimens revealed clustering in a monophyletic clade with T. wickhami with the former barcode sequence. Only 6% (48/758) of the T. wickhami tested positive for GRBV, but none of the heads with salivary glands (0%, 0/50) of the dissected specimens tested positive for GRBV, indicating no virus acquisition. In contrast, half of the dissected heads with salivary glands of S. festinus (52%, 12/23), from the same collection vineyard sites, tested positive for GRBV. Together, our findings confirmed the presence of T. wickhami in northern California vineyards and suggested a dubious role of this treehopper as a vector of GRBV.

Background/Objectives: The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. Methods: Based on morphological traits such as nut size, nut shape, and the number of leaflets, five genotypes were chosen and samples were collected for the current study. For molecular analysis, gDNA was isolated from the fresh leaves, and the five most effective angiosperm-specific markers, ITS2, rbcLa, rbcLc, rpoC1, and UBE3, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and UBE3 were recorded as the most efficient markers followed by rbcLa, rbcLc, and rpoC1. Results: During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on UBE3 clustered with (KF454101.1-Juglans regia) and (KC870919.1-J. regia) with bootstraps of 56 and 100, respectively. Genotype-3 based on rbcla clustered in a major clade with J. regia L., cultivars (MN397935.1 J. regia 'Vina') and (MN397934.1-J. regia 'Serr'), (MN397933.1 J. regia 'Pedro'), (MN397932.1 J. regia 'Lara'), (MN397931.1 J. regia 'Howard'), and (MN397930.1 J. regia 'Hartley') with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on rbclc and rpoC1 clustered with (MN397935.1 J. regia 'Vina') and (MN397934.1 J. regia 'Serr'), across the database sequences. To clarify the taxonomic status of cryptic walnut genotypes, it is necessary to combine diverse DNA barcodes. The results of ITS2 and UBE3, followed by rbcL barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. Conclusions: It has been determined that the genotypes of walnuts in the study area are both J. regia L. and its cultivars and that the accuracy of discrimination regarding the genus Juglans L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of Juglans taxa and its population. [ABSTRACT FROM AUTHOR]

The Hypnodendrales form the sister group to all other pleurcarpous mosses in the superorder Hypnanae. Many species are closely associated with the cool temperate rainforests of Australasia, while others are widespread in higher altitude wet forests across tropical South-East Asia. Several of the Malesian entities are taxonomically ambiguous, having been variously treated as species complexes or as single very variable species. Here we use dated phylogenies to examine the timing of diversification of major clades within the Hypnodendrales, test the hypothesis that widespread, taxonomically problematic species have diversified relatively recently in South-East Asia from within clades of southern temperate origin, and address taxonomic questions within Hypnodendron. A chronogram with broad taxonomic sampling is constructed, followed by a second dated phylogeny with dense sampling from Hypnodendron vitiense , a representative morphologically variable species found throughout Eastern Australia and South-East Asia. The crown group Hypnodendrales are found to have originated in the mid-Cretaceous, although they share a Most Recent Common Ancestor (MRCA) with the other Hypnanae in the late Jurassic. Extant members of Hypnodendron share a MRCA ±28 Mya, a little before the start of the collision of Australia with the Sunda plate that initiated the Australasian–Malesian floristic interchange. Within H. vitiense , there is strong phylogenetic structure consistent with the diversification and isolation of populations in South-East Asia within the last 10 Mya. The New Zealand endemic H. marginatum is found to be derived from within an Australasian clade of H. vitiense , this in turn being distinct from a South-East Asian/tropical Australian H. vitiense clade. Our results suggest that the phylogeography of this prominent group of mosses closely mirrors that of the rainforest ecosystems of which they are a part. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is an important zoonotic parasite that is mainly prevalent in China, Korea, Vietnam and the Russian Far East. To explore the genetic variation and population structure of C. sinensis, an in silico analysis was conducted based on mitochondrial cytochrome c oxidase subunit 1 (COX1), ribosomal internal transcribed spacer 1 (ITS1) and ribosomal internal transcribed spacer 2 (ITS2) sequences. The sequences obtained from NCBI were truncated for further analyses, including haplotype network, phylogenetic, gene flow, diversity and neutrality analyses. The results showed that there were 20, 11 and 4 haplotypes for COX1, ITS1 and ITS2, respectively. The results of both the haplotype network and phylogenetic analyses indicated that the haplotypes for each type of sequence from the same country were not all clustered together. Haplotype diversity values were all lower than 0.5. Values of nucleotide diversity were higher than 0.005, except for ITS2. Tajima's D and Fu's Fs values were all negative, and p-values showed significant differences, indicating that the population of C. sinensis is growing. Fst values were all lower than 0.05. In conclusion, this study found that there are specific variations of C. sinensis in different countries, and the population of this parasite is growing with less genetic variation. The findings provide a crucial foundation for understanding the molecular epidemiology and population dynamics of C. sinensis. [ABSTRACT FROM AUTHOR]

