Partial DNA Sequences Research Articles

Molecular Epidemiology of Theileria annulata in Cattle from Two Districts in Punjab (Pakistan)

Simple SummaryPakistan is a tropical country where climate is favourable for tick growth and hence its incidence of tick-borne diseases is high, affecting the output of the livestock sector. In the present study the infection rate of Theileria annulata, the causative agent of bovine theileriosis, was compared in apparently healthy cattle enrolled from two different regions in Pakistan. Parasite prevalence was found to be higher in Dera Ghazi Khan District than in Lodhran. The infection rate was higher in cattle that were infested with ticks and in those animals that were housed indoors at dairy farms with other animals. This prophylactic detection of parasite will help to design strategies to control tick and tick-borne diseases in study areas.The present study was designed to report the molecular prevalence of T. annulata in cattle blood samples collected from Punjab in Pakistan. A total of 428 cattle blood samples were collected from Districts Lodhran (n = 218) and Dera Ghazi Khan (n = 210). The prevalence of T. annulata was determined by the amplification of a fragment from its cytochrome b gene and parasite prevalence was significantly higher (p = 0.03) in the blood samples of cattle collected from Dera Ghazi Khan (70/210; 33%) as compared to Lodhran (52/218; 24%). Presence of T. annulata was also confirmed by the amplification of a fragment from their 30 kDa gene. The amplified PCR products of both genes were confirmed by DNA sequencing and these partial DNA sequences were submitted to GenBank. Phylogenetic analysis revealed that amplified partial gene sequences resembled previously reported T. annulata sequences in cattle from India, China, Iran, Tunisia, Turkey and Egypt. The incidence of T. annulata infection was higher in Sahiwal cattle (p = 0.04) than the other enrolled cattle breed from Dera Ghazi Khan. Female cattle from Lodhran (p = 0.02), while males (p = 0.02), animals housed in close compounds (p = 0.04), animals with a tick burden (p = 0.005) and farms with only cattle (p = 0.01) in Dear Ghazi Khan were found to be more susceptible to T. annulata infection. We recommend that large-scale tick and tick-borne disease control strategies be implemented in both districts under investigation, especially in Dera Ghazi Khan.

New species of the genus Spio (Annelida, Spionidae) from the southern and western coasts of Korea.

A new spionid polychaete, Spiopigmentatasp. nov., is described from the southern and western coasts of Korea. This new species differs from its congeners by the combination of the following morphological characteristics: the presence of orange-brown pigmentation on the anterior part of the prostomium, black pigmentation on the peristomium and along the body, U-shaped nuchal organs, a comparatively long extension of metameric dorsal ciliated organs, three pairs of white dots per chaetiger, two to three posterior abranchiate chaetigers, and the presence of tridentate neuropodial hooded hooks. The partial 16S ribosomal DNA (rDNA) and nuclear 18S rDNA sequences of the new species and Spio sp. 2 reported by Abe and Sato-Okoshi (2021) from Japan showed high similarity, indicating that these two specimens belong to the same species. A detailed description and illustrations of the new species, together with molecular information, are provided.

First molecular detection of Anaplasma species in cattle from Kyrgyzstan; molecular identification of human pathogenic novel genotype Anaplasma capra and Anaplasma phagocytophilum related strain

Anaplasmosis is a rickettsial infection with significant effects on human and animal health, and the discovery of new species or genotypes with zoonotic potential in recent years has increased this importance. The aim of this study was to provide the first assessment of the molecular etiology and prevalence of bovine anaplasmosis in Kyrgyzstan (specifically in the Chuy, Talas, Djalal-Abad, Naryn, and Issyk-Kul regions). The prevalence of bovine anaplasmosis was determined as 1.7% (6/358). PCR and partial DNA sequencing results of the 16S ribosomal RNA (rRNA) gene revealed that Anaplasma centrale, A. phagocytophilum like-1, and the human pathogenic novel genotype A. capra are circulating in cattle herds in Kyrgyzstan. Six DNA nucleotide sequences obtained in this study were deposited in GenBank under the following accession numbers: A. centrale (MW672117, MW672118, MW672119, MW672120), A. phagocytophilum (MW672121), and A. capra (MW672115).

