Partial Cox1 Research Articles

Mitogenomic analysis of the position of the Azygiidae and constituent genera, with a new species of Azygia

The Azygiidae Looss, 1899 is a family of digeneans with a Holarctic distribution in which members of some genera mature in marine elasmobranchs while others occur only in freshwater teleosts. Some have questioned whether the marine genus Otodistomum Stafford, 1904 indeed belongs to the same family as the freshwater azygiid genera, namely Azygia Looss, 1899, Proterometra Horsfall, 1933, and Leuceruthrus Marshall and Gilbert, 1905. We present phylogenetic analyses based on mitochondrial genomes, rDNA operons, and partial Cox1 cytochrome c oxidase I sequences from North American and Asian species that support the monophyly of Azygiidae, and placement of Azygioidea in the suborder Hemiurata, in contrast to recently published mitochondrial genome phylogenies. All phylogenies indicate that Azygia includes Leuceruthrus, which we therefore propose to be a junior synonym, together with suppression of the Leuceruthrinae. The status of Proterometra was equivocal, with support in some but not all analyses of 28S, but not in other markers. We describe a new species of Azygia from northeastern North America. Our results confirm the morphological variability of adults in Azygia, with worm size positively correlated with host size in Azygia angusticauda. Phylogenies suggest a marine origin for the Azygiidae, and a Palearctic origin for freshwater azygiids, with a single trans-Atlantic radiation to the Nearctic.

Prevalence and genetic diversity of the lung nematode Eucoleus aerophilus in red foxes (Vulpes vulpes) in Central Europe (Poland) assessed by PCR and flotation

Eucoleus aerophilus is the causative agent of respiratory capillariosis in wild and domestic carnivores and has sporadically affected humans. To date, this parasite has been detected in 38 countries, confirming its worldwide distribution. The aim of this study was to evaluate the prevalence of E. aerophilus in red fox populations from Central Europe and to describe the sequence variations in the partial cox1 gene of this parasite recovered from stool samples. An investigation was carried out using 342 samples of red foxes faeces investigated via seminested PCR and coproscopy. PCR results confirmed the presence of E. aerophilus DNA in 230 samples and E. boehmi DNA in 14 samples. Molecular analysis of the retrieved sequences revealed 22 haplotypes of E. aerophilus, EaPL1–EaPL22, and 4 haplotypes of E. boehmi, EbPL1–EbPL4. Coproscopic examination revealed that eggs of the Capillariidae family were most prevalent in all regions, with a mean prevalence of 73%. Furthermore, the following eggs were detected: Taeniidae (21.3%), Toxocara spp. (26.3%), Toxascaris leonina (5.3%), Trichuris vulpis (1.5%), trematodes (23.4%), hookworms (2.3%) and Mesocestoides spp. (1.8%). This study evaluated the prevalence of E. aerophilus in red fox populations in selected regions of Poland. To the best of our knowledge, this is the first molecular study on E. aerophilus in Poland.

Microscopic detection and molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa): first report from Greece.

The genus Sarcocystis includes protozoan parasites with an indirect life cycle. Sarcocystis spp. can infect various animal species and humans, causing sarcocystosis, a parasitosis of economic importance and zoonotic concern. Wild boars can act as intermediate hosts for Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis that infects humans by consumption of raw or undercooked infected swine meat. In the present study, the diaphragmatic muscle tissue of 123 wild boars hunted in Greece was examined to determine the frequency of Sarcocystis spp. The samples were examined by tissue compression and molecular techniques. Under light microscopy, 34 out of 123 (27.6%) wild boars tested positive for Sarcocystis spp., while a higher infection prevalence (75%) was revealed by multiplex PCR performed in 100 of the samples. The partial mtDNA cox1 gene (~ 1100bp) of 20 samples tested positive for S. miescheriana by multiplex PCR was amplified and sequenced. Sarcocystis miescheriana was identified as the only species involved in these infections. This is the first study on the prevalence of Sarcocystis spp. in wild animals in Greece. Further, large-scale surveys are needed to assess the prevalence and species of this parasite in Greece and to design efficient control and preventive measures in a One Health perspective.