Abstract Background The genus Physalis belongs to the Solanaceae family and has different species with important nutritional and medicinal values. Species within this genus have limited morphological differences, a characteristic that hinders accurate identification, safe utilization and genetic conservation of promising genotypes. In addition, to prevent the perceived loss of Physalis diversity due to habitat destruction, species delimitation needs attention. In this study, we used the sequence and structural information of the internal transcribed spacer 2 (ITS2) barcode to efficiently identify and discriminate Physalis species from a collection of 34 Physalis accessions. Methodology Physalis plant samples were collected from eight Counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. A total of 34 Physalis accessions were identified and accessed for diversity based on the ITS2 barcode region. The sequence similarity of the ITS2 genes was analyzed through the Basic Local Alignment Search Tool (BLAST), the nearest Kimura-2-parameter (K2P) genetic distances were calculated and a phylogenetic tree was constructed using the Bayesian inference (BI) method in MrBayes 3.2.7a software. The differences in the ITS2 secondary structure between the species were analyzed. Results The success rate of PCR amplification and sequencing was 75% and 67%, respectively. The analyzed ITS2 sequences displayed significant inter-specific divergences, clear DNA barcoding gaps and high species identification efficiency. Based on the constructed phylogenetic tree, three Physalis species (Physalis peruviana, Physalis purpurea and Physalis cordata) were identified and were clustered in a homogenized distribution. High genetic diversity (0.36923) and genetic distance (0.703) were observed between Physalis peruviana and Physalis cordata. The highest genetic nucleotide diversity (0.26324) and distance (0.46) within species was obtained for Physalis peruviana. The differences in the secondary structures generated from this study discriminated between the Physalis species. Conclusions Our study demonstrated that ITS2 is a potential DNA barcode for effective identification and discrimination of Physalis species. The results of this study provide insights into the scientific basis of species identification, safe utilization, genetic conservation and future breeding strategies for this important nutritional and medicinal plant species.

Abstract Rhopalocercous Cercaria duplicata von Baer, 1827 develops in an intermediate host, the unionid bivalve Anodonta anatina (L.), but its adult form has been unknown. We examined eight fish species occurring in the presence of a highly infested population of A. anatina in the Zesławice reservoir (S Poland). Gravid Phyllodistomum specimens were obtained from the ureters of ide, Leuciscus idus (L.) and common rudd, Scardinius erythrophthalmus (L.). One of the rudd specimens was doubly infected, a trematode was also found in the urinary bladder. In addition, a gravid Phyllodistomum specimen was found in the ureter of a tench Tinca tinca (L.), caught in Lake Ilmėdas (Lithuania). In order to clarify the phylogenetic position of larval and adult gorgoderids and to establish their life cycle, ITS2 and 28S rDNA sequences were analysed. The analysis showed that adult Phyllodistomum specimens located in the ureters are conspecific with C. duplicata. The trematode found in the urinary bladder of S. erythrophthalmus was P. folium (Olfers, 1816). It is suggested that adult stages of C. duplicata should be referred to as Phyllodistomum duplicatum n. comb. The intercaecal position of the uterus and the deeply-lobed ovary are the main features distinguishing it from other Phyllodistomum species. Host specificity and ecology are discussed.

Over three-quarters of Earth's surface exhibits extreme environments where life thrives under harsh physicochemical conditions. While prokaryotes have often been investigated in these environments, only recent studies have revealed the remarkable adaptability of eukaryotes, in particular fungi. This study explored the mycobiota of two meromictic hypersaline lakes, Ursu and Fără Fund, in Transylvania (Romania). The intrinsic and extrinsic fungal diversity was assessed using amplicon sequencing of environmental DNA samples from sediments, water columns, surrounding soils, and an associated rivulet. The fungal communities, illustrated by the 18S rRNA gene and ITS2 region, exhibited contrasting patterns between the lakes. The ITS2 region assessed better than the 18S rRNA gene the fungal diversity. The ITS2 data showed that Ascomycota was the most abundant fungal group identified in both lakes, followed by Aphelidiomycota, Chytridiomycota, and Basidiomycota. Despite similar α-diversity levels, significant differences in fungal community structure were observed between the lakes, correlated with salinity, total organic carbon, total nitrogen, and ammonium. Taxonomic profiling revealed depth-specific variations, with Saccharomycetes prevalent in Ursu Lake's deeper layers and Lecanoromycetes prevalent in the Fără Fund Lake. The functional annotation using FungalTraits revealed diverse ecological roles within the fungal communities. Lichenized fungi were dominant in Fără Fund Lake, while saprotrophs were abundant in Ursu Lake. Additionally, wood and soil saprotrophs, along with plant pathogens, were more prevalent in the surrounding soils, rivulet, and surface water layers. A global overview of the trophic relations in each studied niche was impossible to establish due to the unconnected graphs corresponding to the trophic interactions of the analyzed fungi. Plotting the unweighted connected subgraphs at the genus level suggests that salinity made the studied niches similar for the identified taxa. This study shed light on the understudied fungal diversity, distribution, and ecological functions in hypersaline environments. [ABSTRACT FROM AUTHOR]

The article "Exploring biodiversity of Uruguayan vascular plants through DNA barcoding" published in Frontiers in Genetics on October 15, 2024, discusses the use of DNA barcoding to study the biodiversity of Uruguayan vascular plants. The study aims to address the decline in biodiversity in Uruguay by establishing a DNA barcode national network. By analyzing 171 sequences from four loci for 50 vascular plant species, the researchers identified suitable markers for plant DNA barcoding and contributed to the Uruguayan Barcode of Life Initiative. The study highlights the importance of creating a comprehensive public database for species identification and biodiversity research. [Extracted from the article]

The chestnut gall wasp (CGW), Dryocosmus kuriphilusYasumatsu, 1951 (Cynipini, Cynipoidea, Hymenoptera), is a globally invasive pest on chestnuts (Castanea spp., Fagaceae). Since its first reported occurrence in the state of Georgia in 1974, CGW has been reported to have spread to 16 additional states in the eastern United States. Despite its wide occurrence, investigations of the invasion and colonization history of CGW have not been conducted. To address this shortcoming, we screened one mitochondrial (cytochrome c oxidase subunit 1; COI) and one nuclear gene (internal transcribed spacer 2; ITS2) to examine the genetic variation across four discrete CGW populations from four states (New Jersey, Pennsylvania, Maryland, Tennessee). We tested whether CGW in the eastern U.S. originated from one or multiple introductions followed by subsequent dispersal. Additional insights came from comparing the sequences with two CGW populations sampled from its native range in mainland China, and one population each from Taiwan and Japan. Sequences revealed that both genes exhibit the same haplotype across all sampled specimens of all locations. The COI haplotype in this study is the dominant haplotype recorded in China, while the haplotype of ITS2 has been reported in both Japan and Europe. The uniformity of genetic structure suggests that CGW invasion in the U.S. could have either single or multiple origins, or both, accompanied with the common haplotype on each occasion. Further insights into the invasion history and expansion of CGW in the U.S. will require a combination of sampling more populations and utilizing a next generation sequencing strategy. [ABSTRACT FROM AUTHOR]

