Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Insights into the Geographical Origins of the Cabo Verde Green Monkey.

The green monkey Chlorocebus sabaeus, L. 1766, native to West Africa, was introduced to the Cabo Verde Archipelago in the 16th century. Historical sources suggest that, due to the importance of Cabo Verde as a commercial entrepôt in the Atlantic slave trade, establishing the precise place of origin of this introduced species is challenging. Non-invasive fecal samples were collected from feral and captive green monkey individuals in Cabo Verde. Two mitochondrial fragments, HVRI and cyt b, were used to confirm the taxonomic identification of the species and to tentatively determine the geographic origin of introduction to the archipelago from the African continent. By comparing the new sequences of this study to previously published ones, it was shown that Cabo Verde individuals have unique haplotypes in the HVRI, while also showing affinities to several populations from north-western coastal Africa in the cyt b, suggesting probable multiple sources of introduction and an undetermined most probable origin. The latter is consistent with historical information, but may also have resulted from solely using mtDNA as a genetic marker and the dispersal characteristics of the species. The limitations of the methodology are discussed and future directions of research are suggested.

The A2 haplotype of Echinococcus multilocularis is the predominant variant infecting humans and dogs in Yili Prefecture, Xinjiang.

Alveolar echinococcosis (AE), caused by Echinococcus multilocularis, is an important zoonotic disease. Yili Prefecture in Xinjiang is endemic for AE, however the molecular variability of E. multilocularis in this region is poorly understood. In this study, 127 samples were used for haplotypes analysis, including 79 tissues from humans, 43 liver tissues from small rodents, and 5 fecal samples from dogs. Genetic variability in E. multilocularis was studied using complete sequences of the mitochondrial (mt) genes of cytochrome b (cob), NADH dehydrogenase subunit 2 (nad2), and cytochrome c oxidase subunit 1 (cox1), using a total of 3558 bp per sample. The Asia haplotype 2 (A2) was the dominant haplotype, with 72.15% (57/79) prevalence in humans, 2.33% (1/43) in small rodents, and 80.00% (4/5) in dogs, followed by A5, the second most common haplotype, which infected 27.91% (12/43) small rodents. Haplotype network analysis showed that all haplotypes clustered together with the Asian group. Pairwise fixation index (FST) values showed lower level of genetic differentiation between different regions within the country. Compared with the sequences of E. multilocularis from North America and Europe, all concatenated sequences isolated from Yili Prefecture were highly differentiated and formed a single population. The A2 haplotype, analyzed using the cob, nad2, and cox1 genes of E. multilocularis, is the predominant variant in humans and dogs in Yili Prefecture.

Larger particle size distribution of environmental RNA compared to environmental DNA: a case study targeting the mitochondrial cytochrome b gene in zebrafish (Danio rerio) using experimental aquariums.

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (ß) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Molecular characterization of Haemoproteus enucleator with emphasis on the host and geographic distribution.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan and highly diverse blood parasites of birds that have been neglected in avian medicine. However, recent discoveries based on molecular diagnostic markers show that these pathogens often cause marked damage to various internal organs due to exo-erythrocytic development, sometimes resulting in severe and even lethal avian haemoproteosis, including cerebral pathologies. Molecular markers are essential for haemoproteosis diagnostics, but the data is limited, particularly for parasites transmitted in tropical ecosystems. This study combined microscopic and molecular approaches to characterize Haemoproteus enucleator morphologically and molecularly. Blood samples were collected from the African pygmy kingfisher Ispidina picta in Cameroon, and the parasite was identified using morphological characters of gametocytes. The analysis of partial cytochrome b sequences (cytb) identified a new Haemoproteus lineage (hISPIC03), which was linked to the morphospecies H. enucleator. Illustrations of blood stages were provided and the phylogenetic analysis showed that the new lineage clustered with five other closely related lineages belonging to the same morphospecies (hALCLEU01, hALCLEU02, hALCLEU03, hISPIC01, and hALCQUA01), with a maximum genetic distance between these lineages of 1.5 % (7 bp difference) in the 478 bp cytb sequences. DNA haplotype network was developed and identified geographic and host distribution of all lineages belonging to H. enucleator group. These lineages were almost exclusively detected in African kingfishers from Gabon, Cameroon, South Africa, and Botswana. This study developed the molecular characterization of H. enucleator and provides opportunities for diagnostics of this pathogen at all stages of its life cycle, which remains undescribed in all its closely related lineages.