Unexpected parasitism of Douglas-fir seed chalcid limits biocontrol options for invasive Douglas-fir in New Zealand

Douglas-fir seed chalcid (DFSC) Megastigmus spermotrophus, a small (3 mm long) host-specific seed-predatory wasp, was accidentally introduced into New Zealand in the 1920s. Concern over DFSC reducing Douglas-fir seed production in New Zealand led to an attempt at biocontrol in 1955 with the release, but failed establishment, of the small (2.5 mm long) parasitoid wasp, Mesopolobus spermotrophus. We investigated why DFSC causes little destruction of Douglas-fir seed in New Zealand (usually <20%) despite the apparent absence of major natural enemies. Douglas-fir seed collections from 13 New Zealand sites yielded the seed predator (DFSC) but also potential parasitoids, which were identified using morphology and partial COI DNA sequencing. DFSC destroyed only 0.15% of Douglas-fir seed. All parasitoids were identified as the pteromalid wasp, Mes. spermotrophus, the host-specific biocontrol agent released in 1955. Total parasitism was 48.5%, but levels at some sites approached 90%, with some evidence of density-dependence. The discovery of the parasitoid Mes. spermotrophus could indicate that the biocontrol agent released in 1955 did establish after all. Alternatively, Mes. spermotrophus could have arrived accidentally in more recent importations of Douglas-fir seed. The high level of parasitism of DFSC by Mes. spermotrophus is consistent with DFSC being under successful biological control in New Zealand. Suppression of DFSC populations will benefit commercial Douglas-fir seed production in New Zealand, but it also represents the likely loss of a potential biological control agent for wilding Douglas-fir.

Higher Genetic Diversity of the Common Sea Cucumber Holothuria (Halodeima) atra in Marine Protected Areas of the Central and Southern Ryukyu Islands

Recently, sea cucumbers (Echinodermata: Holothuroidea) have been over-exploited in many areas of the world, including in the Ryukyu Islands, southern Japan, due to increases in their economic importance. Nevertheless, management and protection of sea cucumbers are insufficient worldwide. The black sea cucumber Holothuria (Halodeima) atra Jaeger, 1833, inhabits a large range across the Indo-West Pacific Ocean and is a widely harvested species. Here we conducted population genetic analyses on H. atra using partial mitochondrial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) to examine 11 different populations around three island groups in the middle Ryukyus; Okinawajima Island, the Kerama Islands, and the Sakishima Islands, all within Okinawa Prefecture. We found 27 haplotypes for COI and 16 haplotypes for 16S. Locations within national and quasi-national parks (Zamami Island, Keramas, and Manza, Okinawajima; managed by the national Ministry of Environment and Okinawa Prefecture, respectively) had the highest number of haplotypes, whereas locations with less management and more anthropogenic pressure had lower numbers The mean of all samples' genetic diversity indices was moderate with regards to both haplotype and nucleotide diversity. According to our results, Zamami Ama was the most genetically diverse location based on both markers used, likely because it is located within Kerama-Shoto National Park with comparatively stricter regulations than most other locations. Based on our COI sequences, three-quarters of the locations with the highest haplotype diversity were found to be distant from Okinawajima Island, indicating that the genetic diversity of H. atra was reduced around Okinawajima Island. Our results possibly reflect negative impacts from anthropogenic pressures such as over-harvesting and coastal development, although future comprehensive research including sequences of nuclear loci is needed to confirm this hypothesis.

Distribution of stem-end rot on the canopy in ‘Hass’ avocado trees in two coastal areas in Peru

Stem-end rot (SER) of avocado is caused by several fungal species, and it is presented worldwide. This plant disease currently affects several avocado producer regions in Peru, causing fruit rot, impacting the industry negatively. Research about SER distribution in the canopy of avocado trees is limited. Thus, the present study aimed to compare which areas in the canopy are prone to have more SER in ‘Hass’ avocado harvested fruit in two different coastal areas in Peru. The experiment was conducted in the northern (Barranca) and southern (Cañete) of Lima. ‘Hass’Avocado fruits from both producer areas were collected to identify the causal agent; Lasiodiplodia theobromae was isolated from infected fruits. Identification was conducted based on morphological features and a partial DNA sequence of the translation elongation factor 1-α gene (tef1-α). The results showed that fruits inside the tree canopy were prone to have a higher disease incidence than the fruits located in the external site (P<0.001). Besides, internal-site fruits displayed a higher percentage of infected fruit for each grade disease (P<0.001) than external-site fruits, except for grade 0 (fruits without symptoms) and grade 1. Finally, the results suggested that the altitude where the fruit is positioned on the canopy could influence the incidence of SER, where fruits located in the high part revealed less incidence than the low section. The results are valuable for enhancing management strategies and avoiding postharvest loss of avocado fruits in our region.