The animal gut microbiome can have a strong influence on the health, fitness, and behavior of its hosts. The composition of the gut microbial community can be influenced by factors such as diet, environment, and evolutionary history (phylosymbiosis). However, the relative influence of these factors is unknown in most bird species. Furthermore, phylosymbiosis studies have largely focused on clades that diverged tens of millions of years ago, and little is known about the degree of gut microbiome divergence in more recent species radiations. This study explores the drivers of microbiome variation across the unique and recent Hawaiian honeycreeper radiation (Fringillidae: Drepanidinae). Fecal samples were collected from 14 extant species spanning the main islands of the Hawaiian archipelago and were sequenced using three metabarcoding markers to characterize the gut microbiome, invertebrate diet, and plant diet of Hawaiian honeycreepers. We then used these metabarcoding data and the honeycreeper host phylogeny to evaluate their relative roles in shaping the gut microbiome. Microbiome variation across birds was highly individualized; however, source island had a small but significant effect on microbiome structure. The microbiomes did not recapitulate the host phylogenetic tree, indicating that evolutionary history does not strongly influence microbiome structure in the honeycreeper clade. These results expand our understanding of the roles of diet, geography, and phylogeny on avian microbiome structure, while also providing important ecological information about the diet and gut microbiota of wild Hawaiian honeycreepers. [ABSTRACT FROM AUTHOR]

Simple Summary: The South American fruit fly Anastrepha fraterculus (Wiedmann) is found from northern Mexico to northern Argentina, where it causes damage to many different wild and cultivated fruits. It is a not a single species, but a complex of practically identical species. Eight morphological types (morphotypes) have been identified. To facilitate the identification of the species, alternative, non-morphological methods, such as those based on genome sequences, are necessary. The sterile insect technique is an efficient method used to combat these pests, which involves the release of many sterile male insects into the wild. Mass-reared sterile field-released males mate with females, which lay inviable eggs, thereby reducing the population. This approach is only successful when the released males are sexually compatible with the females of the target population. Hence, accurate identification is necessary for its success. We evaluated the use of the internal transcribed spacer 2 (ITS2) sequence for discriminating members of the A. fraterculus cryptic species complex and a related species, Anastrepha schultzi Blanchard. The ITS2 sequence successfully discriminated between different morphotypes and provides a basis for the development of keys for discrimination of the species within the complex. ITS2 also represents an important marker for the poorly studied species A. schultzi. The South American fruit fly Anastrepha fraterculus (Wiedmann) has a vast range extending from northern Mexico, through Central America, to South America where it is an extremely polyphagous pest of wild and cultivated fruits. It is a complex of cryptic species currently composed of eight recognised morphotypes: "Mexican", "Venezuelan", "Andean", "Peruvian", "Ecuadorian", and the three Brazilian morphotypes "Brazilian-1", "Brazilian-2", and "Brazilian-3". Molecular markers that can identify the member species of the complex are crucial for the implementation of effective pest control measures, such as the sterile insect technique. The object of this study was to evaluate the use of the internal transcribed spacer 2 (ITS2) sequence for discriminating several members of the A. fraterculus cryptic species complex (Mexican, Peruvian, and Brazilian-1) and a related species, Anastrepha schultzi Blanchard. The analysis highlighted significant genetic differentiation between the evaluated morphotypes, allowed their discrimination within the A. fraterculus cryptic species complex, and provided new insights into their genetic relationships. The ITS2 marker provides a basis for the development of taxonomic keys for the discrimination of the cryptic taxa within the A. fraterculus cryptic species complex. ITS2 also represents an important marker for the poorly studied species A. schultzi. [ABSTRACT FROM AUTHOR]

This study investigates anopheline species diversity in the Andaman and Nicobar Islands, employing morphological and molecular methods, focusing on the D3 domain of 28S rRNA (D3) and second internal spacer (ITS2). Ten Anopheline species were identified morphologically and confirmed with molecular markers. While the D3 region demonstrated low level of inter- and intra-specific genetic distance in all the species, ITS2 revealed clear barcoding gap. Among the ten species, A. barbirostris exhibited significant diversity when compared with the sequences from other countries available in GenBank. Further analyses of additional samples of A. barbirostris were carried out using ITS2 and cytochrome oxidase I (COI) markers. Limited variations among the sequences from the islands were observed, suggesting a prevalent single molecular form. However, when compared with the GenBank sequences, our samples formed a separate cluster closely related to the A3 species. The genetic distance between our samples and the A3 cluster was 0.02 for COI but very high (0.104) for ITS2, suggesting a potentially new molecular form or species in the island region. This warrants a more comprehensive and detailed analysis of A. barbirostris in these islands at both genetic and morphometric levels. Overall, these observations added-up the new knowledge in the understanding of anopheline diversity in the Andaman and Nicobar archipelago and highlight the necessity for continuous molecular investigations to unravel complexities within mosquito population dynamics. [ABSTRACT FROM AUTHOR]

