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Background: The current study aimed to determine the prevalence of cutaneous leishmaniasis (CL) in Al-Muthanna province (Iraq) and to characterize the Leishmania species that cause cutaneous lesions through conventional polymerase chain reaction techniques in some patients during the first 7 months of the year 2020. Materials and Methods: Medical information on patients with CL was obtained from archived records at the Al-Muthanna Health Office's Public Health Department (2015–2020). In the Al-Hussein Teaching Hospital laboratory, 95 CL samples were collected and examined microscopically for molecular characterization using Giemsa staining. Results: Between 2015 and 2020, 2,325 patients (1,184 men and 1,141 women) were enrolled. Although CL occurred across all age groups, those aged range of 5–14 years had the highest proportion of infections (53.0%). This study found that most infections occurred between December and February, peaking in January. Only 63 of 95 CL samples were positive for the Internal Transcribed Spacer 1 region. L. tropica was found in 39 samples (61.9%), whereas L. major was found in 24 samples (38.1%), in CL patients. Although dermal lesions develop in all body regions, a single lesion is the most common. The upper limbs (13 of 16 samples, 33.3%)were infected with L. tropica, whereas the lower limbs (9 of 14 samples, 37.5%) were infected with L. major. In contrast to L. major, most L. tropica lesions occur in urban areas. Conclusion: Our study indicates that CL is endemic in the Al-Muthanna province and that two Leishmania spp. coexist in the province. Molecular diagnosis is a vital component in determining many clinical symptoms of the Leishmania parasite as well as implementing suitable therapeutic, epidemiological, and control strategies. [ABSTRACT FROM AUTHOR]

The gut microbiome's imbalance has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), yet the contribution of the gut mycobiome remains largely unclear. This study delineates the gut mycobiome profile in PAH and examines its interplay with the bacterial microbiome alterations. Fecal samples from monocrotaline-induced PAH rats and matched controls were subjected to internal transcribed spacer 1 (ITS1) sequencing for fungal community assessment and 16S ribosomal RNA (rRNA) gene sequencing for bacterial community characterization. Comparative analysis revealed no significant disparities in the overall mycobiome diversity between the PAH and control groups. However, taxonomic profiling identified differential mycobiome compositions, with the PAH group exhibiting a significant enrichment of genera such as Wallemia, unidentified_Branch02, Postia, Malassezia, Epicoccum, Cercospora, and Alternaria. Conversely, genera Xeromyces, unidentified_Plectosphaerellaceae, and Monilia were more abundant in the controls. Correlations of Malassezia and Wallemia abundance with hemodynamic parameters were observed. Indications of bidirectional fungal–bacterial community interactions were also noted. This investigation reveals distinct gut mycobiome alterations in PAH, which are intricately associated with concurrent bacterial microbiome changes, suggesting a possible contributory role of gut fungi in PAH pathophysiology. These findings underscore the potential for novel gut mycobiome-targeted therapeutic interventions in PAH management. [ABSTRACT FROM AUTHOR]

The gut microbiome’s imbalance has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), yet the contribution of the gut mycobiome remains largely unclear. This study delineates the gut mycobiome profile in PAH and examines its interplay with the bacterial microbiome alterations. Fecal samples from monocrotaline-induced PAH rats and matched controls were subjected to internal transcribed spacer 1 (ITS1) sequencing for fungal community assessment and 16S ribosomal RNA (rRNA) gene sequencing for bacterial community characterization. Comparative analysis revealed no significant disparities in the overall mycobiome diversity between the PAH and control groups. However, taxonomic profiling identified differential mycobiome compositions, with the PAH group exhibiting a significant enrichment of genera such as Wallemia, unidentified_Branch02, Postia, Malassezia, Epicoccum, Cercospora, and Alternaria. Conversely, genera Xeromyces, unidentified_Plectosphaerellaceae, and Monilia were more abundant in the controls. Correlations of Malassezia and Wallemia abundance with hemodynamic parameters were observed. Indications of bidirectional fungal–bacterial community interactions were also noted. This investigation reveals distinct gut mycobiome alterations in PAH, which are intricately associated with concurrent bacterial microbiome changes, suggesting a possible contributory role of gut fungi in PAH pathophysiology. These findings underscore the potential for novel gut mycobiome-targeted therapeutic interventions in PAH management.