Population Genetic Structure of Sargassum horneri, the Dominant Species of Golden Tide in the Yellow Sea

Sargassum horneri golden tides are increasingly becoming a marine ecological problem in the Yellow Sea (YS) and East China Sea. To understand the genetic relationship between the attached S. horneri along the China coast and the floating biomass in the YS, we used partial rbcL, ITS2, cox1, cox3, and cob-cox2 to analyze the population genetic evolution of 165 Sargassum samples. The results showed that all samples were a single species of S. horneri. Partial sequences of each gene had major haplotypes, and other haplotypes evolved from the occurrence of base mutations. The cob-cox2 gene haplotype research showed only the attached samples from ZJ, LN, and KR contained Hap3, and the distribution proportions of Hap2 and Hap4 in SS and the YS were closest to the distribution of the attached samples from SD. These novel findings provided information about the genetic evolutionary relationship between attached S. horneri along the coast of China and floating S. horneri in the YS, as well as new ideas for tracing the source of floating S. horneri in the YS from a molecular biological perspective.

Uncovering further diversity of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) in South American bats

Ochoterenatrema Caballero, 1943 is a genus of lecithodendriid digeneans that prior to this study included 8 species parasitic in bats in the Western Hemisphere. Species of Ochoterenatrema possess a unique morphological feature in form of the pseudogonotyl on the sinistral side of the ventral sucker. In this study, we describe 2 new species of Ochoterenatrema from bats in Ecuador. The new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles, length of oesophagus, sucker ratio and the body shape, among other features. We have generated partial nuclear 28S rDNA and mitochondrial cox1 gene DNA sequences from both new species. The newly obtained sequences were used to differentiate among species and study the phylogenetic interrelationships among Ochoterenatrema spp. The internal topology of the clade was weakly supported, although the cox1 tree was much better resolved than the 28S tree. Comparison of sequences revealed 0–1.2% interspecific divergence in 28S and 3.3–20.5% interspecific divergence in cox1 among Ochoterenatrema spp. The new findings demonstrate that bats in South America likely harbor multiple additional undescribed species of Ochoterenatrema. More extensive sampling from broader geographic and host ranges, especially in North America, should allow for a better understanding of the evolution of host associations and morphological traits of this lineage of lecithodendriid digeneans.

Description of Rhipicephalus hibericus sp. nov. (Ixodoidea: Ixodidae), a species of the Rhipicephalus sanguineus group in southwestern Europe

We describe all the life stages of Rhipicephalus hibericus n. sp., provide the types, and present molecular support for a new species of the Rhipicephalus sanguineus sensu lato group, present in southwestern Europe, that has been historically confused with Rhipicephalus turanicus Pomerantzev, 1940. A new name is proposed for this taxon because it was impossible to ascribe to types of already described species in the group, deposited for more than 100 years in natural history institutions. The males have a dorsum showing deep and coarse punctations (absent in Rhipicephalus sanguineus sensu stricto) and adanal plates with large punctations (absent in R. sanguineus s.s.); the tail of the spiracular plate is as wide as the closest festoon (half the width in R. sanguineus s.s.). Females have large punctations in dorsal fields, a wide spiracular plate, and a “V” shaped genital opening; such a combination of characters cannot be found in other species of the group. Immatures are described from specimens collected on hosts (Rodentia and Eulipotyphla). Both larvae and nymphs are markedly smaller than R. sanguineus s.s. Nymphs display long, backward-projected auriculae; larvae are almost half the size of R. sanguineus s.s. The new species can hybridize with R. sanguineus s.s. in laboratory colonies producing an unfertile F2, laying brown and dry eggs that did not hatch. Phylogenetic analysis of partial coxI gene sequences placed R. hibericus in a well-supported clade with other sequences of R. sanguineus s.l. from Portugal, as a sister clade of R. sanguineus s.s. The new species does not belong to the R. turanicus group of species. Both 12S and 16S partial gene sequences were not as precise in the correct phylogenetic placement of R. hibericus, in part probably due to the existence of erroneously identified sequences in GenBank©. This description, together with the previous reinstatement of Rhipicephalus secundus and Rhipicephalus rutilus, and the description of the neotypes of R. sanguineus s.s. should help researchers to adequately identify their collections. Our findings demonstrate that R. turanicus is absent in southwestern Europe. Old collections should be re-examined to provide the actual range of the new species.