For the first time, the cultivated diversity of microalgae in eroded soils in the northern part of the Fergana Valley in Uzbekistan has been studied based on both morphological and molecular genetic analysis. Ten strains of green microalgae (Chlorophyta) and one charophyte strain (Charophyta) were revealed. Only seven strains could be identified at the species level: Chlorella vulgaris, Chromochloris zofingiensis, Deuterostichococcus epilithicus, Pseudomuriella schumacherensis, and Pseudostichococcus monallantoides. Another four strains were identified only at the genus level and require further study: Bracteacoccus sp., Chlorosarcinopsis sp., Klebsormidium sp., and Tetratostichococcus sp. The low species diversity in the microalgae is likely due to both the low fertility of the eroded soils on the slopes, and the limitations of the culture-based approach that only reveals a fraction of the overall microbial diversity. Microalgal colonization of eroded soils in the arid foothill zone can be facilitated by various adaptations, such as small cell size and the production of extracellular polysaccharides, mycosporine-like aminoacids, and secondary carotenoids. The present work may contribute to the further development of highly functional microalgal consortia, which can lead to improvements and sustainable development of low-productivity, arid, and degraded terrestrial ecosystems. [ABSTRACT FROM AUTHOR]

The ecological success of shallow water corals hinges on their association with photosynthetic Symbiodiniaceae algae. This is affected by environmental drivers among which sea temperature is pivotal. In 2016, a prolonged heat wave challenged New Caledonia reefs triggering a severe bleaching event. Here, we tracked 72 coral colonies comprising two species of Pocillopora and Porites from a cross-shelf gradient during the event and subsequent recovery period. Symbiodiniaceae association over time was assessed using the ITS2 marker. Bleaching prevalence and photosynthetic efficiency showed that 83% of Pocillopora and 29% of Porites colonies were affected, with corals from a mid-shelf site having been most impacted. The majority of tracked colonies recovered by December 2016, with a recorded 33% mortality of Pocillopora, while Porites showed higher resilience. Consistent with previous studies, genotyping data suggest stable, species- and site-specific associations between corals and Symbiodiniaceae. [ABSTRACT FROM AUTHOR]

Abstract Biomphalaria straminea is one of the three snails that act as the intermediate hosts of Schistosoma mansoni and is responsible for maintaining high rates of schistosomiasis in some areas of northeastern Brazil. The principal morphological characteristic of B. straminea is the marked vaginal wrinkling, although it is also present in congeneric species, a group known as the B. straminea complex. Based on the morphological and molecular analyses, we investigated the intraspecific variation of B. straminea. The specimens were obtained from 10 sites in Brazil, and the shells were analyzed, as well as 16 morphological structures of the reproductive system. The COI and ITS2 sequences were used for phylogenetic analysis, genetic divergence, and haplotype network (COI). We observed a large intraspecific variation in the morphological structures examined. The genetic divergence also demonstrated significant intraspecific variability in B. straminea: 0–3% in ITS2 sequences, and 0–6% in COI sequences. Analysis of the distribution of COI haplotypes recovered 16 haplotypes and haplotype diversity of 0.9088. These results indicate phenotypic variability that is not constrained to a locality or strictly controlled genetically by B. straminea, which may have driven a misidentified of these species.

Abstract Background The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. Methods Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. Results In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. Conclusions An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Abstract Background The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex. Methods The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex. Results Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers. Conclusions The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS. Graphical Abstract

Endophytic fungus is crucial for maintaining plant health and defense mechanisms, acting as protective barriers against pathogens, and producing medicinally beneficial bioactive compounds. Genome sequencing and metagenomics have significantly enhanced the understanding of fungal diversity and metabolic capabilities, enabling the identification of new genes and substances. Traditional culture-dependent methods have been complemented by culture-independent techniques, offering a more comprehensive view of fungal diversity. Using both culture-dependent and culture-independent techniques, the present research investigation explored the diversity of endophytic fungi encountered in the foliage of Hardwickia binata. The study examined the topographical characteristics and nutritional content of soil samples collected from the locality of the selected plant sample, H. binata, to better comprehend the effects on the plant’s growth. The balanced nutrient constituted approximately a pH of 7.2, which suggested an alkaline nature and promoted plant development. The ratio of nitrogen, phosphorous, and potassium remained 3:1:1. A total of 25 fungal isolates, categorized into 17 morphotypes, were obtained using the culture-dependent approach; Curvularia and Nigrospora emerged as the most common genera. Furthermore, the prediction of the ITS2 secondary structure supports the identification of species, highlighting a wide variety of fungal species present in H. binata. The culture-independent approach generated 69,570 high-quality sequences, identifying 269 Operational Taxonomic Units (OTUs). The dominant Ascomycota phylum, along with various genera, indicated a rich fungal community associated with H. binata. This study advances the understanding of the endophytic fungus communities that are associated with H. binata and the nature of soil ecology. The findings emphasize the significance of holistic techniques in the study of microbial dynamics within plant systems as well as their implications for ecosystem management and plant health.

Background: The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. Methods: Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. Results: In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. Conclusions: An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon. [ABSTRACT FROM AUTHOR]

Introduction: Rhubarb is a frequently used and beneficial traditional Chinese medicine. Wild resources of these plants are constantly being depleted, meaning that rhubarb products have been subjected to an unparalleled level of adulteration. Consequentially, reliable technology is urgently required to verify the authenticity of rhubarb raw materials and commercial botanical drugs. Methods: In this study, the barcode-DNA high-resolution melting (Bar-HRM) method was applied to characterize 63 rhubarb samples (five Polygonaceae species: Rheum tanguticum, Rh. palmatum, Rh. officinale, Rumex japonicus and Ru. sp.) and distinguish the rhubarb contents of 24 traditional Chinese patent medicine (TCPM) samples. Three markers, namely ITS2, rbcL and psbA-trnH, were tested to assess the candidate DNA barcodes for their effectiveness in distinguishing rhubarb from its adulterants. A segment from ITS2 was selected as the most suitable mini-barcode to identify the botanical drug rhubarb in TCPMs. Then, rhubarbs andTCPMsamples were subjected to HRM analysis based on the ITS2 barcode. Results: Among the tested barcoding loci, ITS2 displayed abundant sites of variation and was effective in identifying Polygonaceae species and their botanical origins. HRM analysis based on the ITS2 mini-barcode region successfully distinguished the authenticity of five Polygonaceae species and eight batches of TCPMs. Of the 18 TCPM samples, 66.7 % (12 samples) were identified as containing Rh. tanguticum or Rh. officinale. However, 33.3 % were shown to consist of adulterants. Conclusions: These results demonstrated that DNA barcoding combined with HRM is a specific, suitable and powerful approach for identifying rhubarb species and TCPMs, which is crucial to guaranteeing the security of medicinal plants being traded internationally. [ABSTRACT FROM AUTHOR]