The Carpathians represent not only a European hotspot of plant diversity for both species richness and endemism, but also an important stepping-stone area in historical migrations between the flora of the Asian and European mountain systems and a starting point of postglacial recolonizations for many species. Yet, until recent years, phylogeographical studies for alpine or arctic-alpine plants were focused on the Alps, whereas peripheral mountain ranges, including the Carpathians, were either neglected or insufficiently sampled. In this study, we aimed to complement the Alpine phylogeographic structure of an emblematic high-alpine European endemic taxon, Eritrichium nanum, by focusing on the Carpathian range of the species. We sampled nine populations from the South-Eastern Carpathians and performed ITS1 sequencing and AFLP fingerprinting. In case of ITS1 region, all the populations, no matter of their geographic origin, presented the same ribotype. The AFLP analysis indicated that, within the Carpathians, the extant populations of E. nanum comprised two major allopatric lineages. One important result of the research was the discovery that the species' sole important genetic break was located in the Southern Carpathians, separating populations of the Retezat Mountains from all the others in the Carpathians. [ABSTRACT FROM AUTHOR]

The Carpathians represent not only a European hotspot of plant diversity for both species richness and endemism, but also an important stepping-stone area in historical migrations between the flora of the Asian and European mountain systems and a starting point of postglacial recolonizations for many species. Yet, until recent years, phylogeographical studies for alpine or arctic-alpine plants were focused on the Alps, whereas peripheral mountain ranges, including the Carpathians, were either neglected or insufficiently sampled. In this study, we aimed to complement the Alpine phylogeographic structure of an emblematic high-alpine European endemic taxon, Eritrichium nanum, by focusing on the Carpathian range of the species. We sampled nine populations from the South-Eastern Carpathians and performed ITS1 sequencing and AFLP fingerprinting. In case of ITS1 region, all the populations, no matter of their geographic origin, presented the same ribotype. The AFLP analysis indicated that, within the Carpathians, the extant populations of E. nanum comprised two major allopatric lineages. One important result of the research was the discovery that the species’ sole important genetic break was located in the Southern Carpathians, separating populations of the Retezat Mountains from all the others in the Carpathians.

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality. [ABSTRACT FROM AUTHOR]

SUMMARY: Correctly identifying species of Chlorella‐like microalgae is difficult because of their morphological simplicity and high phenotypic plasticity. The use of molecular tools has revolutionized research on algal diversity, enabling such advances as the discovery of numerous new taxa. This article presents the results of a study of strains that are newly isolated from a freshwater Lake Prudovikov (Samara region, Russian Federation). These strains had the typical Chlorella morphology, exhibiting spherical cells and a cup‐shaped parietal chloroplast. The chloroplast contained a single pyrenoid enveloped by starch grains. However, 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strains are strongly allied with the well‐supported genus Micractinium. Based on the results of a comparative analysis of the morphological, molecular and environmental characteristics of the studied strains (employing a polyphasic approach), we propose that they are new species of Micractinium: Micractinium kostikovii sp. nov. [ABSTRACT FROM AUTHOR]

Premise: Foliar fungal endophytes vary in their distributions across landscapes or plant host taxa, indicative of specialized ecologies and host specific adaptations. Accounts of specialization, however, depend on the taxonomic breadth and geographic range of the host plants included in each study. A broad region‐scale study or deep sampling of diverse potential host species still remains relatively rare but is becoming increasingly possible with high‐throughput sequencing. Methods: Amplicon sequencing was used to rapidly identify the fungal endophytic community among six pine (Pinus, Pinaceae) species co‐occurring across northeastern United States and to test for site and host specialization. We focused on the endophytic genus Lophodermium (Rhytismataceae), whose species members are thought to specialize on different pine species, to test if amplicon sequencing could rapidly verify previously implied or discover new patterns of host specificity. Results: While amplicon sequencing could analyze more samples at greater depths and recover greater numbers of unique Lophodermium taxa than when endophyte communities were surveyed with traditional culturing methods, patterns of specialization were not better supported. This may be because amplicon sequencing can indiscriminately capture non‐host specific organisms found incidentally from plant tissues or because we have overestimated host‐specificity in the past with biased culturing techniques. Conclusions: Amplicon sequencing can quickly identify patterns of host specificity by allowing large‐scale surveys but has limitations in quantifying the level of intimacy of these relationships. [ABSTRACT FROM AUTHOR]

