Gene Fragments Research Articles

BPP: a platform for automatic biochemical pathway prediction.

A biochemical pathway consists of a series of interconnected biochemical reactions to accomplish specific life activities. The participating reactants and resultant products of a pathway, including gene fragments, proteins, and small molecules, coalesce to form a complex reaction network. Biochemical pathways play a critical role in the biochemical domain as they can reveal the flow of biochemical reactions in living organisms, making them essential for understanding life processes. Existing studies of biochemical pathway networks are mainly based on experimentation and pathway database analysis methods, which are plagued by substantial cost constraints. Inspired by the success of representation learning approaches in biomedicine, we develop the biochemical pathway prediction (BPP) platform, which is an automatic BPP platform to identify potential links or attributes within biochemical pathway networks. Our BPP platform incorporates a variety of representation learning models, including the latest hypergraph neural networks technology to model biochemical reactions in pathways. In particular, BPP contains the latest biochemical pathway-based datasets and enables the prediction of potential participants or products of biochemical reactions in biochemical pathways. Additionally, BPP is equipped with an SHAP explainer to explain the predicted results and to calculate the contributions of each participating element. We conduct extensive experiments on our collected biochemical pathway dataset to benchmark the effectiveness of all models available on BPP. Furthermore, our detailed case studies based on the chronological pattern of our dataset demonstrate the effectiveness of our platform. Our BPP web portal, source code and datasets are freely accessible at https://github.com/Glasgow-AI4BioMed/BPP.

Inhibition of Botrytis cinerea and Escherichia coli by Lactic Acid Bacteria on Leafy Vegetables

The evaluation of the potentiality of lactic acid bacteria (LAB) strains isolated from fermented products to inhibit Botrytis cinerea and Escherichia coli O157:H7 growth on spinach and lettuce was conducted. From a total of forty LAB strains tested, three were selected due to their high inhibitory effect on plant pathogenic fungi. The identification of these isolates based on a 16S rRNA gene fragment sequence analysis confirmed the genus of Levilactobacillus sp. and Lactiplantibacillus sp. An effective method of coating LAB isolates on the lettuce and spinach surface was developed. The leaves were immersed in bacterial suspension (5.0 × 106 cfu mL−1) for 4 s and drained on tissue paper. LAB survived on lettuce and spinach leaves for 8 days at 6 log10 cfu g−1. Additionally, these bacteria decreased the number of filamentous fungi on the leaves. These isolates were found to inhibit the growth of B. cinerea and E. coli O157:H7 in vitro conditions in growing microbiological media. Their efficacy was confirmed in vivo conditions. These isolates inhibited the development of grey mould caused by B. cinerea on lettuce leaves. Two LAB isolates reduced the abundance of the pathogenic bacterium E. coli on spinach leaves by about 0.7 log10 cfu g−1. In glasshouse conditions, LAB stimulated the growth of examined plants. The lactic acid bacteria used in this study showed the capacity to be used as possible alternatives to chemical compounds in the protection of leafy vegetables against grey mould and for a decrease in E. coli O157:H7 contamination.

Presence of the knockdown resistance (kdr) mutations in the head lice (Pediculus humanus capitis) population in the North of Iran.

Head lice infestation continues to be a major health problem worldwide. In order to solve this problem, frequent use of pediculocides has caused growing resistance among head lice population. This study aims to investigate the presence of the knockdown resistance (kdr) mutation in the head lice population in northern Iran. Adult head lice were collected from 115 infested individuals referring to the health centers in different parts of Mazandaran province, northern Iran. 38 samples were randomly selected, and Polymerase chain reaction (PCR) was used to amplify a 332-bp and ⁓ 900-bp fragment of the α-subunit of the voltage-sensitive sodium channel (VSSC) gene. According to the analysis of a 332-bp fragment of the VSSC gene, the frequency of kdr T917I mutation including homozygous susceptible (SS), heterozygous resistant (RS), and homozygous resistant (RR) were 45.83%, 12.5%, and 41.66%, respectively. The total frequency of the resistance allele was 54.16%. The results of the 900-bp fragment of the VSSC gene showed two new mutations in the IIS1-2 extracellular loop (H813P) and IIS2 (S825R) and old well-known kdr mutations (M815I-T917I-L920F). The results of Hardy-Weinberg's exact equilibrium test showed that the frequency of genotypes in the studied areas is different from expectations. Moreover, a positive inbreeding coefficient value (F is >0) was found in studied areas which indicated an excess of homozygotes. Overall, the results showed a high frequency of resistant alleles in the northern region of the country. Therefore, it is necessary to develop appropriate control programs for the treatment of pediculosis.