Molecular phylogenetic analysis of Echinococcus granulosus sensu lato infecting sheep in Italy.

Cystic Echinococcosis (CE), caused by the larval form of Echinococcus granulosus sensu lato, is a neglected zoonosis still threatening public health worldwide. In Italy different epidemiological scenarios were reported depending on the geographical area and associated socio-economic activities. Although in northern Italy the occurrence of E. granulosus is considered sporadic, in the southern regions and, particularly in Sardinia, CE prevalence reaches high levels. We analysed CE cysts collected from infected sheep from various areas of mainland Italy and the Sardinia island, with the main objective to investigate intergenotypic and intragenotypic variations at national level. CE cysts were collected from slaughtered sheep following post mortem inspection at local abattoirs. Total genomic DNA was extracted and amplification and sequencing of the partial mitochondrial genes nad5 and cox1 were performed. A Bayesian phylogenetic tree was estimated on a nad5 dataset (n=260) composed of E. granulosus samples from this study (n=126) and all the nad5 haplotypes available in GenBank (n=134). In addition, haplotype network, diversity and neutrality analysis were performed on nad5 and cox1 sequences of Italian origin obtained in this study. E. granulosus sensu stricto (s.s.) was found to be the only Echinococcus species infecting sheep in Italy, mainly represented by G1 genotype (76%) and, to a lower extent, by G3 genotype (24%). Phylogenetic analyses revealed 40 nad5 and 33 cox 1 haplotypes, and the presence of two founder haplotypes, belonging to G1 and G3 genotype, showing 100% similarity with DNA sequences from different geographic regions. The lack of geographical segregation, high haplotype and low nucleotide diversity coupled with significant negative values of Tajima's D and Fu's Fs observed in this study indicated high genetic variation and demographic expansion of E. granulosus s.s. in Italy.

A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov.

BackgroundThe geographic distribution and host-parasite interaction networks of Sarcocystis spp. in small mammals in eastern Asia remain incompletely known.MethodsExperimental infections, morphological and molecular characterizations were used for discrimination of a new Sarcocystis species isolated from colubrid snakes and small mammals collected in Thailand, Borneo and China.ResultsWe identified a new species, Sarcocystis muricoelognathis sp. nov., that features a relatively wide geographic distribution and infects both commensal and forest-inhabiting intermediate hosts. Sarcocystis sporocysts collected from rat snakes (Coelognathus radiatus, C. flavolineatus) in Thailand induced development of sarcocysts in experimental SD rats showing a type 10a cyst wall ultrastructure that was identical with those found in Rattus norvegicus from China and the forest rat Maxomys whiteheadi in Borneo. Its cystozoites had equal sizes in all intermediate hosts and locations, while sporocysts and cystozoites were distinct from other Sarcocystis species. Partial 28S rRNA sequences of S. muricoelognathis from M. whiteheadi were largely identical to those from R. norvegicus in China but distinct from newly sequenced Sarcocystiszuoi. The phylogeny of the nuclear 18S rRNA gene placed S. muricoelognathis within the so-called S. zuoi complex, including Sarcocystisattenuati, S. kani, S. scandentiborneensis and S. zuoi, while the latter clustered with the new species. However, the phylogeny of the ITS1-region confirmed the distinction between S. muricoelognathis and S. zuoi. Moreover, all three gene trees suggested that an isolate previously addressed as S. zuoi from Thailand (KU341120) is conspecific with S. muricoelognathis. Partial mitochondrial cox1 sequences of S. muricoelognathis were almost identical with those from other members of the group suggesting a shared, recent ancestry. Additionally, we isolated two partial 28S rRNA Sarcocystis sequences from Low’s squirrel Sundasciurus lowii that clustered with those of S. scandentiborneensis from treeshews.ConclusionsOur results provide strong evidence of broad geographic distributions of rodent-associated Sarcocystis and host shifts between commensal and forest small mammal species, even if the known host associations remain likely only snapshots of the true associations.Graphical