Mitochondrial DNA Diversity of the Nangan Islanders Living in the Mazu Archipelago of the Taiwan Strait

This study investigates the genetic relationship of the Mazu peoples on the east coast of China in the Taiwan Strait. Using partial and complete mitochondrial DNA (mtDNA) sequences, we compare Mazu with surrounding East Asia populations. Mazu shows no exclusive affinities with either Southeast or Northeast Asia. High genetic diversity and a very high number of exclusive haplogroups of various Asian origins suggest that Mazu resulted from a process of continuous resettlement that started when it first became an archipelago at the end of the last glacial maximum and that continued till the last century. As a result, genetic drift did not contribute to an exclusive Mazu profile. The structure of haplogroups that show signatures of the Neolithic era (N9a10a), or influx from Island Southeast Asia (F1a4a) suggest recent gene flows and Mazu relationship with it's pre-Neolithic era (presence of pre-E1a or R9/pre-F from Liangdao man) was not seen.

Evaluation of methods for plant genomic DNA sequence analysis without DNA and PCR product purification

Genome-editing technologies are widely used to characterize gene functions and improve the features of agricultural plants. Although sequence analysis of gene editing target DNA is the most reliable method of screening gene-edited plants, the current DNA sequence analysis methods are time consuming and labor intensive because they include genomic DNA and polymerase chain reaction (PCR) product purification. In this study, seven methods were performed for sequence analysis of plant genomic DNA with and/or without genomic DNA and PCR product purification. Consequently, good-quality sequencing chromatograms were obtained using all methods. Results showed that the partial genomic DNA sequence of Nicotiana benthamiana and Arabidopsis thaliana could be sufficiently analyzed without plant genomic DNA and PCR product purification. Furthermore, screening of gene-edited N. benthamiana was successful using the present methods. Therefore, the tested methods could reduce the time, simplify the workflow of plant gene analysis, and help in screening gene-edited plants.

Risk factors associated with tick infestations on equids in Khyber Pakhtunkhwa, Pakistan, with notes on Rickettsia massiliae detection

BackgroundStudies on ticks infesting equids are lacking in various parts of the world, including Khyber Pakhtunkhwa (KP), Pakistan. The aim of this study was to investigate the diversity of ticks infesting equids, associated risk factors and rickettsial detection in ticks from equids in KP.MethodsInspection of 404 equid hosts from November 2018 to October 2019 resulted in the collection of 550 ticks. Data on tick-associated risk factors were collected from equid owners by means of a questionnaire. After morphological identification, partial DNA sequences of the tick mitochondrial 16S rRNA gene were used for taxonomic confirmation of species. Partial sequences of the gltA and ompA genes were used for Rickettsia detection in ticks.ResultsA total of 550 tick specimens were collected on 324 (80.2%) of the equids inspected, of which 161 were horses (50%), 145 (45%) were donkeys and 18 were mules (5%). The ticks were identified as belonging to the following five species: Rhipicephalus microplus (341 specimens, 62% of the total ticks), Rh. haemaphysaloides (126, 23%), Rh. turanicus (39, 7%), Rh. sanguineus (s.l.) (33, 6%) and Hyalomma anatolicum (11, 2%). The most prevalent tick life stage was adult females (279, 51%) followed by adult males (186, 34%) and nymphs (85, 15%). Higher tick infestations were observed on male equids (relative risk [RR] 0.7432, P < 0.0005) and adult equids (RR 1.268, P < 0.0020). Ticks were frequently attached to the axial region of horses (55, 21%), sternum of donkeys (44, 21%) and belly of mules (19, 23%) (P < 0.04). Temporal patterns of tick infestation in association with temperature and humidity were highly significant (P < 0.05). Risk factors, such as animal housing (P < 0.0003), living management (P < 0.006), grazing type (P < 0.01) and location in hilly areas (P < 0.02), significantly enhanced the chances for tick infestation. Tick species analyzed in this study were phylogenetically related to species from Afghanistan, China, South Africa and Taiwan. Partial sequences of the gltA and ompA genes obtained from Rh. microplus and Rh. haemaphysaloides were 100% identical to the spotted fever group pathogen Rickettsia massiliae.ConclusionsEquids exposed to significant risk factors were infected by one or more of at least five tick species in KP, Pakistan, and some of the ticks harbored the human pathogen R. massiliae.Graphical abstract