Unexpected arboviruses found in an epidemiological surveillance of acute tropical febrile syndrome in the department of Meta, Eastern Colombia

BackgroundNonspecific acute tropical febrile illnesses (NEATFI) are common in the Latin American tropics. Dengue, Chikungunya, Zika, Mayaro, and Usutu, among others, can coexist in the American tropics. This study aimed to surveil the arboviruses that cause| acute febrile syndrome in patients in the Meta department, Colombia. MethodsBetween June 2021 and February 2023, an epidemiological surveillance study was conducted in the Llanos of the Meta department in Eastern Colombia. ResultsOne hundred patients in the acute phase with typical prodromal symptoms of NEATFI infection who attended the emergency department of the Villavicencio Departmental Hospital were included. ELISA tests were performed for Dengue, Usutu, Chikungunya, and Mayaro. RT-qPCR was performed to detect the arboviruses Usutu, Dengue, Zika, Mayaro, and Oropouche. The seroprevalence for the Chikungunya, Mayaro, and Usutu viruses was 41 % (28/68), 40 % (27/67), and 62 % (47/75), respectively. Seroconversion for Chikungunya was observed in one patient; two seroconverted to Mayaro and one to Usutu. The NS5 gene fragment of the Usutu virus was detected in nine febrile patients. RT-qPCR of the remaining arboviruses was negative. The clinical symptoms of the nine Usutu-positive patients were very similar to those of Dengue, Chikungunya, Zika, and Mayaro infections. ConclusionsThe pervasive detection of unexpected viruses such as Usutu and Mayaro demonstrated the importance of searching for other viruses different from Dengue. Because Usutu infection and Mayaro fever have clinical features like Dengue, a new algorithm should be proposed to improve the accuracy of acute tropical fevers.

Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa

BackgroundHepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species’ reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach.MethodsSpecimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database.ResultsPeripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi.ConclusionsThe present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.).Graphical

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance.

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Static Task Allocation Method for Multi-Machines in Cooperative Operations Combining OGFR-GA and MLW-Prim

Aiming at the operational efficiency of small farm machinery groups in cooperative operations in hilly areas, this study proposes a static task allocation model. This method combines the optimal gene fragment retention method based on a genetic algorithm (OGFR-GA) and the method for generating multi-loop weighted connected graphs based on Prim’s algorithm (MLW-Prim). The collaborative objective function for the harvester group was established by considering factors such as operation time, fuel consumption, and distance. The OGFR-GA was designed and applied multiple times to obtain several optimal gene fragments corresponding to the number of farm machines. These fragments were used as critical paths in the weighted connected graph generated based on farm machinery performance parameters and task parameters. The MLW-Prim method was proposed to construct this weighted connected graph and realize the static task allocation model for multi-machine cooperative operations. Simulation experiments showed that the model combining OGFR-GA and MLW-Prim achieved optimal values with fewer iterations, and reduced both group cost and cost variance compared to traditional algorithms. This method meets the static task allocation needs for multi-machine cooperative operations in agricultural production and can be integrated with intelligent operations in mountainous and hilly regions, laying a theoretical foundation for improving efficiency.

Frequency of Leptospira in the Blood of Patients with Fever of Unknown Origin in Sinaloa, Mexico.

Background: Leptospira is a genus of bacteria that causes the zoonotic disease known as leptospirosis, which mainly affects countries with tropical and subtropical climates. Its prevalence may be underestimated because the initial stage of the infection is characterized by presenting a febrile condition that is easily confused with other diseases, such as dengue. This work reports the frequency of leptospirosis in the blood of patients with febrile symptoms of unknown origin. Materials and Methods: A total of 218 peripheral blood samples were analyzed from volunteer participants from Culiacan Sinaloa in June 2019, one half corresponded to patients with undiagnosed febrile symptoms and the other half to asymptomatic volunteers. Data collected included the age and sex of the participants. Leptospira was detected by qPCR using a fragment of the lipL32 gene from the bacteria's genome as a target. Fisher's exact test was used as a statistical method to estimate the relationship between the infection and the data collected. Results: The study group comprised 134 female and 84 male patients ranging from ages 1 to 92 years, averaging 41 years. In this study, Leptospira infection was identified in the blood of 22/218 participating volunteers (10.09%), of which 20/109 (18.34%) presented febrile symptoms, whereas 2/109 (1.83%) were asymptomatic. The most affected participants were women with ages between 27 and 59 years. However, the analysis of the relationship between infection and the variables studied did not show statistical significance. Conclusions: Leptospirosis was detected in blood samples from patients with undiagnosed febrile illness and asymptomatic symptoms in Sinaloa. The lipL32 gene is useful as a target in identifying Leptospira in human blood in the acute phase of the disease.