Two new species of freshwater crabs of the genus Bottapotamon Türkay & Dai, 1997 (Decapoda: Brachyura: Potamidae) from China

Abstract We report on two new species of BottapotamonTürkay & Dai, 1997: B. lushanensesp. nov. from Lushan Mountain, Jiujiang City, Jiangxi Province and B. yongzhouensesp. nov. from Ningyuan County, Yongzhou City, Hunan Province, China. The two new species are morphologically compared with the other eight congeners, and can be distinguished by characters of the carapace and the male first gonopods. A phylogenetic tree was produced based on sequences of partial COX1. The establishment of the two new species was supported by both the results of the molecular analyses and the morphological descriptions,. This study brings the number of Bottapotamon species to ten.

Detection of Sarcocystis hominis, Sarcocystis bovifelis, Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis sigmoideus sp. nov. in carcasses affected by bovine eosinophilic myositis

Bovine eosinophilic myositis is an inflammatory myopathy characterized by multiple focal or diffuse grey to green patches leading to condemnation of affected carcasses. Although its etiology is still uncertain, there is evidence that Sarcocystis species may play a role in the development of eosinophilic myositis. The goal of the present study was to identify Sarcocystis spp. in intralesional and extralesional tissues of condemned cattle carcasses, in order to evaluate the possible role of different bovine Sarcocystis spp. in the etiology of bovine eosinophilic myositis. Muscle samples (n = 100) of 26 affected carcasses were collected in Northern Italy. One to five samples with lesions and two aliquots of tissue without lesions were collected from each carcass; lesions were grossly categorized in green focal lesions and green diffuse patches. Genomic DNA was extracted and analyzed by multiplex-PCR targeting different Sarcocystis spp. Unidentified species were characterized morphologically (light microscopy, histology), ultrastructurally (scanning and transmission electron microscopy) and on the molecular level (complete 18S rRNA gene and partial cox1 gene sequencing). A bovine eosinophilic myositis prevalence of 0.017% was visually assessed by routine carcass inspection between 2014 and 2019 in Italy (184/1,108,150 slaughtered cattle). Out of 26 carcasses, 25 revealed the presence of at least one Sarcocystis species (96.2%). The presence of Sarcocystis spp. DNA was significantly more frequent in intralesional than in extralesional samples. Considering the different species, Sarcocystis bovifelis and Sarcocystis hominis were significantly more frequent in intralesional (41.7% and 50%, respectively) than in extralesional samples (1.9% and 15.4%, respectively), while there was no significant difference between the presence of Sarcocystis cruzi and Sarcocystis hirsuta in intralesional (27.1% and 2.1%, respectively) and extralesional (30.8% and 1.9%, respectively) samples. The presence of an unnamed Sarcocystis sp. showing thick-walled (3.7–5.4 μm) cysts with densely packed, flattened, undulating and narrow protrusions, which showed an S-shape in side view, was recorded in the diaphragm of two carcasses. Genomic DNA from individual sarcocysts isolated from the diaphragm was successfully amplified and further sequenced. Sequence comparison revealed <94.6% and 83.4% identity at 18S rRNA and cox1 genes, respectively, with other named Sarcocystis spp., while the phylogenetic analysis clearly separated the unnamed Sarcocystis sp. from the other Sarcocystis spp. using cattle as intermediate hosts. The present study contributes to the understanding of the importance of different Sarcocystis spp. in the pathogenesis of bovine eosinophilic myositis. The results emphasize the association of Sarcocystis hominis and Sarcocystis bovifelis with bovine eosinophilic myositis and highlight the presence of a new Sarcocystis sp. using cattle as intermediate hosts. The name Sarcocystis sigmoideus sp. nov. is proposed for the newly described Sarcocystis species.