Detection and molecular characterization of phytoplasmas based on 16s rDNA gene region by phylogenetic and in silico RFLP analysis of local grapevine cultivars in Şanlıurfa and Adıyaman

During the autumn seasons of 2016-2019, several surveys were carried out in grapevine (Vitis vinifera L.) cultivation areas in Şanlıurfa and Adıyaman provinces of South-eastern part of Turkey, to identify grapevine plants exhibiting phytoplasma-like symptoms including yellowing, small leaf formation, chlorosis, short internodes, severe redness and inward curling and to detect and characterize the causal agent responsible for these symptoms. Purified DNA obtained from collected samples was examined for the highly conserved phytoplasma 16S rDNA gene via nested-PCR with the universal phytoplasma-specific primer sets. DNA amplification via nested-PCR/RFLP analyses with some restriction enzymes confirmed the suspected correlations between the disease symptoms observed and phytoplasma presence in the samples. F2n/R2 amplicons of the phytoplasma strains obtained from the samples showed 99.99% homology with each other and 99% homology with phytoplasma DNA partial sequences belonging to some groups deposited in the GeneBank database. Sequencing and phylogenetic analysis revealed that collected isolates have 99% sequence identity with ‘Candidatus phytoplasma solani’ (16SrXII-A) and ‘Ca.P. asteris’ (16SrI-B). According to our knowledge, the results of this study is the first report of the phylogenetic relationship of phytoplasmas infecting different grapevine cultivars based on 16S rDNA gene. Genetic diversity of genes other than 16S rDNA of the pathogen causing these infections is under study.

A new species of Anurophorus (Collembola: Isotomidae) from South Korea, with notes on its molecular data.

A new species, Anurophorus hallaensis sp. nov. was collected from the withered leaves of Korean fir (Abies koreana Wilson), which is distributed at higher than 1,300 m in altitude on Mt. Hallasan (Jeju Island, South Korea). The morphology and two partial gene regions of this species are described herein. Anurophorus hallaensis sp. nov. showed the same formulas of macrochaetae and sensilla on thoracic segment II to abdominal segment IV as those exhibited by A. laricis Nicolet and A. palearcticus Potapov; however, this new species can be distinguished from them by the number of knobbed hairs on ventral side of legs. Partial DNA sequences of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene and ribosomal DNA (28S rDNA) were used as DNA barcodes to distinguish between A. hallaensis sp. nov. and closely related congeners. The results of the present study indicate that the COI and rDNA are useful for species discrimination within the genus Anurophorus. An identification key to the Korean species of Anurophorus is provided.

Description of the Metacercaria of Cardiocephaloides sp. (Digenea, Diplostomoidea), Newly Recorded from the Brain of Gangetic Leaffish (Nandus nandus) and Its Genetic Characterization in India.

Cardiocephaloides comprises strigeid trematodes that represent a small genus. In this study, metacercaria identified as Cardiocephaloides sp. was collected from the Gangetic leaffish Nandus nandus from the Ganga River at Bairaj, Bijnor (29º01'N, 77º45'E) in the state of Uttar Pradesh (U.P.), India. Partial DNA sequences of the internal transcribed spacers (ITS1-5.8S-ITS2) and 28S gene of nuclear ribosomal DNA were generated and compared with available sequences of Cardiocephaloides species from Genbank database. Encysted metacercariae of Cardiocephaloides sp. were collected from Nandus nandus were processed, identified and documented using morphological methods. The ITS1-5.8S-ITS2 cluster and 28S gene of ribosomal DNA of metacercariae were also sequenced and used for phylogenetic analysis. The infections of brain parasites are poorly understood in India and if studies areavailable, theyare not properly described. During this study, the species collected were found belongs to the genus Cardiocephaloides. Metacercariae of Cardiocephaloides sp. is distinguished morphologically from others that also harbor brain by the presence of having an egg shape cyst and bodyelongate oval in shape with well-developed anterior part. The metacercariae are identified by matching of molecular sequence data and is compared to other species of Strigeidae. This is the first record of metacercaria of Cardiocephaloides sp. from India. This molecular data from the present study will provide future comparative insights into species of Cardiocephaloides and its close affiliation to other congeners from different geographical areas.