Occurrence of Fusarium Crown Rot Caused by Fusarium culmorum on Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximately half of the total sown area of grain crops in XUAR, with 1.14 million hectares in 2021. Fusarium crown rot (FCR) of wheat, caused by Fusarium culmorum (W.G. Smith) Sacc., is one of the most devastating soil-borne diseases known to seriously reduce grain yield (Ma et al. 2024; Saad et al. 2023). In 2016, FCR of wheat, caused by F. culmorum, was firstly identified in Henan Province, China (Li et al. 2016). In June 2023, during the investigation of FCR of wheat in Aksu Prefecture, XUAR, FCR on winter wheat (cv. Xindong 20) was found (82.761349°E, 41.612202°N). The grain-filling period for winter wheat in early June coincided with a period of high temperatures and water demand in Aksu Prefecture. Approximately 8% of the Xindong 20 wheat plants exhibited symptoms of white heads and browning at the stem base, with the disease present in 82% of the wheat fields surveyed. To identify the pathogens, 20 samples of diseased stem basal tissue, each 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, followed by three rinses with sterile water. These samples were then plated onto potato dextrose agar (PDA) medium at 25°C for 5 days. A total of 17 isolates with consistent morphological characteristics were obtained using single-spore technique, with an isolation rate of 85%. The isolated strains exhibited rapid growth on PDA, producing fluffy, pale-yellow hyphae, and accumulating a pale-yellow to dark red pigment on the bottom of the medium. On carnation leaf agar (CLA), these strains formed orange colonies due to the aggregation of a large number of macroconidia. The macroconidia were short and thick, with three to four septa and rounded apical cell, averaging 31.94 to 40.96 × 5.62 to 6.71 μm (Magnification of ×400). Microconidia were not observed. These morphological characters were consistent with those of F. culmorum (Leslie and Summerell. 2006). Two isolates (D-9 and D-11) were selected for molecular identification. The EF-1α gene fragment was amplified using primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') as previously described by O'Donnell et al. (1998). The two 665 bp PCR products were sequenced and submitted to GenBank (GenBank Accession No: PP763247 and PP763248) with 99. 7% identity to the published F. culmorum sequences (e.g., OP985478, OP985477, MG195126, KX702638). The molecular identification was further confirmed by F. culmorum species-specific PCR primers FcOIF/FcOIR (Nicholson et al. 1998). The expected PCR products of 553 bp were produced only in F. culmorum. Strains D-9 and D-11 were used to conduct the pathogenicity experiment on 7-day-old winter wheat (cv. Xindong 20) using drip in the lower stem inoculation method with a 10-μl of 106 macroconidia ml-1 suspension, and the control 7-day-old winter wheat were treated with sterile water (Xu et al. 2017). The experiments were replicated five times in a greenhouse at temperatures ranging from 20℃ to 25℃. After 4 weeks, all inoculated wheat seedlings showed stem base browning or even death. No symptoms were observed on the control plants. The fungus was reisolated from all inoculated wheat plants by the method described above and identified by morphological and PCR amplification using F. culmorum species-specific primers FcOIF/FcOIR. No F. culmorum was isolated from the control wheat plants, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F.culmorum causing FCR on winter wheat in XUAR, China. Considering wheat is the predominant grain crop and plays a pivotal role in grain production in China, necessary measures should be taken to prevent the spread of F. culmorum to other regions.

A fragment of the β-glucosidase gene from the rumen fungus Neocallimastix patriciarum J11 encodes a recombinant protein that exhibits activities in β-glucosidase and β-glucanase

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as β-glucosidase. This study involved cloning a β-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-β-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against β-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both β-glucosidase and β-glucanase activities, a characteristic rarely found in β-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing β-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify β-glucan and ferment it to produce ethanol. This gene, with its dual β-glucosidase and β-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast.

