Small Subunit rDNA Gene Research Articles

Intragenomic diversity of the small subunit rDNA gene shows limited impact on the pathogenicity of Blastocystis infection in clinical patients

The clinical significance of Blastocystis sp. remains to be fully elucidated. This study assesses whether Blastocystis subtype diversity can affect the outcome of the infection and the occurrence of clinical manifestations in infected individuals. Stool samples from 219 Blastocystis-positive patients by PCR targeting the ssu rDNA gene were fully genotyped by Sanger sequencing analyses. Co-infections by other parasitic, viral, and bacterial enteropathogens were identified by molecular and culture methods. Sequence analyses revealed the presence of six Blastocystis subtypes including ST1 (21.5 %), ST2 (17.8 %), ST3 (29.7 %), ST4 (22.8 %), ST6 (5.5 %), and ST7 (2.3 %), with a single sample harbouring a ST1+ST3 co-infection (0.5 %). Multivariate risk factor analyses using logistic regression models indicated that neither Blastocystis subtypes nor patient-associated variables including sex, country of origin, travelling history, and presence of nonspecific symptoms were positively associated with a higher likelihood of developing gastrointestinal symptoms (abdominal pain and diarrhoea). However, being of a young age (p-value: 0.003) and experiencing skin pruritus (p-value < 0.001) and eosinophilia (p-value: 0.016) were found to increase the odds of presenting gastrointestinal symptoms. Blastocystis subtypes based on variability within the ssu rDNA gene do not seem to be the main drivers of clinical manifestations in the surveyed clinical population.

Analysis of Molecular Diversity in Castanopsis sieboldii with Felt Disease Caused by Septobasidium sp.

In 2020, within the Dongbaekdongsan area in Jeju Island, a Septobasidium sp. associated with a felt disease in &lt;i&gt;Castanopsis sieboldii&lt;/i&gt; (Makino) Hatus. ex T. Yamaz. &amp; Mashiba was identified. The symptom included the presence of brown, thin, and silk-like mycelial mats attached to the tree's bark, displaying variations in size from large to small. To induce hyphal growth, the samples collected were incubated in a moist chamber, and the newly formed hyphae were subjected to genomic DNA extractions. The nucleotide sequences of the internal transcribed spacer and small subunit rDNA genes were determined, and molecular characteristics among the isolates were investigated through polymerase chain reaction-based restriction fragment length polymorphism analysis. This &lt;i&gt;Septobasidium&lt;/i&gt; sp. exhibited distinct morphological and phylogenetic features compared to those that were previously reported in South Korea. Consequently, this strain is taxonomically classified as a provisionally novel species of &lt;i&gt;Septobasidium&lt;/i&gt;. Furthermore, the observed felt disease exhibited a high degree of host specificity, as it was exclusively identified in &lt;i&gt;C. sieboldii&lt;/i&gt; without occurrence in other tree species at the time of observation.

Morphology and phylogenetic relationships of two Antarctic strains within the genera Carolibrandtia and Chlorella (Chlorellaceae, Trebouxiophyceae)

The genera Carolibrandtia and Chlorella have been described as small green algae with spherical cell shapes that inhabit various environments. Species of these genera are often difficult to identify because of their simple morphology and high phenotypic plasticity. We investigated two small coccoid strains from Antarctica based on morphology, molecular phylogeny by two alignment methods which have been applied to previous phylogenetic studies of the genus Chlorella, and comparison of the secondary structures of nuclear small subunit (SSU) and internal transcribed spacer (ITS) rDNA sequences. Light microscopy of two strains revealed spherical cells containing chloroplasts with pyrenoids, and the morphological characteristics of the strains were nearly identical to those of other Chlorella species. However, based on the phylogenetic analyses of nuclear SSU and ITS rDNA sequences, it was determined that the Antarctic microalgal strains belonged to two genera, as the Chlorella and Carolibrandtia. In addition, the secondary structures of the SSU and ITS2 sequences were analyzed to detect compensatory base changes (CBCs) that were used to identify and describe the two strains. A unique CBC in the SSU rDNA gene was decisive for distinguishing strain CCAP 211/45. The ITS2 rDNA sequences for each strain were compared to those obtained previously from other closely related species. Following the comparison of morphological and molecular characteristics, we propose KSF0092 as a new species, Chlorella terrestris sp. nov., and the reassignment of the strain Chlorella antarctica CCAP 211/45 into Carolibrandtia antarctica comb. nov.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

Molecular, Morphological and Chemical Diversity of Two New Species of Antarctic Diatoms, Craspedostauros ineffabilis sp. nov. and Craspedostauros zucchellii sp. nov.