Background: The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex. Methods: The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex. Results: Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers. Conclusions: The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS. [ABSTRACT FROM AUTHOR]

Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) is widespread in the western Palaearctic Region, but it was recorded in Karelia (Russia) for the first time. This record is one of the northernmost ones in the Palaearctic Region and Russia, updates the northern border of the An. claviger range. Mosquitoes were collected from July to September 2023 in the southern Karelia (the village of Gomselga, Kondopoga District, and Petrozavodsk) using Krishtal trap (from human) and Mosquito Magnet® trap (Pioneer design, Octenol as attractant). Seven females of An. claviger were collected in Gomselga; one specimen was sampled from Petrozavodsk City parks. Morphological identification of eight females was verified by COI and ITS2 sequences. Phylogenetic analysis of ITS2 and COI sequences confirmed the collected specimens to An. claviger s. s., clustering in both cases in a strongly supported clade clearly differentiated from the closely related species An. petragnani. The high diversity of An. claviger haplotypes from Karelia is in agreement with data from other geographical regions and shows that the records of this species in Gomselga and Petrozavodsk are not accidental. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to detect Toxocara cati in cats by microscopic and molecular analysis using PCR, sequencing, and phylogenetic analysis. Materials and Methods: Randomly selected 200 cat feces samples were taken from various private veterinarian clinics in Baghdad. To identify eggs of T. cati by the flotation method, DNA from 100 cat feces was extracted, and one pair of ITS2 region-specific primers was used for polymerase chain reaction, followed by sequencing. Results: Toxocara cati infection rate was found to be 23 out of 100 fecal samples using PCR. Ten DNA product sequence data studies showed 98%-100% similarity to the 5.8S ribosomal RNA gene sequences found in the Gene Bank. The study incidence showed that the overall infection rate by microscopic examination was 23%, with no significant difference between stray cats (27%), and domestic cats (19%). After studying the effect of several epidemiological parameters on the infection rate, it was found that the infection rates of stray and domestic cats were higher in kittens under six months of age, at 46.1% and 27%, respectively, whereas rates were lower for the adult than six months was 11.5% of domestic cats and 14.7% of stray cats. The percentage of stray and domestic male cats that were registered was 35.5%, whereas the female cats registered were 20.6% and 17.5%, respectively. Conclusion: Cats are significant clinical reservoirs for zoonotic parasites. In Iraq, Baghdad has a high incidence of T. cati detections. Compared to conventional methods, PCR is thought to be a more sensitive, accurate diagnostic procedure that confirms the species' identity. [ABSTRACT FROM AUTHOR]

Endophytic fungus is crucial for maintaining plant health and defense mechanisms, acting as protective barriers against pathogens, and producing medicinally beneficial bioactive compounds. Genome sequencing and metagenomics have significantly enhanced the understanding of fungal diversity and metabolic capabilities, enabling the identification of new genes and substances. Traditional culture-dependent methods have been complemented by culture-independent techniques, offering a more comprehensive view of fungal diversity. Using both culture-dependent and culture-independent techniques, the present research investigation explored the diversity of endophytic fungi encountered in the foliage of Hardwickia binata. The study examined the topographical characteristics and nutritional content of soil samples collected from the locality of the selected plant sample, H. binata, to better comprehend the effects on the plant's growth. The balanced nutrient constituted approximately a pH of 7.2, which suggested an alkaline nature and promoted plant development. The ratio of nitrogen, phosphorous, and potassium remained 3:1:1. A total of 25 fungal isolates, categorized into 17 morphotypes, were obtained using the culture-dependent approach; Curvularia and Nigrospora emerged as the most common genera. Furthermore, the prediction of the ITS2 secondary structure supports the identification of species, highlighting a wide variety of fungal species present in H. binata. The culture-independent approach generated 69,570 high-quality sequences, identifying 269 Operational Taxonomic Units (OTUs). The dominant Ascomycota phylum, along with various genera, indicated a rich fungal community associated with H. binata. This study advances the understanding of the endophytic fungus communities that are associated with H. binata and the nature of soil ecology. The findings emphasize the significance of holistic techniques in the study of microbial dynamics within plant systems as well as their implications for ecosystem management and plant health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Methods: Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Results: Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. Conclusions: An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B. [ABSTRACT FROM AUTHOR]

Black corals (Hexacorallia: Antipatharia) are a major component of mesophotic and deep marine ecosystems. Due to their preference for light deprived environments, black corals have historically been considered azooxanthellate, yet recent works have found them in association with dinoflagellates of the family Symbiodiniaceae down to 396 m depth. While corals and Symbiodiniaceae generally establish a symbiotic relationship in shallow water environments, the implications of this association is less well understood at deeper depths, where low light penetration cannot sustain efficient photosynthetic activity for the algae. However, Symbiodinaceae are not obligate autotrophs, and their capacity for heterotrophic feeding categorizes themas mixotrophs. In this study, we investigated the presence and diversity of Symbiodiniaceae associated with the deep-sea black coral Bathypathes thermophila (Antipatharia: Schizopathidae), collected from 204 to 655 m depth in the Saudi Arabian Red Sea. Using high-throughput sequencing of the ITS2 region, we report (1) the deepest record to date of Symbiodiniaceae associated with an anthozoan from 655 m, and (2) the first Red Sea record of Antipatharia in association with Symbiodiniaceae. Our analyses revealed that 14 out of 27 colonies of B. thermophila were associated with Symbiodiniaceae of the genera Cladocopium and Durusdinium. We unveiled 16 novel ITS2 type profiles, possibly unique to black corals and/or to these depths, along with seven profiles that were already known from shallow-water hard corals. No significant pattern was detected in terms of community diversity in relation to depth or sampling locality. Our study supports the existence of black corals-Symbiodiniaceae association and warrants further research to better understand the evolutionary processes and physiological mechanisms driving this association, specifically in light deprived environments. [ABSTRACT FROM AUTHOR]