This is the first study on phylogenetic relationships in the genus Artemia Leach, 1819 using the pattern and sequence of secondary structures of internal transcribed spacer 1 (ITS1). Significant intraspecific variation in the secondary structure of ITS1 rRNA was found in Artemia tibetiana. In the phylogenetic tree based on joined primary and secondary structure sequences, Artemia urmiana and parthenogenetic populations displayed new lineages, and two New World species (Artemia franciscana and Artemia persimilis) were located in a basal clade that was not detected in previous studies. The close evolutionary relationship between A. franciscana and A. persimilis are expressively supported by the previous empirical and experimental investigation on the ability of hybridization (in natural habitats and lab conditions) and analysis on allozyme markers.

A high tolerance to a wide spectrum of environmental factors and excellent taste of its meat explain why the African sharptooth catfish (Clarias gariepinus) is widely used for creating interspecific hybrids with the broadhead catfish (Clarias macrocephalus), a species which is characterized by a high growth rate. However, when the spawning stock on a fish farm is being supplemented with new individuals, there is a serious concern about introducing hybrids instead of purebred individuals. Therefore, it is necessary to develop a quick and reliable method of hybrid identification In the presented study species diversity was assessed based on PCR and sequence analysis of the internal transcribed spacer 1 (ITS1) and cytochrome oxidase subunit 1 (COI) sequences. Additionally, three restriction enzymes (Fok I, Eco47I and Hhal) were selected and analysed with PCR-RFLP of COI sequences. ITS1 sequence comparisons revealed 0.64% diversity between analysed species and were submitted into GenBank database (KJ136021 and KJ136022). Analysis of COI sequences revealed over 14% variation between two species and based on obtained species-characteristic restriction patterns, a PCR-RFLP test has been developed. [ABSTRACT FROM AUTHOR]

Six Trypanosoma evansi isolates collected from ponies (PH1 and PK6), camel (CB2), donkeys (DJ3 and DH4) and cattle (CK5) from Haryana, Rajasthan, Uttar Pradesh and Gujarat states of India were used for molecular characterization of internal transcribed spacer 1 (ITS 1). The DNA was isolated from purified trypanosomes of these six isolates after propagation in mice model. ITS1-PCR of purified parasite DNA yielded an amplification product approximately 540 bp in size. Nucleotide sequence of ITS1 gene of CB2 isolate had 530 bp while CK5, DH4, DJ3, and PH1 isolates had 532 bp, whereas, PK6 isolates had 533 bp size. Blast data of the Indian isolates revealed 99 % homology with other available sequences of T. evansi. Multiple alignment of nucleotide sequence of ITS1 gene variants from Indian T. evansi isolates with selected homologous sequences from GenBank revealed that nucleotide substitution mostly occurred at the position of 101-103, 218-223, 243-244, 301-396 and 470-480. The isolates PH1, CK5, DH4 and DJ3 were found more associated with T. evansi isolates from the Philippines, Thailand, Iran, Egypt and China, whereas, PK6 and CB2 isolates were related to each other and were phylogenetically distant from rest of the Indian isolates used in this study. Based on the ITS1 rDNA sequence, the Neighbour-Joining consensus tree indicated clear evidence of existence of genetic diversity among T. evansi isolates from India. [ABSTRACT FROM AUTHOR]

Babylonia areolata is an aquatic mollusk in the genus of the gastropod family Buccinidae. Genetic variations were observed in the 542 base pairs of the sequences from its cytochrome oxidase subunit I (COI) gene of mtDNA. COI alignments showed that there were two monophyletic clades on the phylogenetic tree, referred as B. areolata. These results point towards the possibility of B. areolata being a single species with strong phylogeographic subdivisions. About 520 base pairs of the internal transcribed spacer 1 (ITS1) sequence were able to distinguish between the shell colors (brown, cream and white) of B. areolata.