The current study focuses on the biological diversity of two strains of Antarctic diatoms (strains IMA082A and IMA088A) collected and isolated from the Ross Sea (Antarctica) during the XXXIV Italian Antarctic Expedition. Both species presented the typical morphological characters of the genus Craspedostauros: cribrate areolae, two “fore-and-aft” chloroplasts and a narrow “stauros”. This classification is congruent with the molecular phylogeny based on the concatenated 18S rDNA-rbcL-psbC alignment, which showed that these algae formed a monophyletic lineage including six taxonomically accepted species of Craspedostauros. Since the study of the evolution of this genus and of others raphe-bearing diatoms with a “stauros” is particularly challenging and their phylogeny is still debated, we tested alternative tree topologies to evaluate the relationships among these taxa. The metabolic fingerprinting approach was implemented for the assessment of the chemical diversity of IMA082A and IMA088A. In conclusion, combining (1) traditional morphological features used in diatoms identification, (2) phylogenetic analyses of the small subunit rDNA (18S rDNA), rbcL and psbC genes, and (3) metabolic fingerprint, we described the strains IMA082A and IMA088A as Craspedostauros ineffabilis sp. nov. and Craspedostauros zucchellii sp. nov. as new species, respectively.

Molecular subtyping of Blastocystis sp. detected in patients at a large tertiary referral hospital in Lusaka, Zambia

BackgroundBlastocystis sp. is a common enteric eukaryote of humans whose pathogenicity is still debatable. However, a number of reported Blastocystis colonization associated with enteric disease exist. In Zambia, presence of the pathogen has previously been reported in children. However, the molecular epidemiology of Blastocystis colonization remains unclarified in Zambia.Methods and resultsArchived stool samples submitted for routine parasitological diagnosis at Zambia’s largest tertiary referral hospital positive for Blastocystis sp. by microscopic examination were selected for the study. Subtyping of the Blastocystis was done based on polymerase chain reactions (PCR) amplification, sequencing and subsequent phylogenetic analysis of the 18S small subunit (SSU) rDNA gene. Four subtypes, ST1 (allele 4), ST2 (allele 12), ST3 (allele 34, 36, 37, 38, 39) and ST6 (allele 122), were identified by molecular procedures in the study, with some Zambian sequences showing close relationships with those detected in non-human primates and common rats.ConclusionsThe study revealed the circulation of multiple Blastocystis subtypes ST1, 20% (9/45), ST2, 15% (7/45), ST3 24.4% (11/45), and ST6, 2.2% (1/45) in the study population. The close clustering of some Zambian sequences with those detected from animals suggests the possibility of the presence of both anthroponotic and zoonotic transmission cycles in the country. Further studies in animal populations are recommended for a better understanding of the epidemiology of Blastocystis and for implementation of effective evidence-based control strategies.

Detection, Molecular Identification and Transmission of the Intestinal Protozoa Blastocystis sp. in Guinea from a Large-Scale Epidemiological Study Conducted in the Conakry Area.

Blastocystis sp. is a single-celled parasite estimated to colonize the digestive tract of 1 to 2 billion people worldwide. Although it represents the most frequent intestinal protozoa in human stools, it remains still under-investigated in countries with a high risk of infection due to poor sanitary and hygiene conditions, such as in Africa. Therefore, the present study was carried out to determine the prevalence and subtype (ST) distribution of Blastocystis sp. in the Guinean population. For this purpose, fecal samples were collected from 500 individuals presenting or not digestive disorders in two hospitals of Conakry. Search for the parasite in stools was performed by real-time PCR targeting the small subunit rDNA gene followed by sequencing of the PCR products for subtyping of the isolates. A total of 390 participants (78.0%) was positive for Blastocystis sp. Five STs were identified in the Guinean cohort (ST1, ST2, ST3, ST4 and ST14) with varying frequency, ST3 being predominant. Among them, ST4 was found in only two patients confirming its global rarity in Africa whereas infections by ST14 were likely the result of zoonotic transmission from bovid. No significant association was detected between Blastocystis sp. colonization or ST distribution and the symptomatic status of Guinean subjects or the presence of digestive symptoms. In contrast, drilling water consumption represented a significant risk factor for infection by Blastocystis sp. Predominance of ST3 coupled with its low intra-ST diversity strongly suggested large-scale human-to-human transmission of this ST within this cohort. In parallel, the highest intra-ST diversity of ST1 and ST2 was likely correlated with various potential sources of infection in addition to anthroponotic transmission. These findings highlighted the active circulation of the parasite in Guinea as reported in some low-income African countries and the necessity to implement prevention and control measures in order to limit the circulation of this parasite in this endemic geographical area.