ABSTRACT While in hospite Symbiodiniaceae dinoflagellates are essential for coral health, ambient free-living counterparts are crucial for coral recruitment and resilience. Comparing free-living and in hospite Symbiodiniaceae communities can potentially provide insights into endosymbiont acquisition and recurrent recruitment in bleaching recovery. In this study, we studied coral-endosymbiotic and ambient free-living Symbiodiniaceae communities in the South China Sea. We collected samples from 183 coral and ambient plankton samples and conducted metabarcoding to investigate the diversity distribution, driving factors, and assembly mechanisms of the two groups of Symbiodiniaceae. Results revealed Cladocopium C1 and Durusdinium D1 as dominant genotypes. We detected a higher genotypic diversity in free-living than in hospite symbiodiniacean communities, but with shared dominant genotypes. This indicates a genetically diverse pool of Symbiodiniaceae available for recruitment by corals. Strikingly, we found that the cooler area had more Symbiodiniaceae thermosensitive genotypes, whereas the warmer area had more Symbiodiniaceae thermotolerant genotypes. Furthermore, in hospite and free-living Symbiodiniaceae communities were similarly affected by environmental factors, but shaped by different assembly mechanisms. The in hospite communities were controlled mainly by deterministic processes, whereas the ambient communities by stochastic processes. This study sheds light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms influencing Symbiodiniaceae inside and outside corals.IMPORTANCESymbiodiniaceae dinoflagellates play a pivotal role as key primary producers within coral reef ecosystems. Coral-endosymbiotic Symbiodiniaceae communities have been extensively studied, but relatively little work has been reported on the free-living Symbiodiniaceae community. Conducting a comparative analysis between sympatric coral-endosymbiotic and free-living Symbiodiniaceae communities can potentially enhance the understanding of how endosymbiont communities change in response to changing environments and the mechanisms driving these changes. Our findings shed light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms shaping free-living and in hospite Symbiodiniaceae communities, with implications in evaluating the adaptive and resilient capacity of corals in response to future climate change.

ABSTRACTHigh-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.

To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B.

Objective: This study aimed to detect Toxocara cati in cats by microscopic and molecular analysis using PCR, sequencing, and phylogenetic analysis. Materials and Methods: Randomly selected 200 cat feces samples were taken from various private veterinarian clinics in Baghdad. To identify eggs of T. cati by the flotation method, DNA from 100 cat feces was extracted, and one pair of ITS2 region-specific primers was used for polymerase chain reaction, followed by sequencing. Results: Toxocara cati infection rate was found to be 23 out of 100 fecal samples using PCR. Ten DNA product sequence data studies showed 98%–100% similarity to the 5.8S ribosomal RNA gene sequences found in the Gene Bank. The study incidence showed that the overall infection rate by microscopic examination was 23%, with no significant difference between stray cats (27%), and domestic cats (19%). After studying the effect of several epidemiological parameters on the infec¬tion rate, it was found that the infection rates of stray and domestic cats were higher in kittens under six months of age, at 46.1% and 27%, respectively, whereas rates were lower for the adult than six months was 11.5% of domestic cats and 14.7% of stray cats. The percentage of stray and domestic male cats that were registered was 35.5%, whereas the female cats registered were 20.6% and 17.5%, respectively. Conclusion: Cats are significant clinical reservoirs for zoonotic parasites. In Iraq, Baghdad has a high incidence of T. cati detections. Compared to conventional methods, PCR is thought to be a more sensitive, accurate diagnostic procedure that confirms the species' identity. [J Adv Vet Anim Res 2024; 11(2.000): 392-397]

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. asper funereus Mulsant, 1862, M. verecundus bulgaricus 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.

Clonorchis sinensis is an important zoonotic parasite that is mainly prevalent in China, Korea, Vietnam and the Russian Far East. To explore the genetic variation and population structure of C. sinensis, an in silico analysis was conducted based on mitochondrial cytochrome c oxidase subunit 1 (COX1), ribosomal internal transcribed spacer 1 (ITS1) and ribosomal internal transcribed spacer 2 (ITS2) sequences. The sequences obtained from NCBI were truncated for further analyses, including haplotype network, phylogenetic, gene flow, diversity and neutrality analyses. The results showed that there were 20, 11 and 4 haplotypes for COX1, ITS1 and ITS2, respectively. The results of both the haplotype network and phylogenetic analyses indicated that the haplotypes for each type of sequence from the same country were not all clustered together. Haplotype diversity values were all lower than 0.5. Values of nucleotide diversity were higher than 0.005, except for ITS2. Tajima’s D and Fu’s Fs values were all negative, and p-values showed significant differences, indicating that the population of C. sinensis is growing. Fst values were all lower than 0.05. In conclusion, this study found that there are specific variations of C. sinensis in different countries, and the population of this parasite is growing with less genetic variation. The findings provide a crucial foundation for understanding the molecular epidemiology and population dynamics of C. sinensis.