Mitochondrial Genomes of the United States Distribution of Gray Fox (Urocyon cinereoargenteus) Reveal a Major Phylogeographic Break at the Great Plains Suture Zone

We examined phylogeographic structure in gray fox (Urocyon cinereoargenteus) across the United States to identify the location of secondary contact zone(s) between eastern and western lineages and investigate the possibility of additional cryptic intraspecific divergences. We generated and analyzed complete mitochondrial genome sequence data from 75 samples and partial control region mitochondrial DNA sequences from 378 samples to investigate levels of genetic diversity and structure through population- and individual-based analyses including estimates of divergence (FSTand SAMOVA), median joining networks, and phylogenies. We used complete mitochondrial genomes to infer phylogenetic relationships and date divergence times of major lineages ofUrocyonin the United States. Despite broad-scale sampling, we did not recover additional major lineages ofUrocyonwithin the United States, but identified a deep east-west split (∼0.8 million years) with secondary contact at the Great Plains Suture Zone and confirmed the Channel Island fox (Urocyon littoralis) is nested withinU. cinereoargenteus. Genetic diversity declined at northern latitudes in the eastern United States, a pattern concordant with post-glacial recolonization and range expansion. Beyond the east-west divergence, morphologically-based subspecies did not form monophyletic groups, though unique haplotypes were often geographically limited. Gray foxes in the United States displayed a deep, cryptic divergence suggesting taxonomic revision is needed. Secondary contact at a common phylogeographic break, the Great Plains Suture Zone, where environmental variables show a sharp cline, suggests ongoing evolutionary processes may reinforce this divergence. Follow-up study with nuclear markers should investigate whether hybridization is occurring along the suture zone and characterize contemporary population structure to help identify conservation units. Comparative work on other wide-ranging carnivores in the region should test whether similar evolutionary patterns and processes are occurring.

Molecular Detection of Toxigenic Clostridioides difficile among Diarrheic Dogs and Cats: A Mounting Public Health Concern.

Nowadays, pet animals are known to be asymptomatic carriers of Clostridioides difficile. This study was conducted to investigate the burden of toxigenic C. difficile among diarrheic dogs and cats using direct PCR on fecal samples to reveal better insights about the epidemiology of such toxigenic strains referring to its public health significance. For this purpose, fecal samples were obtained from 58 dogs and 42 cats experiencing diarrhea. Following DNA extraction, the extracted DNA was examined for the occurrence of C. difficile as well as toxigenic strains through the detection of C. difficile 16S rRNA and toxin encoding genes (tcdA, tcdB, cdtA and cdtB) using PCR. Moreover, partial DNA sequencing of toxigenic strains retrieved from dog and cat was carried out. Of 100 examined diarrheic animals, 90 (90%) were C. difficile positive, including 93.1% and 85.7% of dogs and cats, respectively. In addition, toxigenic strains were detected in 13 animals, giving an overall prevalence 13% with the following prevalence rates among dogs and cats 12.1% and 14.3%, respectively. Furthermore, the phylogenetic analysis of the obtained sequence revealed high genetic relatedness of tcdA sequence obtained from a cat to strains of human diarrheic cases to point out the public health threat of such sequence. In conclusion, the direct detection of toxigenic C. difficile using PCR among dogs and cats highlights the potential role of household pets as a source for such strains to human contacts.