First Report of Fusarium commune Causing Leaf Spot Disease on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae family, known as Baiji in Chinese) is a perennial herb and has been traditionally used for hemostasis and detumescence in China. In April of 2020, a leaf spot disease on B. striata was observed in plant nurseries (∼0.2 h) in Guilin, Guangxi Province, China. Approximately 20% of the plants were symptomatic, of which 150 plants were randomly selected for investigation. Initial symptoms include the appearance of small, circular or irregular light brown spots, randomly scattered on the edges and surfaces of the leaves, which progressively expand into large, suborbicular or irregular-shaped dark brown, necrotic areas. At the severe stage, the lesions coalesced into large necrotic areas and ultimately resulted in leaf abscission. To isolate the pathogen, three representative plants exhibiting symptoms were collected from the nurseries. Leaf tissues (5 × 5 mm) were cut from the margin of necrotic lesions (n = 18), surface-disinfected in 1% sodium hypochlorite (NaOCl) solution for 2 min, then rinsed three times in sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h photoperiod) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to obtain pure cultures. Nine fungal isolates with similar morphological characteristics were obtained. Three single-spore isolates, BJ23.1, BJ55.1, and BJ91.3, were subjected to further morphological and molecular characterisation. Colonies on PDA plates were villose, had a dense growth of aerial mycelia and appeared white (1A1) to yellowish white (3A2). Macroconidia were smooth, hyaline, straight to slightly curved, usually contained three or five septa, and measuring 23.3 to 42.1 × 3.0 to 6.2 μm (mean ± SD: 31.2 ± 5.1 × 4.2 ± 0.6 μm, n = 50). Microconidia were generally cylindrical, straight to slightly curved, aseptate, and measuring 7.2 to 18.8 × 2.5 to 4.3 μm (mean ± SD: 12.1 ± 2.8 × 3.3 ± 0.5 μm, n = 62). Morphological characteristics are similar to those of F. commune (Skovgaard et al. 2003). For molecular identification, the genomic DNA of the isolates BJ23.1, BJ55.1, and BJ91.3 were extracted using the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region of rDNA, partial translation elongation factor-1 alpha (TEF-1α), RNA polymerase second largest subunit (RPB2), and the mitochondrial small subunit rDNA (mtSSU) genes were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu et al. 1999, Reeb et al. 2004), MS1/MS2 (Li et al. 1994), respectively]. The obtained sequences were deposited in NCBI GenBank under the following accession numbers: ITS (MZ424697 to MZ424699), TEF-1α (MZ513467 to MZ513469), RPB2 (MZ513473 to MZ513475), and mtSSU (MZ513470 to MZ513472). BLAST® analysis of the deposited sequences showed 99 to 100% identity with those of F. commune present in GenBank (Accession numbers: DQ016205, MH582348, MH582181, AF077383). In addition, a phylogenetic analysis using concatenated sequences of ITS, TEF-1α, mtSSU genes showed that BJ23.1, BJ55.1, and BJ91.3 located on the same clade with strains of F. commune. Therefore, based on morphological and molecular characteristics, the isolates were identified as F. commune (Skovgaard et al. 2003, Stewart et al. 2006). Pathogenicity was tested using 1.5-year-old B. striata plants. Healthy leaves on plants were inoculated with 5 × 5 mm mycelial discs of strains BJ23.1, BJ55.1, and BJ91.3 from 3-day-old PDA cultures, each isolate was inoculated onto three plants; three other plants inoculated with sterile PDA discs served as controls. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C for 14 days (12-h photoperiod). Three days post-inoculation, leaf spot symptoms appeared on the inoculated leaves. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch's postulates, F. commune was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. F. commune has been reported to cause diseases on some plants, including sugarcane (Wang et al. 2018), maize (Xi et al. 2019) and Wax Gourd (Zeng et al. 2020). To our knowledge, this is the first report of F. commune causing leaf spot disease on B. striata in China. Identification of this pathogen provides the information for further studies to develop management strategies to control the disease.