Molecular phylogenetic relationships based on mitochondrial genomes of novel deep-sea corals (Octocorallia: Alcyonacea): Insights into slow evolution and adaptation to extreme deep-sea environments.

A total of 10 specimens of Alcyonacea corals were collected at depths ranging from 905 m to 1 633 m by the manned submersible Shenhai Yongshi during two cruises in the South China Sea (SCS). Based on mitochondrial genomic characteristics, morphological examination, and sclerite scanning electron microscopy, the samples were categorized into four suborders (Calcaxonia, Holaxonia, Scleraxonia, and Stolonifera), and identified as 9 possible new cold-water coral species. Assessments of GC-skew dissimilarity, phylogenetic distance, and average nucleotide identity (ANI) revealed a slow evolutionary rate for the octocoral mitochondrial sequences. The nonsynonymous ( Ka) to synonymous ( Ks) substitution ratio ( Ka/ Ks) suggested that the 14 protein-coding genes (PCGs) were under purifying selection, likely due to specific deep-sea environmental pressures. Correlation analysis of the median Ka/ Ks values of five gene families and environmental factors indicated that the genes encoding cytochrome b (cyt b) and DNA mismatch repair protein ( mutS) may be influenced by environmental factors in the context of deep-sea species formation. This study highlights the slow evolutionary pace and adaptive mechanisms of deep-sea corals.

A new cavefish of Sinocyclocheilus (Teleostei: Cypriniformes: Cyprinidae) from the Nanpanjiang River in Guizhou, China.

A new species, Sinocyclocheilus xingyiensis, is described based on specimens collected from a karst cave in Guizhou Province, China. The authors used an integrated taxonomic approach, including morphological and molecular data, to identify the new species as a member of the Sinocyclocheilu angularis group, and it can be distinguished from all other members of this group by a combination of the following features: two pairs of long barbels and long pectoral fins, 42-46 lateral-line scales, 7 (13-14) on outer (inner) side of the first gill arch and 35 (14-15 + 4 + 16 - 17) vertebrae. Phylogenetic analyses based on the cytochrome b (cyt b) gene fragment suggest that S. xingyiensis is a sister lineage to Sinocyclocheilus flexuosdorsalis. The genetic distance (Kimura 2-parameter) between the S. xingyiensis and S. angularis groups of Sinocyclocheilus species based on cyt b gene fragment ranged from 1.2% to 15.4%.

Disclosing the hidden nucleotide sequences: a journey into DNA barcoding of raptor species in public repositories.

BACKGROUND: In nucleotide public repositories, studies discovered data errors which resulted in incorrect species identification of several accipitrid raptors considered for conservation. Mislabeling, particularly in cases of cryptic species complexes and closely related species, which were identified based on morphological characteristics, was discovered. Prioritizing accurate species labeling, morphological taxonomy, and voucher documentation is crucial to rectify spurious data. OBJECTIVE: Our study aimed to identify an effective DNA barcoding tool that accurately reflects the efficiency status of barcodes in raptor species (Accipitridae). METHODS: Barcode sequences, including 889 sequences from the mitochondrial cytochrome c oxidase I (COI) gene and 1052 sequences from cytochrome b (Cytb), from 150 raptor species within the Accipitridae family were analyzed. RESULTS: The highest percentage of intraspecific nearest neighbors from the nearest neighbor test was 88.05% for COI and 95.00% for Cytb, suggesting that the Cytb gene is a more suitable marker for accurately identifying raptor species and can serve as a standard region for DNA barcoding. In both datasets, a positive barcoding gap representing the difference between inter-and intra-specific sequence divergences was observed. For COI and Cytb, the cut-off score sequence divergences for species identification were 4.00% and 3.00%, respectively. CONCLUSION: Greater accuracy was demonstrated for the Cytb gene, making it the preferred primary DNA barcoding marker for raptors.