DdRAD sequencing: an emerging technology added to the biosecurity toolbox for tracing the origin of brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae)

BackgroundBrown marmorated stink bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) is native to East Asia but has invaded many countries in the world. BMSB is a polyphagous insect pest and causes significant economic losses to agriculture worldwide. Knowledge on the genetic diversity among BMSB populations is scarce but is essential to understand the patterns of colonization and invasion history of local populations. Efforts have been made to assess the genetic diversity of BMSB using partial mitochondrial DNA sequences but genetic divergence on mitochondria is not high enough to precisely accurately identify and distinguish various BMSB populations. Therefore, in this study, we applied a ddRAD (double digest restriction-site associated DNA) sequencing approach to ascertain the genetic diversity of BMSB populations collected from 12 countries (2 native and 10 invaded) across four continents with the ultimate aim to trace the origin of BMSBs intercepted during border inspections and post-border surveillance.ResultA total of 1775 high confidence single nucleotide polymorphisms (SNPs) were identified from ddRAD sequencing data collected from 389 adult BMSB individuals. Principal component analysis (PCA) of the identified SNPs indicated the existence of two main distinct genetic clusters representing individuals sampled from regions where BMSB is native to, China and Japan, respectively, and one broad cluster comprised individuals sampled from countries which have been invaded by BMSB. The population genetic structure analysis further discriminated the genetic diversity among the BMSB populations at a higher resolution and distinguished them into five potential genetic clusters.ConclusionThe study revealed hidden genetic diversity among the studied BMSB populations across the continents. The BMSB populations from Japan were genetically distant from the other studied populations. Similarly, the BMSB populations from China were also genetically differentiated from the Japanese and other populations. Further genetic structure analysis revealed the presence of at least three genetic clusters of BMSB in the invaded countries, possibly originating via multiple invasions. Furthermore, this study has produced novel set of SNP markers to enhance the knowledge of genetic diversity among BMSB populations and demonstrates the potential to trace the origin of BMSB individuals for future invasion events.

Isolation of an atypical Leptospira strain assigned to the Sejroe serogroup from a water buffalo in Brazil

The isolation of leptospires from buffaloes worldwide is still limited to a few strains. Thus, the aim of this study was to describe the first Leptospira isolate from buffalo urine, assigned to the Sejroe serogroup, which does not belong to the Wolffi subgroup, traditionally isolated in Brazil. A total of 244 urine samples of water buffaloes (Bubalus bubalis) raised in the Brazilian Amazon were subjected to bacteriological culturing and polymerase chain reaction (PCR) for the detection of leptospires. The obtained isolate was characterized by serogrouping using polyclonal antibodies, partial DNA sequencing, Hardjo-Bovis-specific PCR, multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) and experimental infection in hamsters. PCR was performed on the urine samples; 11/244 were positive (4.5 %) for Leptospira, and only one isolate was recovered (0.4 %). Regarding characterization, the isolate was assigned to the Sejroe serogroup with high titers (12,800) for the Saxkoebing and Sejroe serovar antisera. The isolate was negative for Hardjo-Bovis-specific PCR, and the species Leptospira borgpetersenii was identified by DNA sequencing. The MLVA results showed that the VNTR profile of the isolate was 1−2-5, compatible with that of serovars Sejroe/Istrica. In the experimental infection in hamsters, the animals did not develop clinical signs, and no macroscopic lesions were observed on the organs at necropsy; however, the strain was detected in the kidneys, uterus, and testicles of the animals. The isolate described herein highlights infection by Sejroe strains that may be overlooked in buffaloes and that may be different from those normally isolated and used in serological studies.

Molecular Characterization of Blastocystis in Captive and Free-Ranging New World Primates, Platyrrhini.

Blastocystis species are widely distributed micro-eukaryote parasites found in both human and nonhuman primates. Despite having a global distribution, descriptions of Blastocystis subtype diversity in neotropical primates is largely limited to captive animals. The aim of this study was to molecularly characterize the presence of Blastocystis in free-ranging black-headed night monkeys, Aotus nigriceps, and to analyze Blastocystis heterogeneity in primates of the Parvorder Platyrrhini. We analyzed Blastocystis small sub-unit ribosomal DNA (SSUrDNA) from both A. nigriceps and Azara's night monkey, A. azarae boliviensis, in Southeastern Peru. We also included additional Blastocystis sequence from other neotropical primate studies to explore the distribution and host specificity of Blastocystis subtypes (ST) throughout the neotropics. Thirteen percent of A. nigriceps samples were Blastocystis positive. Only ST8 was amplified in A. nigriceps and this partial DNA sequence was highly similar to ST8 sequence previously obtained from a human in Brazil. In our analysis of all available Blastocystis SSU sequences from primates of the Parvorder Platyrrhini, we found 15 monophyletic lineages corresponding to previously described subtypes ST1-ST10, ST12-15, and ST17. Blastocystis SSU sequences amplified from A. nigriceps fecal samples shared high sequence similarity to isolates found in several other neotropical primates, Alouatta palliata, A. caraya, Ateles fusciceps, and Lagothrix. Similar subtypes have been found in human and captive primates which supports the possibility of transmission when in close contact. Expanded sampling of sympatric neotropical primates in the wild will establish whether subtypes and clades are limited to taxonomic group or whether transmission occurs between overlapping species.