Genetic diversity and natural selection analysis of VAR2CSA and vir genes: implication for vaccine development

Variable surface antigens (VSAs) encoded by var and vir genes in Plasmodium falciparum and Plasmodium vivax, respectively, are known to be involved in malaria pathogenesis and host immune escape through antigenic variations. Knowledge of the genetic diversity of these antigens is essential for malaria control and effective vaccine development. In this study, we analysed the genetic diversity and evolutionary patterns of two fragments (DBL2X and DBL3X) of VAR2CSA gene and four vir genes (vir 4, vir 12, vir 21 and vir 27) from different endemic regions, including Southeast Asia and sub-Saharan Africa. High levels of segregating sites (S) and haplotype diversity (Hd) were observed in both var and vir genes. Among vir genes, vir 12 (S = 131, Hd = 0.996) and vir 21 (S = 171, Hd = 892) were found to be more diverse as compared to vir 4 (S = 11, Hd = 0.748) and vir 27 (S = 23, Hd = 0.814). DBL2X (S = 99, Hd = 0.996) and DBL3X (S = 307, Hd = 0.999) fragments showed higher genetic diversity. Our analysis indicates that var and vir genes are highly diverse and follow the similar evolutionary pattern globally. Some codons showed signatures of positive or negative selection pressure, but vir and var genes are likely to be under balancing selection. This study highlights the high variability of var and vir genes and underlines the need of functional experimental studies to determine the most relevant allelic forms for effective progress towards vaccine formulation and testing.

Genome-Wide Identification of APX Gene Family in Citrus maxima and Expression Analysis at Different Postharvest Preservation Times.

Ascorbate peroxidase (APX) is a crucial enzyme involved in cellular antioxidant defense and plays a pivotal role in modulating reactive oxygen species (ROS) levels under various environmental stresses in plants. This study utilized bioinformatics methods to identify and analyze the APX gene family of pomelo, while quantitative real-time PCR (qRT-PCR) was employed to validate and analyze the expression of CmAPXs at different stages of fruit postharvest. This study identified 96 members of the CmAPX family in the entire pomelo genome, with uneven distribution across nine chromosomes and occurrences of gene fragment replication. The subcellular localization includes peroxisome, cytoplasm, chloroplasts, and mitochondria. The CmAPX family exhibits a similar gene structure, predominantly consisting of two exons. An analysis of the upstream promoter regions revealed a significant presence of cis-acting elements associated with light (Box 4, G-Box), hormones (ABRE, TCA-element), and stress-related (MBS, LTR, ARE) responses. Phylogenetic and collinearity analyses revealed that the CmAPX gene family can be classified into three subclasses, with seven collinear gene pairs. Furthermore, CmAPXs are closely related to citrus, pomelo, and lemon, followed by Arabidopsis, and exhibit low homology with rice. Additionally, the transcriptomic heat map and qPCR results revealed that the expression levels of CmAPX57, CmAPX34, CmAPX50, CmAPX4, CmAPX5, and CmAPX81 were positively correlated with granulation degree, indicating the activation of the endogenous stress resistance system in pomelo cells by these genes, thereby conferring resistance to ROS. This finding is consistent with the results of GO enrichment analysis. Furthermore, 38 miRNAs were identified as potential regulators targeting the CmAPX family for post-transcriptional regulation. Thus, this study has preliminarily characterized members of the APX gene family in pomelo and provided valuable insights for further research on their antioxidant function and molecular mechanism.

Development of a CRISPR/Cas9-mediated gene knockout method for functional genomics of the barley spot blotch pathogen Bipolaris sorokiniana

Bipolaris sorokiniana (=Cochliobolus sativus) is an important fungal pathogen that causes spot blotch, common root rot and kernel blight in barley and wheat. In this study, we aimed to develop a CRISPR/Cas9-mediated approach for efficiently knocking out genes of B. sorokiniana. We first assessed the efficiency of Cas9/sgRNA combined with the split marker system for gene replacement. We designed sgRNAs to target a polyketide synthase gene (PKS1) that is required for melanin biosynthesis of the fungus. When the preassembled Cas9/gRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments each harboring 449 bp upstream and 595 bp downstream flanking sequences of PKS1 were co-transformed into the protoplasts of B. sorokiniana, the number of transformants with the PKS1 gene replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs with PCR-amplified HygB gene cassette carrying 40 bp or 60 bp arms homologous to the flanking sequences of the PKS1 target site for fungal transformation and showed that the RNPs significantly enhanced gene disruption efficiency through the short-homology recombination, and more gene knockout mutants were obtained with longer (60 bp) homologous arms than the shorter (40 bp) ones. Finally, we disrupted an uncharacterized non-ribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed the mutants lost virulence on barley cv. Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.