Molecular Characterization of Spirometra erinaceieuropaei from Jungle Cat (Felis chaus) in North of Iran.

The aim of this study is to conduct a molecular characterization of Spirometra tapeworm from jungle cat (Felis chaus) in Guilan Province, north of Iran using DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) and 12S rDNA sequences. Morphological features of the adult tapeworm of Spirometra were evaluated using specific staining and light microscopy. The molecular characterization was performed using partial Cox1 and 12S rDNA regions. Genetic diversity was calculated and phylogenetic trees of the obtained sequences were constructed. Morphological features were compatible with previous description of adult Spirometra erinaceieuropaei. The Cox1 sequence of the specimen showed 100% similarity with S. erinaceieuropaei sequences in GenBank from Korea, China and Iran. Also, the 12S rDNA sequence revealed 99.7% similarity with S. erinaceieuropaei isolates from China and Japan. Intra-species variation within isolates of S. erinaceieuropaei was 0-1.4% and 0-4.6% for Cox1 and 12S rDNA genes, respectively. This is the first report of molecular characterization of S. erinaceieuropaei in jungle cat, F. chaus in Iran. Jungle cat probably plays a major role as reservoir host in maintaining of this parasite in this area with favorable climate condition. Needs for further assessment on the role of appropriate hosts, especially intermediate/paratenic hosts as well as the potential risk of human infectivity with sparganosis is emphasized.

American mitogenome reference for the tropical brown dog tick, Rhipicephalus linnaei (Audouin, 1826)

The brown dog tick, Rhipicephalus linnaei (Audouin, 1826), is distributed across the American continent and is formerly known as the “tropical lineage”. It belongs to the Rhipicephalus sanguineus (Latreille, 1806) species complex, referred to as R. sanguineus (sensu lato). Mitochondrial genome sequences are frequently used for the identification and represent reference material for field studies. In the present study, the entire mitochondrial genomes of R. linnaei (∼15 kb) collected from dogs in Mexico were sequenced and compared with available mitogenomes of R. sanguineus (s.l.). The mitochondrial genome is ∼90% identical to the reference genome of R. sanguineus (sensu stricto, former “temperate lineage”) and > 99% identical to R. linnaei mitogenome derived from the neotype. Two additional mitogenomes were obtained and described as R. linnaei and R. turanicus from dogs in Saudi Arabia. The present study delivers a molecular reference for R. linnaei from America and complements R. linnaei mitogenomes from Africa, Asia and Australia. We propose to consider the complete mitogenome, as the reference for American R. linnaei, even when partial mitochondrial cox1, 12S rRNA or 16S rRNA genes are characterised.

Fatal neural angiostrongyliasis in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis) leading to defining Angiostrongylus cantonensis risk map at a zoo in Australia

Neural angiostrongyliasis (NA) is a parasitic disease caused by Angiostrongylus cantonensis (rat lungworm). This study presents a case of NA in a captive Bolivian squirrel monkey from a zoo in western Sydney, Australia. The objective was to identify the A. cantonensis cox1 haplotype responsible for the infection and compare its mitochondrial DNA (mtDNA) to known Australian mtDNA. An epidemiological investigation was conducted to assess the risk of infection, focusing on the resident rat population in the zoo. Methods involved trapping rats and collecting rat faeces for Angiostrongylus detection, speciation, and cox1 haplotype confirmation. Various techniques were employed, including necropsy, morphological examination, and molecular methods such as ITS-2 qPCR, cox1 sequencing, and ITS-2 metabarcoding. Cluster analysis of rat faeces distribution and Angiostrongylus detection utilised an equal sampling effort (ESE) approach. Gastropods were collected throughout the study for Angiostrongylus surveillance using a hypersensitive qPCR assay. Results revealed significant clustering of rat faeces near exhibits with fresh food provision and absence of predators. Angiostrongylus-positive faeces were uniformly distributed across the zoo property. Mitochondrial DNA analysis confirmed the presence of the Ac13 haplotype of A. cantonensis in the monkey. Morphology, ITS-2 metabarcoding and partial cox1 sequencing detected only A. cantonensis, with the Ac13 cox1 haplotype predominating. A high prevalence of infection (64%, 9/14) was found in brown rats, with quantification of larvae indicating high shedding rates. Co-infections with both Ac13 and local SYD.1 A. cantonensis cox1 haplotypes were observed. Only three gastropods (all of which were Angiostrongylus-negative) were found in the survey. To minimise the risk of exposure for susceptible species, targeted rodent control was implemented in areas with higher exposure risk. A potential strategy (which requires further exploration) to consider for future zoo design was suggested. This study provides insights into the epidemiology and genetic diversity of A. cantonensis in Australia, emphasising the importance of control measures to prevent future outbreaks.