Analysis of Genetic Diversity of cytb gene from Babesia gibsoni Isolates from Naturally Infected Dogs in Karnataka, India.

PURPOSE: The study aimed to investigate genetic diversity in Babesia gibsoni, the causative agent of canine babesiosis, and to assess the presence of atovaquone-resistant isolates in naturally infected dogs. METHODS: A total of 24 blood samples confirmed for B. gibsoni infection was subjected to PCR amplification and sequencing based on cytb gene. Genetic characterization of B. gibsoni as well as attempts to detect the point mutation rendering atovaquone resistance was carried out based on the analysis of nucleotide sequence of cytb gene using bioinformatics software. RESULTS: The findings indicated that the B. gibsoni isolates in the investigation exhibited a high nucleotide identity with the Asian genotype, ranging from 98.41 to 98.69%. Notably, none of the isolates carried cytb gene variants associated with atovaquone resistance. Phylogenetic analysis revealed clustering of most isolates with those from Japan and China, except for one isolate forming a distinct subclade. Haplotype network analysis indicated a high diversity with 22 distinct haplotypes among the B. gibsoni isolates, emphasizing the genetic variability within the studied population. CONCLUSION: In conclusion, the cytb gene exhibited remarkable conservation among the twenty-four B. gibsoni isolates studied and the study represents the first genetic diversity assessment of B. gibsoni using the cytb gene in dogs from India. These findings shed light on the genetic characteristics of B. gibsoni in the region and provide valuable insight for addressing the challenges posed by this life-threatening disease in dogs.

Population genetic characterization of Theileria annulata based on the cytochrome b gene, with genetic insights into buparvaquone susceptibility in Haryana (India).

The present investigation was aimed at population genetic characterization of Theileria annulata on the basis of the cytochrome b (cyt b) gene along with the evaluation of status of buparvaquone resistance in Haryana (India). The sequences originating from China, Egypt, India, Iran, Iraq, Tunisia, Turkey and Sudan were included in the analysis. The maximum likelihood tree based on the Tamura-Nei (TN93+G) model placed all the sequences of T. annulata into a single clade. The median-joining haplotype network exemplified geographical clustering between T. annulata haplotypes originating from each country. Only five haplotypes (7.81 %) were shared between any two countries, while the remaining 59 haplotypes (92.19 %) were singleton and unique to one country. The values of pairwise genetic distance (FST) between all the populations indicated huge genetic differentiation (> 0.25) between different T. annulata populations, barring the FST value between Iraq and Turkey (0.14454) which suggested a moderate differentiation. Contrary to the FST index, the values of gene flow (Nm) between T. annulata populations were very low. The neutrality indices and mismatch distributions indicated a population expansion in the Indian T. annulata population. Furthermore, the secondary structure and homology modeling of the partial cyt b protein is also reported. The molecular analysis of newly generated sequences for buparvaquone resistance revealed that all the isolates were susceptible to buparvaquone treatment. However, two novel mutations at positions V203I and V219I in between the Q01 and Q02 drug-binding regions of the cyt b gene were observed for the first time.

Genetic diversity and divergence among native and translocated populations of the golden flathead goby Glossogobius aureus (Gobiiformes: Gobiidae) in Philippine lakes.