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer ( ITS/ ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large-scale meta-analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity-based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample-rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species. [ABSTRACT FROM AUTHOR]

Populations of nematodes of the superfamily Criconematoidea were obtained and identified morphologically from different geographical areas in the continental United States. This previous information was used to analyze their phylogenetic relationship based on the first internal transcribed spacer ribosomal region. Phylogenetic analysis of the ITS1-rDNA region showed monophyletic and paraphyletic groups in Criconematidae: A) species of Mesocriconema and Criconemoides; and B) Bakernema, Criconema Hemicriconemoides and Xenocriconemella species. Subfamily Hemicycliophorinae, (Caloosia, Hemicaloosia and Hemicycliophora) and family Tylenchulidae (Paratylenchus and Gracilacus) were not resolved because of the high variation of the marker Molecular phylogenetic analysis using the ITS1-rDNA marker rejects the hypothesis of a common ancestor for criconematids with double sheath or cuticle among others characters. This molecular phylogenetic study showed that ITS1-rDNA sequences are too variable to study phylogenetics of Criconematoidea. However, ITS1-rDNA is useful to identify and to characterize species within families. [ABSTRACT FROM AUTHOR]

Abstract: The taxonomic framework of Ligophorus, monogenean specialists of the gills of grey mullets (Mugilidae), is evaluated and its interspecific relationships are assessed for the first time using molecular data. The position of Ligophorus within the paraphyletic Ancyrocephalinae is re-assessed based on newly sequenced species. Furthermore, the relationship between morphometric and genetic interspecific similarities is evaluated. Partial 28S and complete ITS1 rDNA sequences from representatives of 14 of the 16 nominal species of Ligophorus from the Mediterranean, Black and Azov Seas were analysed together with published sequences of members of the Dactylogyridae. The phylogenetic analyses of the Dactylogyridae (i) confirmed the position of Ligophorus within the marine Ancyrocephalinae; (ii) revealed a sister relationship between Ergenstrema and Ligophorus, whose species are all exclusive parasites of grey mullets; and (iii) substantiated the affinities of Ergenstrema with the marine Ancyrocephalinae. The phylogenetic analysis restricted to Ligophorus confirmed the distinct status of the included species. The ITS1 region provided the highest divergence between species and phylograms with the strongest branch support. Both the 28S and ITS1 phylograms revealed two main clades. One included species from hosts with Mediterranean and NE Atlantic distribution and another was formed by species parasitising several Liza spp., including Lz. haematocheilus from the Northwestern Pacific, and Mugil cephalus, which suggests an origin outside the Mediterranean for the latter clade. The phylogenetic evidence presented herein indicated that a combination of host-switching and lineage duplication events accounted for the diversification of this genus in the Mediterranean basin. The agreement between molecular and morphological interspecific similarities observed in Ligophorus supports the validity of morphometric characters used for species identification. [Copyright &y& Elsevier]

Abstract: Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People''s Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10−3 ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis. [Copyright &y& Elsevier]

The article presents an analysis of an aborted southern white rhinoceros fetus that was infected by Neospora caninum. Based on the study on the cause of abortion, it was revealed that the aborted seven-month gestation male fetus has necrosis in the hepatic parenchyma which was associated with low numbers of lymphocytes, plasma cells, and neutrophils. According to the authors, the presence of Neospora zoites within necrotic hepatic lesion indicates that the abortion of the rhinoceros fetus was due to the protozoal infection. It also explains that the neonatal death could be the result of transplacental vertical transmission from the dam of the aborted fetus which may have been exposed to the infection.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman® MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low. [ABSTRACT FROM AUTHOR]