Introduction: The Red Sea is a narrow rift basin characterized by latitudinal environmental gradients which shape the diversity and distribution of reefdwelling organisms. Studies on Symbiodiniaceae associated with select hard coral taxa present species- specific assemblages and concordant variation patterns from the North to southeast Red Sea coast at depths shallower than 30 m. At mesophotic depths, however, algal diversity studies are rare. Here, we characterize for the firsttime host-associated algal communities of a mesophotic specialist coral species, Leptoseris cf. striatus, along the Saudi Arabian Red Sea coast. Methods: We sampled 56 coral colonies spanning the eastern Red Sea coastline from the Northern Red Sea to the Farasan Banks in the South, and across two sampling periods, Fall 2020 and Spring 2022. We used Next Generation Sequencing of the ITS2 marker region in conjunction with SymPortal to denote algal assemblages. Results and discussion: Our results show a relatively stable coral species-specific interaction with algae from the genus Cladocopium along the examined latitudinal gradient, with the appearance, in a smaller proportion, of presumed thermally tolerant algal taxa in the genera Symbiodinium and Durusdinium during the warmer season (Fall 2020). Contrary to shallow water corals, our results do not show a change in Symbiodiniaceae community composition from North to South in this mesophotic specialist species. However, our study highlights for the first time that symbiont communities are subject to change over time at mesophotic depth, which could represent an important phenomenon to address in future studies. [ABSTRACT FROM AUTHOR]

The intensification of agriculture is impacting farmland and is recognised as a main driver for biodiversity loss in these environments. Many farmland bird populations have been declining in recent decades because of agricultural intensification. This is the case of the Red-legged Partridge Alectoris rufa, a game bird that inhabits farming landscapes and has recently suffered drastic population declines in Spain. To fully understand the effects that agricultural intensification has on these birds, it is crucial to deepen the knowledge regarding their feeding habits and how these may vary seasonally. The aim of this study was to provide a detailed and reliable description of wild partridges' plant diet composition. For this purpose, we collected faecal samples from 35 partridge flocks during autumn, winter and spring, and used DNA metabarcoding to identify plants consumed by this species. We used nuclear and chloroplastic barcodes for the amplification of ITS2 and rbcL gene regions, respectively. Our results showed that the diet of partridges was highly diverse, mainly consisting of cereals, weeds and legumes. The most frequently consumed genus was Hordeum, followed by Lolium, Papaver, Medicago and Avena. We further showed that diet composition varied significantly amongst seasons, with a higher proportion of cultivated plants (> 25%) being consumed during autumn and winter. During the cereal sowing period, cropped plants might be consumed as sown seeds, which are often coated with pesticides and could represent an important risk. In spring, before breeding, the diet was mostly composed of weeds. Plants of the Papaver genus could be consumed for anti-parasitic purposes, and as a source of antioxidants and carotenoids to increase ornamental coloration, especially during pairing and laying. Our results highlight the importance of maintaining heterogenous farming landscapes with fallow lands and field margins as sources of weeds to help the conservation of this species. [ABSTRACT FROM AUTHOR]

Ligula intestinalis (Lineaus1758) is one of the most common parasites of freshwater fish. This study was conducted to survey the possibility of infection of freshwater breams (Abramis brama) caught from Siahdarvishan River and their morphological and molecular characteristics were investigated. 75 breams, after dissection, all parts of their abdominal cavity completely were examined for the presence of the L. intestinalis plerocercoids. Then, the morphometric characteristics of parasites were investigated. In the next step, the definitive identification of the genus and species of parasitic samples was confirmed by identification keys. For molecular analysis, the samples were stored at -20°C. Then, molecular studies were done on 10 plerocercoids obtained from 10 separate fish by analysis of ITS2 gene. From 75 A. brama samples, 18 fish (24%) were infected by L. intestinalis. In this study, the minimum, mean and maximum size of the isolated plerocercoids were 8, 26 and 74cm, respectively. Sequencing of the ITS2 gene of L. intestinalis isolates with GenBank samples showed that these isolates had genetic differences in different hosts and based on the comparison of the ITS2 gene locus, and the presence of host-dependent species in the genus Ligula is probable. [ABSTRACT FROM AUTHOR]

Features of shell and genitalia as well as nucleotide sequences of selected mitochondrial and nuclear genes of specimens of Monacha cantiana from ten northern French and two Dutch populations were compared with the same features of British and Italian populations. They were found to be very similar to populations previously identified as belonging to the CAN-1 lineage of M. cantiana. This confirms previous suggestions that M. cantiana was introduced to western Europe (England, France and the Netherlands) in historical times. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Derogenes ruber Lühe, 1900, the type-species of the genus Derogenes Lühe, 1900, is a poorly known derogenid digenean. The original description of this species was not illustrated and aspects of the morphology of the parasite from the type-host remain scarce. Available records of this species were brief and/or lacked illustrations and were based on morphology alone. Additionally, molecular data for Derogenes spp. are warranted to untangle species complexes as they provide a better assessment of interspecific genetic divergence. Methods: Derogenes ruber is redescribed based on newly collected specimens from the gall bladder of its type-host Chelidonichthys lastoviza (Bonnaterre, 1788) collected in the Western Mediterranean off the Algerian coast during 2017–2019 and molecular data are provided using a partial fragment of the nuclear 28S ribosomal RNA gene (28S rRNA), the internal transcribed spacer 2 (ITS2) and a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Results: We herein provide a detailed illustrated redescription and morphometric data of D. ruber from its type-host C. lastoviza. We report a new geographical record (off Algeria) for it. Derogenes ruber is also genetically characterised for the first time. Species/lineages of Derogenes were recovered in five strongly supported reciprocally monophyletic clades: (i) D. ruber from C. lastoviza off Algeria; (ii) D. lacustris from Galaxias maculatus (Jenyns) off Argentina; (iii) Lineage "D. varicus DV1" (D. varicus sensu stricto) from fish hosts in the White and Barents seas and the North Sea; (iv) Lineage "D. varicus DV2" from mollusc hosts in the White Sea; and (v) Lineage "D. varicus DV3" from Eumicrotremus fedorovi Mandrytsa. in the Pacific Ocean. Hence, comparison of the newly generated sequences with other available data for Derogenes species supports the distinction of D. ruber confirming its taxonomic status and helping assess interspecific variation. Comparison of D. ruber with the closely related species Derogenes latus revealed overlaps in morphometric data and the validity of the latter species is questioned. Conclusion: The combination of morphological and molecular data provided for D. ruber provides a firm foundation for further investigations of Derogenes spp. Although we do describe herein material of D. ruber from the type-host, given that the occurrence of a single Derogenes species in various hosts has been challenged by molecular data, and both D. lacustris and D. varicus sensu stricto had been genetically proven to occur in various hosts, D. ruber and D. latus may be indeed synonymous. Additional sequencing effort on Derogenes spp. will strengthen systematic comparative studies and evolutionary relationships within the Derogenidae in general. [ABSTRACT FROM AUTHOR]