Phylogenetic analysis of eight species of Anomopoda based on transcriptomic and mitochondrial DNA sequences.

Anomopoda is the widespread planktonic microcrustacean, which plays a crucial role in aquatic ecosystem. There are few studies about the evolutionary relationships among various Anomopoda basing on molecular data. In the present study, phylogenetic analysis of eight Anomopoda was carried out. Firstly, the culture system was developed to breed cladocerans. By using this system, eight species (Daphnia magna, D. pulex, D. sinensis, Ceriodaphnia reticulata, Moina micrura, Scapholeberis kingi, Simocephalus vetulus and Eurycercus lamellatus) were purified and cultured stably in the laboratory. Then, transcriptomic sequences and partial mitochondrial DNA sequences were both used to reconstruct the phylogenetic tree among 8 species. Transcriptomic sequences were sequenced on Illumina Hiseq 2500 platform. After assembly and annotation, transcriptomic sequences were spliced together and aligned for phylogenetic analysis. Basing on the orthologous genes derived from transcriptomic sequences, the phylogenetic analysis showed that 4 genera of Daphniidae were clustered into one group, and among the 4 genera, Ceriodaphnia was closer to Daphnia than Simocephalus, while Scapholeberis was farthest from other species. In addition, Eurycercidae was closer to Daphniidae than Moinidae. The phylogenetic trees based on both 12S rRNA and 16S rRNA sequences were similar with that based on transcriptomic sequences. Meanwhile, the phylogenetic tree based on 16S rRNA sequences was more suitable than that based on 12S rRNA sequences. These results suggested that the phylogenetic analysis basing on the transcriptomic sequences was available in cladocerans, which will help us to effectively understand the phylogenetic relationships among various cladocerans.

Interrelationships of Anenterotrema (Digenea: Dicrocoeliidae) from Neotropical bats (Mammalia: Chiroptera) with description of a new species from Molossus molossus in Brazil.

Anenterotrema is a small genus of dicrocoeliids (Digenea: Dicrocoeliidae) containing 6 species found in Neotropical bats. Members of this genus are characterized by the lack of digestive system organs and, unlike the majority of dicrocoeliids, parasitize the intestine of their definitive hosts. In this study, we have morphologically examined newly collected specimens belonging to four species of Anenterotrema from Brazil, Ecuador, and Panama. Based on the data in original descriptions and our analysis of quality new specimens, we resurrected Anenterotrema freitasi, previously synonymized with A. eduardocaballeroi. We also described a new species of Anenterotrema from Molossus molossus in the Brazilian Amazon. The new species differs from congeners in several morphological features, most prominently in the size and structure of its highly muscular, protuberant ventral sucker. It is also characterized by the lack of the semi-circular thickening of the tegument around the oral sucker typical for some members of the genus. We used partial DNA sequences of the large ribosomal subunit gene (28S) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to test the monophyly of Anenterotrema, and study the interrelationships and determine the inter- and intraspecific variation of three Anenterotrema spp. collected from different bat species in Brazil, Ecuador and Panama.

Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in different Brazilian biomes

BackgroundEntamoeba species harbored by humans have different degrees of pathogenicity. The present study explores the intra- and interspecific diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in different Brazilian regions.MethodsCross-sectional studies were performed to collect fecal samples (n = 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis.ResultsLight microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6–10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four different species were identified: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n = 32) were characterized; however, new ribosomal lineages were not identified. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histolytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the first containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecific diversity for tetra- and octonucleated Entamoeba spp. (H ± SD = 0.9625 ± 0.0126). The intraspecific diversity varied according to species or subtype: E. dispar and E. histolytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni.ConclusionsTetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a field to be further explored and provides information with important implications for public health.Graphical