Mitochondrial cytochrome oxidase I (COI) and ribosomal internal transcribed spacer 1 (ITS-1) sequences were analysed in 12 Romanian Bythinella populations. Phylogenetic relationships were inferred using maximum parsimony, maximum likelihood and Bayesian techniques. For COI, the Kimura two-parameter (K2P) distances and haplotype networks were computed. Two sympatric and four allopatric groups were distinguished. The K2P distances are similar to those for congeneric rissooids, so each of the six groups represents a species. Two are identified as Bythinella molcsanyi H. Wagner, 1941, and Bythinella dacica Grossu, 1946. The other four groups cannot be ascribed to any known Bythinella. The occurrence of six species, each with a low haplotype number and high interspecific differences between haplotypes, is explained by: (1) a relatively long history of Bythinella in the territory of a Pleistocene glacial refugium; (2) the discontinuous character of the northernmost refugium, promoting speciation, but local extinction and subsequent recolonization; (3) unstable, post-glacial microhabitat conditions; and (4) the fragmented distribution of Romanian Bythinella. [ABSTRACT FROM AUTHOR]

Abstract: Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for γ-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK21, KH-R). The best growth of tachyzoites was observed in BHK21 cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK21 and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany. [Copyright &y& Elsevier]

Abstract: A TaqMan PCR assay was developed for the detection of Trypanosoma evansi. The assay targets the internal transcribed spacer 1 (ITS-1) region of rRNA. The ITS-1 region of eleven strains of T. evansi from widely separated geographical regions were sequenced and alignments compared. Primers and probe for the test were designed from these sequence data. The assay was tested using blood from infected rats and was found to be sensitive, detecting less than one genomic equivalent of T. evansi. The assay has been tested against 10 different species of trypanosomes found in native animals in Australia and did not detect any of these trypanosome species. Time course experiments using rats infected with T. evansi were performed to compare the TaqMan assay with the Haematocrit centrifugation test (HCT) and the mouse inoculation (MI) assay. The assay was more sensitive than the HCT but not as sensitive as the MI. The TaqMan assay has the ability to rapidly detect T. evansi and determine the number of organisms present in a blood sample from an infected animal. This is the first time a TaqMan assay has been developed for the detection of T. evansi. [Copyright &y& Elsevier]

Babylonia areolata is an aquatic mollusk in the genus of the gastropod family Buccinidae. Genetic variations were observed in the 542 base pairs of the sequences from its cytochrome oxidase subunit I (COI) gene of mtDNA. COI alignments showed that there were two monophyletic clades on the phylogenetic tree, referred as B. areolata. These results point towards the possibility of B. areolata being a single species with strong phylogeographic subdivisions. About 520 base pairs of the internal transcribed spacer 1 (ITS1) sequence were able to distinguish between the shell colors (brown, cream and white) of B. areolata. [ABSTRACT FROM AUTHOR]

Bauhinia blakeana Dunn is the Hong Kong Special Administrative Region emblem and a popular horticultural species in many Asian countries. It was first described as a new species from Hong Kong almost a century ago. This plant is sterile and has long been considered a hybrid, possibly from two related species, B. purpurea and B. variegata. However, not much evidence based on molecular methods was available to support this hypothesis. In this study, sequences of internal transcribed spacer 1 ( ITS1), rbcL and atpB-rbcL intergenic spacer for five Bauhinia species and two varieties of one of the species were determined and compared. There were two types of ITS1 sequences in B. blakeana, one indistinguishable from that of B. purpurea and the other one identical to that of B. variegata. This confirmed that B. blakeana was a hybrid of these two species. Chloroplast atpB-rbcL intergenic spacer sequence of B. blakeana was identical to that of B. purpurea, indicating that B. purpurea was the female parent. The hybridization event seemed to occur only recently and was a rare incident. Its occurrence was likely facilitated by interspecific pollen competition. It appeared that human efforts played a crucial role in the preservation and ubiquity of B. blakeana. [ABSTRACT FROM AUTHOR]

Summary: The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece. [Copyright &y& Elsevier]