Alphacoronaviruses from bats captured in European Russia in 2015 and 2021 are closely related to those of Northern Europe

IntroductionBats are considered the natural reservoirs of several viruses including coronaviruses (CoVs), two genera of which, alpha- and betacoronaviruses, infect humans. Despite widespread screening of bat samples for novel viruses, information on the diversity of coronaviruses in bats inhabiting Russian territory remains scarce. Here, we analyzed the presence and diversity of alphacoronaviruses (Alpha-CoVs) in bats from European Russia.MethodsFourty three fecal samples from bats of 8 species: P. nathusii, P. kuhlii, M. brandtii, M. daubentonii, N. noctula, V. murinus, M. dasycneme, and P. auritus were taken to study.Results and discussionWe detected Alpha-CoV RdRp gene fragments in 30% (13/43) of samples examined in 75% (6/8) of species sampled. Phylogenetic analysis of RdRp showed that most of the identified Alpha-CoV sequences fall into clades within the Pedacovirus subgenus, with minor clusters of nyctacoviruses or myotacoviruses. We assert that closely related pedacoviruses have been circulating for a long time (from 2015 to 2021) in a large region from European Russia to Northern Europe. We propose that closely related pedacoviruses collected from common areas represent a separate species, which we name NE-Alpha coronavirus, with its host being bats of the genus Pipistrellus that inhabit a region from the European part of Russia to Northern Europe. Among the animals sampled, 4.6% (2/43) carried two Alpha-CoVs related to different subgenera (pedacovirus/myotacovirus or pedacovirus/nyctacovirus) simultaneously. We confirmed the presence of two different Alpha-CoV subgenera related to pedacovirus and nyctacovirus in P. kuhlii captured in 2021 and kept in captivity using whole genome sequencing of these viruses. The presence of two or more coronaviruses in one individual animal host is an essential prerequisite for recombination to occur. We also obtained two Alpha-CoV whole genomes from two specimens of P. nathusii captured in 2015. The genomic organization of BatCoV/MOW15-21 and BatCoV/MOW15-23 was similar to other Alpha-CoVs, but the assembled genomes contained a long insertion in the ORF1ab gene which has not been described in other Alpha-CoVs, except for a single sequence from P. nathusii captured in the Netherlands. We propose that the insertion encodes a previously undescribed domain of unknown function, probably related to the SEA domain superfamily.

Comparison of Vaginal microbiota in HPV-negative and HPV-positive pregnant women using a culture-based approach

The purpose of this study was to conduct a comparative analysis of the composition of the dominant groups of vaginal microorganisms in healthy pregnant women and pregnant women infected with HPV using a microbiological culture-based method. The MALDI TOF MS method and 16S rRNA gene fragment sequencing were used to identify microorganisms isolated from healthy pregnant women (n=32) and pregnant women infected with HPV (n=24). It was found that vaginal secretion samples from both groups contained bacteria of 4 phyla: Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota, and Ascomycota fungi. The most common microbial community in healthy pregnant women being CST I (p=0.0007), and CST V in pregnant women infected with HPV (p=0.0001). At the genus level, a total of 25 taxa were found in all samples, with Lactobacillus being the dominant genus overall. Escherichia (p<0.0001) and Prevotella (p=0.0001) concentrations were higher in HPV infected patients. When calculating the Pearson correlation coefficient for the phyla, it was found that Bacillota correlated negatively with HPV genotypes 16 and 51 (p≤0.05), but positively with HPV genotype 59 (p≤0.05), just like Actinomycetota (p≤0.05). Bacteroidota correlated positively with HPV genotype 56 (0.001<p<0.01), and Ascomycota correlated positively with HPV genotypes 39 and 51 (p≤0.05; 0.001<p<0.01). Pearson correlation coefficients between bacteria genera and HPV genotypes were statistically significant for the following genera: Lactobacillus, Streptococcus, Enterococcus, Gardnerella, Escherichia, Prevotella. The data obtained in our study indicates that the culture-based method is informative when assessing the qualitative and quantitative composition of the microbiota.