The golden flathead goby Glossogobius aureus is a native species in the Philippines, Australia, Japan, Taiwan, and many other countries in Asia. In the Philippines, it is an important food fish as it is commonly caught in major lakes. In this study, a total of 307 specimens morphologically identified as G. aureus were sampled from nine major lakes in the Philippines and were sequenced for their mitochondrial cytochrome b (cyt b) gene. Two hundred sixty of the 307 cyt b sequences had sequence similarities of ≥ 99% with G. aureus reference sequence in GenBank, while the remaining 47 (all from Lake Lanao) had sequence similarities of only 95% and were thus designated as Glossogobius cf. aureus and treated as a separate population. The sequences were then analyzed to examine the pattern of genetic diversity, relatedness, divergence, and demographic history among native and translocated populations of the species. Twenty-nine haplotypes were recovered, of which four haplotypes were shared among three to seven populations. Only one haplotype each was found in the native population in Lake Buhi and translocated population in Lake Paoay. Low haplotype and low nucleotide diversities were found for the populations in Laguna de Bay, Lanao, Bato, Buhi, Paoay, and Sebu lakes, which indicate founder event for the introduced populations in Lanao, Paoay, and Sebu lakes and recent genetic bottleneck for the native populations in Laguna de Bay, Bato, and Buhi. In contrast, high haplotype but low nucleotide diversities were found for the native populations of Taal, Naujan, and Buluan lakes, signifying a recent bottleneck followed by population expansion. Pairwise FST values showed generally large (FST = 0.168-0.249) to very large (FST = 0.302-1.000) genetic divergence between populations except between Laguna de Bay and Lake Bato, Laguna de Bay and Lake Buhi, and Lake Bato and Lake Buhi populations, which showed nonsignificant genetic differentiation. Lake Buluan and Lake Sebu populations showed moderate genetic differentiation (FST = 0.098). Neutrality tests showed significant negative Tajima's D and Fu's FS values only for the population from Laguna de Bay, which suggests that the population is undergoing expansion. These results are important for establishing scientifically sound strategies for effective conservation and sustainable exploitation of G. aureus in the Philippines.

Study of Local Black Iraqi Goats Genotypes for the Cytb Gene.

Goats are the earliest domesticated ruminants. The local goat, Capra hircus, is considered one of the most important animals globally to provide good livestock production under harsh environmental conditions. This study aimed to detect the genetic structures of the local Iraqi goats bred in the central and southern regions of the country and investigate the possibility of benefiting from their genetic structures to construct improvement programs for increasing the productivity of these animals. To this end, blood samples were taken from 15 domestic black goats. A total of 10 ml of each animal's blood was placed in plastic containers of 10 ml. The DNA was extracted and sent to the laboratories of Juan Ju University, People's Republic of China, to analyze the sequences of the nitrogenous bases of the Cytochrome b (Cytb) gene. The results showed the presence of a genetic morphology for a segment of 670 base pairs for all the studied samples, and 15 sequences of this strain were recorded in the gene bank under the following accession numbers (LC496353.1:1-LC496367.1:1). The sequences of the nitrogenous bases of this segment of the gene, which were registered in the gene bank of some international goat breeds, were used for comparison with the sequences of black Iraqi goats to analyze the phylogenetic tree, calculate the genetic distance, study haplotypes, and calculate neutrality. The results showed the presence of one mutation in the studied segment of the Cytb gene, with a size of 670 bp. The mutation in base 46 of the studied gene converted from the purine group to the pyrimidine group (the shift from the nitrogen leaders A

Single nucleotide polymorphism rs527236194 of the cytochrome B gene (MT-CYB) is associated with alterations in sperm parameters.

BACKGROUND: The mitochondrial genome is substantially susceptible to mutations and has high polymorphism due to structural features, location, and lack of recombinant variability, as its inheritance is strictly maternal. All of these events can be accompanied by the accumulation of mitochondrial single nucleotide polymorphisms (mtSNPs) in the sperm. The aim of this research was to analyze the influence of mutations in the MT-CYB gene on sperm quality. METHODS AND RESULTS: We conducted a case‒control study to identify mutations in the mitochondrial cytochrome B (MT-CYB) gene in men with asthenoteratozoospermia (89 cases) and oligoasthenoteratozoospermia (65 cases). The comparison group consisted of 164 fertile men. Somatic cell lysis followed by mtDNA extraction was conducted to analyze three mtDNA polymorphisms, rs28357373 (T15629C (Leu295=), rs527236194 (T15784C (p.Pro346=), rs2853506 (A15218G, p.Thr158Ala). Detection and genotyping of polymorphic loci in the MT-CYB gene was performed using the TaqMan allelic discrimination assay. To verify mutations in the MT-CYB gene, automated Sanger DNA sequencing was used. We found that rs527236194 was associated with asthenoteratozoospermia. rs28357373 in the MT-CYB gene did not show any polymorphism in the analyzed groups, which indicates a rare frequency of the TT genotype in our region. Rs28357373 and rs2853506 are not associated with male sperm abnormalities in the Volga-Ural region. CONCLUSION: The association of the rs527236194 polymorphic variant with sperm parameter alterations suggests its role in the pathophysiology of male infertility and requires further investigation in larger samples.