Many species of tachinid flies are viewed as generalist parasitoids because what is apparently a single species of fly has been reared from many species of caterpillars. However, an ongoing inventory of the tachinid flies parasitizing thousands of species of caterpillars in Area de Conservación Guanacaste, northwestern Costa Rica, has encountered >400 species of specialist tachinids with only a few generalists. We DNA-barcoded 2,134 flies belonging to what appeared to be the 16 most generalist of the reared tachinid morphospecies and encountered 73 mitochondrial lineages separated by an average of 4% sequence divergence. These lineages are supported by collateral ecological information and, where tested, by independent nuclear markers (28S and ITS1), and we therefore view these lineages as provisional species. Each of the 16 apparently generalist species dissolved into one of four patterns: (i) a single generalist species. (ii) a pair of morphologically cryptic generalist species, (iii) a complex of specialist species plus a generalist, or (iv) a complex of specialists with no remaining generalist. In sum, there remained 9 generalist species among the 73 mitochondrial lineages we analyzed, demonstrating that a generalist lifestyle is possible for a tropical caterpillar parasitoid fly. These results reinforce the emerging suspicion that estimates of global species richness are likely underestimates for parasitoids (which may constitute as much as 20% of all animal life) and that the strategy of being a tropical generalist parasitic fly may be yet more unusual than has been envisioned for tachinids. [ABSTRACT FROM AUTHOR]

Abstract: The internal 16S/23S rDNA (rrs/rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain (Ochrobactrum intermedium LMG 3301T), and 2–3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA–DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing ITS1c ( ITS1) similarity fell within a species, and ITS1c ( ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more. [Copyright &y& Elsevier]

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns. [ABSTRACT FROM AUTHOR]

Abstract: Two expansion segments of the large ribosomal subunit (28S-D2 and 28S-D3) and the ribosomal first internal transcribed spacer (ITS-1) were sequenced to investigate the potential of each region for defining species limits and for inferring phylogenetic relationships within the aphelinid genus Encarsia (Hymenoptera, Chalcidoidea). Alignment of the ITS-1 region was complicated by the presence of high levels of length polymorphism. Secondary-structure models of the ITS-1 molecule are proposed which led to an improved alignment and the inclusion of some highly polymorphic sequences. The additional information obtained from the secondary-structure models can provide a justification for the exclusion of hypervariable regions from the phylogenetic analysis. The D3 region is the most conserved, ITS-1 the most variable of the three gene regions investigated. Phylogenetic analyses suggest the D2 region to be most suitable not only for inferring relationships, but also for taxonomic and diagnostic purposes at species level. The ITS-1 region is best suited for inferring relationships between closely related species or among populations of the same species. The combined analysis using all three regions led to a better tree and a reduced number of equally most parsimonious trees. See also Electronic Supplement at: http://www.senckenberg.de/odes/06-07.htm [Copyright &y& Elsevier]

Abstract: Cryptic species of the digeneans, Cercaria batillariae (Heterophyidae) and an undescribed philophthalmid, were detected using polymerase chain reaction-based restriction fragment-length polymorphism methodology and sequence analyses. These digeneans were all collected from the same species of gastropod first intermediate host, Batillaria cumingi (=Batillaria attramentaria). The mitochondrial cytochrome oxidase subunit 1 gene (approximately 800bp) and nuclear internal transcribed spacer 1 gene (approximately 400bp) were used for species level discrimination. Restriction fragment-length polymorphism analyses of cytochrome oxidase subunit 1 gene showed that C. batillariae included 10 distinguishable fragment patterns, and the philophthalmid included five patterns. On the basis of subsequent sequence analyses, the restriction fragment length polymorphism patterns of C. batillariae were grouped into eight phylogenetically distinct lineages and those of the philophthalmid into three phylogenetically distinct lineages. There was no evidence of gene flow among the different lineages due to the lack of heterozygosity within the observed internal transcribed spacer 1 gene fragment patterns. This suggests that all of these lineages are different species. Most of these species were widespread, but some exhibited restricted geographic distributions. We discuss the implications of these findings for host specificity of these trematodes. These results demonstrate the utility of genetic analysis to distinguish species of morphologically similar trematodes. Hence, trematode species diversity may often be underestimated when species identifications are limited to morphological features. [Copyright &y& Elsevier]

We developed multiplex polymerase chain reaction methods to identify five Orius (Heteroptera: Anthocoridae) species that occur commonly in Japan: Orius sauteri, Orius minutus, Orius strigicollis, Orius nagaii, and Orius tantillus. The method amplified internal transcribed spacer 1 of the nuclear ribosomal DNA by using five primers simultaneously and produced species-specific banding patterns upon agarose gel electrophoresis. Reliability of the method was tested for 350 individuals of 23 strains, and consistent results were obtained. Dichotomous keys are also provided for easy and quick species identification. [Copyright &y& Elsevier]

Studies multiple polymorphic sites at the ITS and ATAN Loci in cultured isolates of Perkinsus marinus. Etiologic agent of Dermo disease on oysters; Distribution of the parasite.