A phylogenetic overview of the Hydnaceae (Cantharellales, Basidiomycota) with new taxa from China.

The family Hydnaceae (Cantharellales, Basidiomycota) is a group of fungi found worldwide which exhibit stichic nuclear division. The group is highly diverse in morphology, ecology, and phylogeny, and includes some edible species which are popular all over the world. Traditionally, Hydnaceae together with Cantharellaceae, Clavulinaceae and Sistotremataceae are four families in the Cantharellales. The four families were combined and redefined as “Hydnaceae”, however, a comprehensive phylogeny based on multiple-marker dataset for the entire Hydnaceae sensu stricto is still lacking and the delimitation is also unclear. We inferred Maximum Likelihood and Bayesian phylogenies for the family Hydnaceae from the data of five DNA regions: the large subunit of nuclear ribosomal RNA gene (nLSU), the internal transcribed spacer regions (ITS), the mitochondrial small subunit rDNA gene (mtSSU), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-alpha gene (TEF1). We also produced three more phylogenetic trees for Cantharellus based on 5.8S, nLSU, mtSSU, RPB2 and TEF1, Craterellus and Hydnum both based on the combined nLSU and ITS. This study has reproduced the status of Hydnaceae in the order Cantharellales, and phylogenetically confirmed seventeen genera in Hydnaceae. Twenty nine new taxa or synonyms are described, revealed, proposed, or reported, including eight new subgenera (Cantharellus subgenus Magnus, Craterellus subgenus Cariosi, subg. Craterellus, subg. Imperforati, subg. Lamelles, subg. Longibasidiosi, subg. Ovoidei, and Hydnum subgenus Brevispina); seventeen new species (Ca. laevihymeninus, Ca. magnus, Ca. subminor, Cr. badiogriseus, Cr. croceialbus, Cr. macrosporus, Cr. squamatus, H. brevispinum, H. flabellatum, H. flavidocanum, H. longibasidium, H. pallidocroceum, H. pallidomarginatum, H. sphaericum, H. tangerinum, H. tenuistipitum and H. ventricosum); two synonyms (Ca. anzutake and Ca. tuberculosporus as Ca. yunnanensis), and two newly recorded species (H. albomagnum and H. minum). The distinguishing characters of the new species and subgenera as well as their allied taxa are discussed in the notes which follow them. The delimitation and diversity in morphology, ecology, and phylogeny of Hydnaceae is discussed. Notes of seventeen genera which are phylogenetically accepted in Hydnaceae by this study and a key to the genera in Hydnaceae are provided.

First Report of Fusarium miscanthi Causing Ear Rot on Maize in China.