Resistance risk and resistance-related point mutations in cytochrome b of florylpicoxamid in Colletotrichum scovillei.

Anthracnose caused by Colletotrichum scovillei is one of the most destructive diseases of chili worldwide. Florylpicoxamid is a new quinone inside inhibitor (QiI) fungicide, which shows intensively inhibitory activity against C. scovillei. Currently, florylpicoxamid is in the registration process to control chili anthracnose in China. This study investigated the risk of resistance and resistance genetic mechanism of C. scovillei to florylpicoxamid. Baseline sensitivity of 141C. scovillei isolates to florylpicoxamid was established with an average EC50 value of 0.2328 ± 0.0876 µg/mL. A total of seven stable florylpicoxamid-resistant mutants were obtained with resistance factors ranging from 41 to 276. The mutants showed similar or weaker traits in mycelial growth, sporulation, conidial germination and pathogenicity than their parental isolates. Generally, the resistance risk of C. scovillei to florylpicoxamid would be moderate. In addition, there was no cross-resistance between florylpicoxamid and the commercially available fungicides tested. A37V and S207L mutations in the cytochrome b protein were detected in four high-resistance and three moderate-resistance mutants, respectively, of which, S207L is a new mutation. Molecular docking showed that the two mutations conferred different resistance levels to florylpicoxamid. These results provide a new perspective for QiI fungicide-resistance mechanism and may help in the reasonable use of florylpicoxamid against chili anthracnose in the future.

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella.

BACKGROUND: Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. METHODS: We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. RESULTS: We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. CONCLUSIONS: Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.

Phylogenetic and network analysis of Pediculus humanus in Nigeria reveal the presences of clade E body lice and novel haplotypes.

An investigation was conducted for the first time to determine the prevalence and genetic diversity of human lice, for the first time in Nigeria, using conventional PCR and sequencing methods. Three mitochondrial genes, cytochrome oxidase subunit 1 (cox1), cytochrome b (cytb), and 12S rRNA of Nigerian human lice, were amplified, sequenced, and analyzed. Overall, high prevalence (72.5%; 103/142) of lice infestation was recorded among the examined volunteers. Head lice infestation was more common 63 (61.2%) than body lice infestation 34 (33.0%). Co-infestation with both head and body lice was recorded in six humans (5.8%). The Nigerian human lice specimens were placed mostly into clade A with few in clade E, including body lice for the first time. Six, three, and eight haplotypes of Nigerian human lice were obtained for the cytb, cox1, and 12S rRNA genes, respectively. Additionally, one (E51), three (A31, A32, and E5), and six (A20, A21, A23, A24, A30, and E1) novel haplotypes were recorded for cox1, cytb, and 12S rRNA, respectively, from the Nigerian specimens which were corroborated by the ML phylogenetic trees and MJ network analyses. Genetic diversity indices indicate minimal variation in the parameters analyzed among the clades of the three genes. However, a statistically significant Snn test, negative Tajima's D test for clade A (cox1 and 12S rRNA genes), and negative Fu and Li's D test in clade A for cox1 gene indicate a geographical structure and the signature of population expansion of the Nigerian human lice. The findings from this study provide additional data on the human lice structure in Africa.

Atovaquone exposure and Pneumocystis jirovecii cytochrome b mutations: French data and review of the literature.