The objective of this study was to confirm the presence of seven species of Eimeria involved in chicken coccidiosis in Australia by comparing internal transcribed spacer 1 (ITS-1) sequences, ITS-1 polymerase chain reaction (PCR) methods and to apply phylogenetic analysis to assess evolutionary relationships of Australian isolates. Twenty-two distinct ITS-1 regions of 15 Australian Eimeria isolates were sequenced, and analysed using maximum parsimony, distance and maximum likelihood methods. Poor bootstrap support, resulting from high ITS-1 sequence heterogeneity between all species groups, resulted in polychotomy of the Eimeria species in all three trees generated by these analyses. Percentage identity analyses revealed two distant ITS-1 lineages in both E. mitis and E. maxima at the same levels that separate the two species E. tenella and E. necatrix. One E. maxima lineage consisted of Australian isolates, the other American isolates, with one European sequence (originating from the same isolate) in each lineage. One Australian E. praecox sequence was only distantly related (33% variation) to three E. praecox sequences from Australian and European isolates. Short and long ITS-1 variants were isolated from both E. tenella (cloned line) and E. necatrix isolates with deletions (106 and 73 bp, respectively) in the short variants within the 3′ region of the ITS-1 sequence. ITS-1 sequences of strains of both E. brunetti and E. acervulina species varied the least. Apart from E. maxima, all of the ITS-1 sequences of the six remaining individual species clustered to the exclusion of other species in all phylogenetic trees. Published ITS-1 tests for E. necatrix, E. acervulina, E. brunetti and E. tenella, combined with three new tests for E. mitis, E. praecox and Australian E. maxima amplified all respective Australian isolates specifically in a nested format using conserved ITS-1 PCR products as template to improve the sensitivity. All PCR tests were confirmed against a collection of 24 Australian chicken Eimeria isolates and contaminating species were detected in some instances. In conclusion, once the genetic variation between species and strains is determined, the ITS-1 is a good target for the development of species-specific assays, but the ITS-1 sequences alone do not seem suitable for the confirmation of phylogenetic inferences for these species. This study reports the first attempt at the analysis of the phylogeny and sequence comparison of the Eimeria species involved in chicken coccidiosis in Australia. [Copyright &y& Elsevier]

AbstractThe phylogenetic relationships of East and West African members of the Simulium damnosum complex were studied by sequence analyses of the mitochondrial 16s ribosomal RNA (rRNA) gene. Results suggest that: (i) the S. damnosum complex is divided into an East and a West African clade, and (ii) S. pandanophilum and the cytoform ‘Kiwira’ form an East African subbranch distinct from the ‘Sanje’ group. In contrast to former assumptions from cytogenetic analyses, our molecular data do not support a direct relationship between the East African S. kilibanum and the West African S. squamosum. Length differences of the rDNA internal transcribed spacer 1 (ITS-1) turned out to be useful to distinguish between cytoforms. [ABSTRACT FROM AUTHOR]

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

Objective: To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania ., Methods: Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 ( ITS1 ) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti , Toxoplasma gondii , Plamodium vivax , P. ovale , P. falciparum , P. malariae , L. donovani and L. infantum ., Results: After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum , and no cross-reactions were observed with B. microti , T. gondii , P. vivax , P. ovale , P. falciparum or P. malariae . The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA., Conclusions: A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum , which is effective for field screening of leishmaniasis.