Maize (Zea mays L.), an important food and feed crop worldwide, can be infected by Fusarium pathogens that can contaminate grain with mycotoxins. From August to October in 2018 and 2019, a field survey for maize ear rot was conducted in 76 counties of Guizhou province. The incidence ranged from 3% to 15% at individual fields in different areas. A total of 175 diseased maize ears with similar symptoms, including kernels covered with white, pink or salmon-colored mold or exhibiting a white streaking ("starburst") symptom, were collected from fields. Symptomatic kernels were surface-sterilized by soaking for 30 s in 70% alcohol and for another 2 min in 2% sodium hypochlorite solution, followed by five rinses with sterile water. Each kernel was cut into half and placed on potato dextrose agar (PDA). After incubation at 28 °C in the dark for 5 days, colonies displaying morphological characteristics of Fusarium were transferred to fresh PDA (Leslie and Summerell 2006). Single-sporing was conducted to purify the putative Fusarium colonies. A total of 120 isolates belonged to 16 Fusarium species were determined and F. meridionale was the dominant species. Five isolates from Huaxi district of Guiyang City were identified as F. miscanthi (Gams et al. 1999). Colonies on PDA were white and floccose, and pigmentation as viewed from the underside of the Petri dish was violet. The average growth rate was 7.5-8.0 mm/day at 28 °C in the dark. In cultures grown on PDA, 0-1-septate microconidia were produced in slimy heads. Microconidia were clavate to fusiform with a truncate base and a broadly rounded tip, 4.8-13.3 μm × 1.8-3.3 μm (n=110). In cultures grown on half-strength CMC broth (Xu et al. 2010), macroconidia were mostly 3-septate, almost straight for most of the length, with a slightly foot-shaped basal cell and curved apical cell that gradually tapered, 17.8-71.3 μm × 2.0-4.3 μm (n=78). The identity of the fungus was confirmed by sequence comparison of the partial translation elongation factor-1α (TEF-1α), RNA polymerase II subunit (RPB2), mitochondrial small subunit rDNA (mtSSU) and β-tubulin genes (Mirete et al. 2004; O'Donnell et al. 2010; O'Donnell and Cigelnik 1997). BLASTn searches of GenBank, using the partial TEF-1α (MN750829), RPB2 (MN750834), mtSSU (MT594104) and β-tubulin (MT584781) sequences of representative isolate GYHXB03 as the queries, revealed 99.84%, 99%, 100% and 100% sequence identity, respectively, to F. miscanthi NRRL 26231 accessions AF324331, JX171634, AF060371 and AF060384. Inoculum of isolate GYHXB03 was prepared (Xu et al. 2010), and a pathogenicity test was conducted on maize hybrid "Shundan7" and repeated twice. A 106/mL spore suspension (2 mL) or sterile water was injected into each of 8 maize ears through the silk channel at the blister stage of reproductive development in field (Duan et al. 2019). After three weeks, typical Fusarium kernel rot symptoms the same as those previously shown in the field was observed on all pathogen-inoculated plants, while the controls were asymptomatic. The pathogens re-isolated from two diseased kernels were identified as F. miscanthi based on morphology and TEF-1α and mtSSU analyses. F. miscanthi was first isolated from Miscanthus sinensis in Denmark (Gams et al. 1999), and also identified from M. × giganteus rhizomes in Belgium (Scauflaire et al. 2013). To our knowledge, this is the first report of F. miscanthi causing maize ear rot in China. This disease should be monitored in Guizhou due to its threat to maize production.

Cryptosporidium ratti n. sp. (Apicomplexa: Cryptosporidiidae) and genetic diversity of Cryptosporidium spp. in brown rats (Rattus norvegicus) in the Czech Republic.

The diversity and biology of Cryptosporidium that is specific for rats (Rattus spp.) are not well studied. We examined the occurrence and genetic diversity of Cryptosporidium spp. in wild brown rats (Rattus norvegicus) by microscopy and polymerase chain reaction (PCR)/sequencing targeting the small subunit rDNA (SSU), actin and HSP70 genes. Out of 343 faecal samples tested, none were positive by microscopy and 55 were positive by PCR. Sequence analysis of SSU gene revealed the presence of Cryptosporidium muris (n = 4), C. andersoni (n = 3), C. ryanae (n = 1), C. occultus (n = 3), Cryptosporidium rat genotype I (n = 23), Cryptosporidium rat genotype IV (n = 16) and novel Cryptosporidium rat genotype V (n = 5). Spherical oocysts of Cryptosporidium rat genotype I obtained from naturally-infected rats, measuring 4.4-5.4 μm × 4.3-5.1 μm, were infectious to the laboratory rats, but not to the BALB/c mice (Mus musculus) nor Mongolian gerbils (Meriones unguiculatus). The prepatent period was 3 days post infection and the patent period was longer than 30 days. Naturally- and experimentally-infected rats showed no clinical signs of disease. Percentage of nucleotide similarities at the SSU, actin, HSP70 loci between C. ratti n. sp. and the rat derived C. occultus and Cryptosporidium rat genotype II, III, IV, and V ranged from 91.0 to 98.1%. These genetic variations were similar or greater than that observed between closely related species, i.e. C. parvum and C. erinacei (93.2-99.5%). Our morphological, genetic and biological data support the establishment of Cryptosporidium rat genotype I as a new species, Cryptosporidium ratti n. sp.

Prevalence, Molecular Characterization and Risk Factors of Blastocystis sp. from Farmed Pigs in Yunnan Province, Southwestern China.