Solanaceae is one of the prominent plant families that provide medicine and food. For the diagnosis of its species (Physalis peruviana and Physalis angulata), the use of molecular markers with reliable and precise nuclear ribosomal DNA (nrDNA) ITS sequences has helped in the presumption of molecular evolution and phylogenetic studies of the plants. It precisely measured the variance between two samples of Physalis L. Purified fragments' sequencing using BLAST to align each sequence, evaluate its relationship with other sequences, and confirm the species of different sequences. The genetic analysis program, MEGA V.11, aided in performing multiple sequence alignment and phylogenetic analysis. The first sample showed a relation to the species Physalis peruviana voucher Smith 217 (WIS) placed in the GenBank with the serial number DQ314161.1 by 96.91%, which belonged to a strain located in the USA. The researcher registered the species as Physalis peruviana isolate AH-ZE1, with serial number OQ616506.1. However, the second sample, after contrasting it with the global species sequences, indicated a connection to the species Physalis angulata isolate LHR28I deposited in the Genome Bank with the serial number MK412130.1 at a rate of 98.02%, which belonged to a Spanish strain. The species' registration by the researcher continued as Physalis angulata isolate AHZE1, sequence number OQ616509.1. The current study results revealed the diagnosis of two species of Physalis identified for the first time in Iraq, as they incur registration in the National Center for Biotechnology Information. The recording depended on the molecular characteristics' differences in the ribosomal DNA (rDNA) region, knowing the evolutionary relationship between the Physalis species and comparing them to the sequences found in the GenBank for previously defined types. The results also showed that the ITS2 region provided success ability. Therefore, using other DNA barcodes as auxiliary factors to distinguish between Physalis species would be beneficial. [ABSTRACT FROM AUTHOR]

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. asper funereus Mulsant, 1862, M. verecundus bulgaricus 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 previouslydefined 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. [ABSTRACT FROM AUTHOR]

Plants of the genus Physalis are of economic interest because of their fleshy edible fruits with high nutritional value. Some species have high medicinal value with a long history of ethno-medicinal use to treat diverse diseases. There is therefore a need to correctly discriminate the different species of Physalis for proper utilization. Although most Physalis species have unique morphologies, their vegetative stages are identical, making it difficult to accurately identify them based on morphological characteristics. DNA barcoding has the potential to discriminate species accurately. In this study, ribulose bisphosphate carboxylase large (rbcL) and internal transcribed spacer 2 (ITS2) regions were used to discriminate Physalis species and to reveal their phylogenetic relationships and genetic diversity. Physalis plant samples were collected from seven counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. Genomic DNA was isolated from leaf samples of 64 Physalis accessions and used for PCR amplification and the sequencing of rbcL and ITS2 barcode regions. The discriminatory ability of the barcodes was based on BLASTn comparison, phylogenetic reconstruction and cluster analysis, and the determination of inter- and intra-specific distances. The nucleotide polymorphism, genetic diversity and distance of the identified Physalis species were determined using DnaSP and MEGA 11.0 software. Species discrimination was more robust using ITS2 sequences. The species identified and discriminated by ITS2 sequences were Physalis purpurea, Physalis peruviana and Physalis cordata. The rbcL sequences were only able to identify Physalis to the genus level. There was high interspecific and low intraspecific divergence within the identified Physalis species based on ITS2 sequences. The ITS2 barcode is an ideal DNA barcode for use in the discrimination of species, as well as in genetic diversity studies of Physalis accessions in Kenya.

Abstract Objectives Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species. Results Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu. Data description Our data offers valuable insights into honey’s geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.

DNA barcoding is a straightforward strategy that uses short orthologous genetic sequences and standard genomes to specify species. This technique has the capability of molecular identification, detection of living species and discovery of unfamiliar species, preservation of genetic resources, identification of genetic diversity and phylogenetic characterization, and testing of differentiated existing plant species, as well as assuring the safety and efficacy of pharmaceuticals. In this experiment, the allelic diversity of 7 classes of medicinal plants viz. fenugreek, local fenugreek, waybread, cumin, flax, fixweed and sesame, from Ilam and Khuzestan provinces; west and south of Iran, respectively, was carried out employing this technique and with the aid of primers designed and established on ITS nuclear barcodes (ITS1 and ITS2 genes). The results indicated that there was a great difference between the fragments and the duplicated sequences of ITS1 and ITS2 barcodes in different samples. Moreover, the nucleotide searching of the sequences showed that there was a very high similarity (more than 90%) between the acquired sequences and their equivalents in the NCBI database. The nucleotide sequence of the ITS1 gene of fenugreek showed the highest similarity (78.2%) with native fenugreek. Regarding the ITS1 gene, more amount of G~C content than A~T was observed and, in the waybread plants the amount of C base was higher than G, and for native fenugreek, the amount of A~T content was more than G~C. In the case of ITS2 position, in all examined samples (except the fixweed plant, which had higher A~T), the values ​​of G~C content were higher than A~T. The output of the cluster analysis with the UPGMA algorithm showed the precise grouping and separation of species and the high potential of these sequences using the barcode system in the phylogenetic evaluation of medicinal plant species.