• Recent outbreak results in population expansion with high diversity of L. major • High genetic diversity of the samples but overall a low divergence between haplotypes • Results suggest a genetic flow between the Zagora and both Ouarzazate and Tinghir Cutaneous leishmaniasis (CL) is an infectious disease caused by various Leishmania species. It is among the most neglected tropical diseases and has been considered a major health threat over the past decades in the country. Its zoonotic form caused by Leishmania (L) major is the most prevalent in Morocco. This study investigated the population structure of L. major in southeastern Morocco. Samples (n = 67) were collected from patients with CL in five different endemic areas located in three provinces (Ouarzazate, Tinghir, and Zagora). These samples were then sequenced using two nuclear markers: internal transcribed spacer 1 (ITS1) and a fragment of the virulence factor GP63. Next, the sequences were edited and analyzed. Molecular diversity indices showed a high population genetic diversity but an overall low haplotype diversity. Our results suggest small population differentiation, indicating a low geographic structure. Tajima's D and Fu's Fs tests both suggested recent population expansion based on the significant deviations from neutrality in both tests for all populations except Tinghir, which may be due to a small sample size. Based on our findings, the region is experiencing rapid population expansion caused by recent CL outbreaks, and one of them has been recently studied. In addition, analysis of molecular variance and F ST suggested gene flow between Zagora and both Ouarzazate and Tinghir. Nonetheless, no gene flow was observed between Tinghir and Ouarzazate. To the best of our knowledge, this is the first analysis of the population structure of L. major in Morocco. The results of this study provide crucial background information for epidemiological studies by showing the presence of gene flow between populations and clonal expansion in cases of an outbreak. This will drive authorities to reconsider the implemented control strategies. Image, graphical abstract [ABSTRACT FROM AUTHOR]

An emerging threat to agriculture, Meloidogyne enterolobii Yang & Eisenback, 1983, is a tropical species and considered to be the most damaging root-knot nematode (RKN) in the world because of its wide host range, aggressiveness, and ability to overcome resistance to RKN in many crops. It was first detected in the United States on ornamental plants in Florida in 2001 but has since been identified in North Carolina, South Carolina, and Louisiana. Several thousand RKN populations were collected from North Carolina field crops, ornamental plants, and turfgrasses for species identification in the Nematode Assay Laboratory in the North Carolina Department of Agriculture & Consumer Services. From 2006 to 2019, root systems showing galling symptoms were dissected under the microscope, and females were obtained for DNA analysis. When only soil samples were submitted, the second-stage juveniles or males were used instead. Molecular characterization was performed via polymerase chain reaction with species-specific primers and DNA sequencing on the ribosomal DNA 18S-ITS1-5.8S and 28S D2/D3 and mitochondrial DNA CoxII-16S. One hundred thirty-five representative RKN populations from North Carolina were characterized and identified as M. enterolobii. Six populations from China where the species was originally described were included in this study for identity confirmation and comparison. As of December 2019, M. enterolobii has been confirmed from a limited number of fields in 11 North Carolina counties: Columbus, Craven, Greene, Harnett, Johnston, Lenoir, Nash, Pitt, Sampson, Wayne, and Wilson. Currently, M. enterolobii is the most important emerging RKN species in the United States and causes severe damage to agronomic and horticultural crops, especially sweetpotato in North Carolina.

Deep-sea demersal fishes of the Bothrocara hollandi species complex are distributed in the Japan Sea, the Okhotsk Sea, and the northwestern Pacific Ocean. Based on the nucleotide sequences of the nuclear Internal Transcribed Spacer 1 (ITS1) region and the microsatellite analysis, cryptic speciation resulting in the existence of two species (sp. 1 and sp. 2) in the Japan Sea was indicated for this species complex. In the Japan Sea off the San-in district, the westernmost part of the Japanese mainland, the frequency of sp. 2 individuals was highest at a depth of ∼400 m and decreased at both greater and lesser depths. Complete genetic deviation was observed between the individuals of the Japan Sea and the other sea areas, with the exception of a single sp. 2 individual, which shared an ITS1 sequence with an individual from the Pacific Ocean. Furthermore, a microsatellite analysis showed that the individuals of the other sea areas were more closely related to sp. 2 individuals. Two species were thought to have deviated from each other after their isolation from the individuals of the sea areas outside of the Japan Sea, through the occurrence of habitat fragmentation and bottleneck events in the Japan Sea during the glacial periods. Group A, one of two mitochondrial haplotype groups that were reported for the Japan Sea individuals in the previous studies, may have evolved within the lineages of sp. 2.