Exploring gene sequences and phylogenetic relationships of four terrestrial planarian species (Platyhelminthes; Tricladida; Geoplanidae) in Europe.

Partial mitochondrial cox1 gene sequences from four recently recognised European species of terrestrial planarians, and ribosomal ITS1 sequences for two of them, are presented: Marionfyfea adventor, Artioposthia exulans (both introduced from New Zealand), Australopacifica atrata (from Australia) and specimens putatively identified as Microplana edwardsi, presumed to be native to the UK. The sequences are compared with those from other terrestrial planarian species and analysed phylogenetically. Results indicate that the sister group of M. adventor comprises a clade constituted by at least the genus Arthurdendyus. The phylogenetic position of Ar. exulans remains less certain, Australopacifica atrata might be closely related to the species Parakontikia ventrolineata and Endeavouria septemlineata. The specimens of M. cf. edwardsi are distinct from all other Microplana species for which sequences are available.

The systematic position of Cryptopotamonanacoluthon (Kemp, 1918), with the description of a new species of Sinolapotamon Tai & Sung, 1975 (Crustacea, Decapoda, Brachyura, Potamidae) from southern China.

The systematics of the potamid freshwater crab Cryptopotamonanacoluthon (Kemp, 1918) is clarified, and its generic position in Sinolapotamon Tai & Sung, 1975, is confirmed based on morphological comparisons, geographical information and phylogenetic analyses. A new species of Sinolapotamon, Sinolapotamoncirratumsp. nov. is described from the Guangxi Zhuang Autonomous Region of China. Sinolapotamoncirratumsp. nov. is distinguished from its congeners by the combination of characters of its carapace, third maxilliped, anterolateral margin, and unique male first gonopod. Phylogenetic analyses based on partial COX1, 16S rRNA and 28S rRNA genes also support the species as new.

Comparison between p-distance and single-locus species delimitation models for delineating reproductively tested strains of pennate diatoms (Bacillariophyceae) using cox1, rbcL and ITS.

Several automated molecular methods have emerged for distinguishing eukaryote species based on DNA sequence data. However, there are knowledge gaps around which of these single-locus methods is more accurate for the identification of microalgal species, such as the highly diverse and ecologically relevant diatoms. We applied genetic divergence, Automatic Barcode Gap Discovery for primary species delimitation (ABGD), Assemble Species by Automatic Partitioning (ASAP), Statistical Parsimony Network Analysis (SPNA), Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP) using partial cox1, rbcL, 5.8S + ITS2, ITS1 + 5.8S + ITS2 markers to delineate species and compare to published polyphasic identification data (morphological features, phylogeny and sexual reproductive isolation) to test the resolution of these methods. ASAP, ABGD, SPNA and PTP models resolved species of Eunotia, Seminavis, Nitzschia, Sellaphora and Pseudo-nitzschia corresponding to previous polyphasic identification, including reproductive isolation studies. In most cases, these models identified diatom species in similar ways, regardless of sequence fragment length. GMYC model presented smallest number of results that agreed with previous published identification. Following the recommendations for proper use of each model presented in the present study, these models can be useful tools to identify cryptic or closely related species of diatoms, even when the datasets have relatively few sequences.

Oswaldocruzia ukrainae (Nematoda: Molineidae)-A Parasite of European Green Toad Bufotes viridis: Morphological and Molecular Data.