Pneumocystis jirovecii is a transmissible fungus responsible for severe pneumonia (Pneumocystis pneumonia [PCP]) in immunocompromised patients. Missense mutations due to atovaquone selective pressure have been identified on cytochrome b (CYB) gene of P. jirovecii. It was recently shown that atovaquone prophylaxis can lead to the selection of specific P. jirovecii CYB mutants potentially resistant to atovaquone among organ transplant recipients. In this context, our objectives were to provide data on P. jirovecii CYB mutants and the putative selective pressure exerted by atovaquone on P. jirovecii organisms in France. A total of 123 patients (124 P. jirovecii specimens) from four metropolitan hospitals and two overseas hospitals were retrospectively enrolled. Fourteen patients had prior exposure to atovaquone, whereas 109 patients did not at the time of P. jirovecii detection. A 638 base-pair fragment of the CYB gene of P. jirovecii was amplified and sequenced. A total of 10 single nucleotide polymorphisms (SNPs) were identified. Both missense mutations C431T (Ala144Val) and C823T (Leu275Phe), located at the Qo active site of the enzyme, were significantly associated with prior atovaquone exposure, these mutations being conversely incidental in the absence of prior atovaquone exposure (P < 0.001). Considering that the aforementioned hospitals may be representative of the national territory, these findings suggest that the overall presence of P. jirovecii CYB mutants remains low in France.

The mutations C431T (Ala144Val) and C823T (Leu275Phe) at the cytochrome b (CYB) active site of Pneumocystis jirovecii are associated with patient prior exposure to atovaquone. Conversely, these mutations are incidental in the absence of exposure. Overall, the presence of P. jirovecii CYB mutants remains low in France.

Purifying selection leads to low protein diversity of the mitochondrial cyt b gene in avian malaria parasites.

BACKGROUND: Mitochondrial respiration plays a central role in the survival of many eukaryotes, including apicomplexan parasites. A 479-bp fragment from the mitochondrial cytochrome b gene is widely used as a barcode to identify genetic lineages of avian malaria parasites Plasmodium and related haemosporidians. Here we looked for evidence of selection in the avian Plasmodium cyt b gene, using tests of selection and protein structure modeling. We also tested for the association between cyt b polymorphism and the host specificity of these parasites. RESULTS: Based on 1,089 lineages retrieved from the Malavi database, we found that the frequency of the most conserved amino acids in most sites was more than 90%, indicating that the protein diversity of the avian Plasmodium cyt b barcode was low. The exceptions were four amino acid sites that were highly polymorphic, though the substitutions had only slight functional impacts on the encoded proteins. The selection analyses revealed that avian Plasmodium cyt b was under strong purifying selection, and no positively selected sites were detected. Besides, lineages with a wide host range tend to share cyt b protein haplotypes. CONCLUSIONS: Our research indicates that purifying selection is the dominant force in the evolution of the avian Plasmodium cyt b lineages and leads to its low diversity at the protein level. Host specificity may also play a role in shaping the low mitochondrial diversity in the evolution of avian malaria parasites. Our results highlight the importance of considering selection pressure on the cyt b barcode region and lay a foundation for further understanding the evolutionary pattern of mitochondrial genes in avian malaria.

The recent introduction of Angiostrongylus cantonensis and its intermediate host Achatina fulica into Guadeloupe detected by phylogenetic analyses.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is the main pathogen responsible for eosinophilic meningitis in humans. One of its intermediate snail hosts, Achatina fulica, was already present in many countries around the world before it appeared in the West Indies in the late 1980s. In the French territories in the Caribbean and northern South America, the first cases of human neuroangiostrongyliasis were reported in Martinique, Guadeloupe and French Guiana in 2002, 2013 and 2017, respectively. In order to better characterize angiostrongyliasis in Guadeloupe, particularly its geographical origin and route of introduction, we undertook molecular characterization of adult worms of Angiostrongylus cantonensis and its intermediate host Achatina fulica. METHODS: Genomic DNA of adult Angiostrongylus cantonensis and Achatina fulica was extracted and amplified by polymerase chain reaction (PCR) targeting the mitochondrial genes cytochrome B and C for A. cantonensis and 16S ribosomal RNA for A. fulica. The PCR products were sequenced and studied by phylogenetic analysis. RESULTS: Cytochrome B and cytochrome C molecular markers indicate a monophyletic lineage of A. cantonensis adult worms in Guadeloupe. Two sequences of A. fulica were identified. CONCLUSIONS: These results confirm the recent introduction of both Angiostrongylus cantonensis and Achatina fulica into Guadeloupe. Achatina fulica in Guadeloupe shares a common origin with those in Barbados and New Caledonia, while Angiostrongylus cantonensis in Guadeloupe shares a common origin with those in Brazil, Hawaii and Japan.