Prevalence, molecular characterisation and phylogenetic analyses of hydatid cysts and cysticercus tenuicollis isolates and first report of E. canadensis (G6/G7) in wild boars in Bingol province, Türkiye

Wild boars can directly or indirectly transmit numerous zoonotic helminths to humans in rural communities as they serve as reservoir hosts. This study was conducted to determine the occurrence and molecular characterisation of cystic metacestode forms of helminth parasites in wild boar and to identify haplotypes. To this end, 23 wild boar carcasses shot by hunters during the 2023 hunting season were necropsied and all internal organs were examined. Cysticercus tenuicollis (n = 07) and hydatid cyst (n = 10) isolates were obtained from the examined boars. Species identification of Cysticercus and hydatid cyst isolates was performed by amplification of partial fragments of the cox1 gene. According to BLAST search, all sequences of C. tenuicollis isolates were identified as Taenia hydatigena. Out of the hydatid cyst isolates, seven were classified as Echinococcus granulosus sensu stricto (G1/G3) and one sample was identified as Echinococcus canadensis (G6/G7). All isolates of E. granulosus s.s. (G1/G3) were re-amplified with the NADH dehydrogenase subunit 5 (nad5) in order to distinguish between G1 and G3 genotypes. Based on the sequence analysis, it was found that five of the E. granulosus s.s. isolates were classified as G1, while two were classified as G3. Based on the results of this study, it can be concluded that the G1 genotype is the most prevalent genetic variant among wild boar populations in Bingol province, Türkiye. In this study, a total of five novel haplotypes were identified. A previously unidentified haplotype was revealed through the haplotype analysis of E. canadensis (G6/G7). All isolates in the haplotype network of T. hydatigena were shown to exhibit distinct and geographically specific haplotypes. According to the findings of the study, wild boars include a substantial amount of genetic variety in E. granulosus s.s. And T. hydatigena.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang etal. 1993.

First record of the rust fungus Uromyces gageae on Yellow Star-of-Bethlehem (Gagea lutea) in the Netherlands.

Yellow Star-of-Bethlehem (Gagea lutea) is a rare and threatened bulbous plant in the Netherlands, with its largest stronghold in the northern province of Drenthe. In 2022, numerous plants within a population of G. lutea were found to be infected by a rust fungus, which was identified as Uromyces gageae based on morphological characteristics. Further examination of collected teliospores revealed differences from Uromyces acutatus, a closely related rust species known to infect Ornithogalum and Gagea species. Rust symptoms on G. lutea plants were observed within the same population in April 2023, suggesting that teliospores surviving winter conditions serve as a viable source for recurrent infection. DNA of U. gageae and U. acutatus extracted from teliospores was used to obtain partial ribosomal DNA (rDNA) gene fragments by PCR. Amplicon sequencing revealed nucleotide variation between both rust species and verified the identity of the rust fungus on G. lutea as U. gageae. This confirmation substantiates the first documentation of U. gageae in the Netherlands. This study raises new avenues for research on the distribution and host range of U. gageae, as well as additional studies on the population dynamics of this potentially rare, wild plant-rust interaction.

Larval morphology, vocalisations, and phylogenetic position of Crossodactylus dantei (Anura: Hylodidae): new insights into a little-known anuran species

Aspects of the biology and natural history of the endemic anuran Crossodactylus dantei remain unknown even almost 30 years since its description. In this study, we describe its larval stage, along with the advertisement and territorial calls, and assess its phylogenetic positioning based on a fragment of the 16S rRNA mitochondrial gene. The larval stage of C. dantei follows the morphological pattern of Hylodidae, corroborating the relatively uniform larval morphology of the family. However, it is still possible to observe characters that distinguish C. dantei from other species in the genus, such as the elliptical body in dorsal view, nostrils located closer to the snout than to the eyes, an elongated pre-nasal arena, and the absence of taenia tecti medialis and taenia tecti transversalis in the chondrocranium. Its advertisement call is characterised by a sequence of about 23 pulsed notes, with ascending amplitude modulation, and has a mean duration of 2.5 seconds, with a dominant frequency of 3356 Hz. The territorial call has a mean duration of 23 seconds, a dominant frequency of 3160 Hz, and two distinct types of notes. Phylogenetic analysis indicated the existence of two main lineages in the genus. Crossodactylus dantei was recovered with high statistical support as a sister species to the clade composed of C. gaudichaudii and C. timbuhy. Furthermore, the group formed by these three species was recovered as the sister lineage of C. trachystomus. In summary, the information presented here represents a significant advance in the knowledge of this enigmatic and rare species of the northern Atlantic Forest, providing baseline data for future ecological and evolutionary studies of this unique frog. Keywords: tadpole, oral anatomy, chondrocranium, calls, phylogeny, Atlantic Forest

Island hitchhikers: pathogen agents of Madeira and Azores ticks

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.