Nematodes of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent molecular analysis of Oswaldocruzia nematodes revealed that only Oswaldocruzia filiformis, which possesses high morphological variability, parasitizes amphibians and reptiles in European Russia. Here we present the study of Oswaldocruzia nematodes from the European green toad Bufotes viridis (Anura, Bufonidae) collected at different localities of the Middle Volga region in 2018-2022. We analyzed the morphological characteristics of the Oswaldocruzia spp. taxonomy together with novel molecular phylogenetic data. The data on phylogenetic analysis (based on partial CoxI mtDNA gene sequences) showed that Bufotes viridis is parasitized by two Oswaldocruzia species, the host-specific parasite Oswaldocruzia ukrainae and species generalist Oswaldocruzia filiformis. Broad morphological variability was revealed in O. ukrainae nematodes both from the same host specimen and from various toad individuals from different localities. Our results highlight the need for further biodiversity research of morphologically similar Oswaldocruzia species from amphibians and reptiles in the Western Palearctic using molecular genetic methods.

Non-invasive investigation of Polychromophilus parasite infections in bat populations in Serbia using bat flies

BackgroundHaemosporidian parasites of the genus Polychromophilus infect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only five Polychromophilus morphospecies have been described to date. The two predominant species, Polychromophilus melanipherus and Polychromophilus murinus, are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of either Polychromophilus species to infect other host families is poorly characterized.MethodsWe collected 215 bat flies from two bat species, Miniopterus schreibersii and Rhinolophus ferrumequinum, which sometimes form mixed clusters in Serbia. Miniopterus schreibersii is known to be frequently infected with P. melanipherus, whereas R. ferrumequinum has been observed to be incidentally infected with both Polychromophilus species. All flies were screened for Polychromophilus infections using a PCR targeting the haemosporidian cytb gene. Positive samples were subsequently sequenced for 579 bp of cytochrome b (cytb) and 945 bp of cytochrome oxidase subunit 1 (cox1).ResultsPolychromophilus melanipherus DNA was detected at six out of nine sampling locations and in all three examined bat fly species collected from M. schreibersii (Nycteribia schmidlii, n = 21; Penicillidia conspicua, n = 8; Penicillidia dufourii, n = 3). Four and five haplotypes were found for cytb and cox1, respectively. Evidence for multiple Polychromophilus haplotypes was found in 15 individual flies. These results point to a high diversity of P. melanipherus parasites in Miniopterus hosts and efficient transmission throughout the study area. A single Phthiridium biarticulatum bat fly collected from R. ferrumequinum screened positive for P. melanipherus, but only yielded a partial cox1 sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite.ConclusionsThe results of this study provide new insights into the prevalence and distribution of Polychromophilus parasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation of Polychromophilus infections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats.Graphical

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid

Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

Mitogenomic and nuclear ribosomal transcription unit datasets support the synonymy of Paragonimus iloktsuenensis and P. ohirai (Paragonimidae: Platyhelminthes).

The complete mitogenome (mtDNA) of nominal Paragonimus iloktsuenensis (Paragonimidae: Trematoda) and the nuclear ribosomal transcription unit (rTU) coding region (rTU*: from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene, excluding the external spacer region) of this species and of P. ohirai were obtained and used to further support the previously suggested synonymy of these taxa in the P. ohirai complex. The complete mitogenome of P. iloktsuenensis was 14,827bp long (GenBank: ON961029) and nearly identical to that of P. ohirai (14,818bp; KX765277), with a 99.12% nucleotide identity. The rTU* was 7543bp and 6932bp in these two taxa, respectively. All genes and spacers in the rTU were identical in length, with exception of the first internal transcribed spacer, which contained multiple tandem repeat units (6.7 for P. iloktsuenensis and 5.7 for P. ohirai). There was near 100% identity for the rTU genes. The phylogenetic topology inferred from the mtDNA and from individual gene regions (partial cox1 of 387bp and the ITS-2 of 282bp - 285bp) indicated a very close relationship consistent with synonymy of P. iloktsuenensis and P. ohirai. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics of the genus Paragonimus and family Paragonimidae.