Blastocystis is an enteral eukaryote that omnipresent existence in animals and humans global. The objective of the current study investigated the frequency and subtypes to evaluate risk factors for Blastocystis infection in pigs in southwestern China. The 866 fecal samples were collected from farmed pigs in Yunnan province, which were determined by the molecular method of the nested PCR and phylogenetic analysis established on the small subunit rDNA (SSU rDNA) gene fragment. A total of 433 specimens were detected Blastocystis positive, and the prevalence of is 50.0% (433/866). The significant differences in prevalence were also observed in pigs from regions (P < 0.01, df = 8, χ2 = 29.17) and age groups (P < 0.01, df = 2, χ2 = 66.6). Older pigs had more Blastocystis infection than younger ones. Moreover, three zoonotic subtypes were identified by DNA sequence analysis, included ST1 (7.39%, 32/433), ST3 (4.39%, 19/433) and ST5 (88.22%, 382/433). The results suggested a high prevalence and diversified subtypes in farmed pigs in Yunnan province, southwestern China, which indicated pigs are most likely to potential reservoirs and zoonotic transmission of Blastocystis for human infection. To our knowledge, the study is the first large-scale systematic statement of Blastocystis colonization in Yunnan province, Southwestern China, which contributed to the in-depth study of genetic characteristics and prevention, control and treatment of Blastocystis in pigs in Yunnan province and other regions.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Ultrastructure, phylogeny and histopathology of two novel haplosporidians parasitising amphipods, and importance of crustaceans as hosts.

This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.

Ultrastructure and phylogenetic characterization of the microsporidian parasite Heterosporis lessepsianus n. sp. (Microsporidia: Glugeidae) infecting the lizardfish Saurida lessepsianus (Pisces: Synodontidae) inhabiting the Red Sea

Heterosporosis is an increasingly important microsporidian disease distributed worldwide, impacting wild and farmed-raised fish in both fresh and marine water environments. Twenty three out of 130 (17.69%) of the lizardfish Saurida lessepsianus were found to be naturally infected with microsporidian parasites. The rate of parasitic infection was increased during winter reaching 29.23% (19/65) and fall to 6.15% (4/65) in summer. The infection was recorded as numerous macroscopic whitish cysts reached 3.8–6.5 mm in diameter embedded in the abdominal cavity, skeletal muscles and mesenteric tissues of the infected fish inducing an enormous hypertrophy of infected tissues. Light microscopic examination revealed that parasitic foci were encapsulated by a host–derived fibrous membrane containing different developmental stages of the parasite. Spores were oval to pyriform in shape. Transmission electron microscopic study showed the presence of smooth membranes of the sarcoplasmic reticulum forming a thick, amorphous coat surrounding the various developmental stages of the examined parasite (meronts, sporont, sporoblasts, and spores). Mature spores were electron dense and uninucleate. The anchoring disk was found in a central position at the anterior end of the spore and a large vacuole was located at the posterior end. There was a definite number (7–8) of the polar filament turns. Molecular analysis based on the 16 small subunit (SSU) rDNA gene was performed to determine the phylogenetic position of the present parasite species. A 615 bp region of the 16SSU rDNA gene of the studied parasite was sequenced and deposited in GenBank under the accession number MF769371. Multiple sequence alignment demonstrated a high degree of similarity (>82%) with other twenty microsporidian species isolated from different aquatic hosts. The most closely related sequence was provided by the GenBank entry JF745533 for Heterosporis saurida isolated from the marine fish Saurida undosquamis with the highest percentage of identity (98%) and lowest divergence value (0.9). The ultrastructural characteristics and phylogenetic analysis support the recognition of a new species, herein named Heterosporis lessepsianus sp. n.

Integrative taxonomic approach of trypanosomes in the blood of rodents and soricids in Asian countries, with the description of three new species.

Trypanosoma lewisi (Kinetoplastea: Trypanosomatida: Trypanosomatidae) with a cosmopolitan distribution is the type species of the subgenus Herpetosoma, which includes ca. 50 nominal species isolated mainly from rodents. Since members of Herpetosoma in different host species have an almost identical morphology of bloodstream forms, these trypanosomes are referred to as 'T. lewisi-like', and the molecular genetic characterization of each species is necessary to verify their taxonomy. In the present study, we collected blood samples from 89 murid rodents of 15 species and 11 soricids of four species in Indonesia, Philippines, Vietnam, Taiwan, and mainland China for the detection of hemoprotozoan infection. T. lewisi and T. lewisi-like trypanosomes were found in the blood smears of 10 murid animals, which included Bandicota indica (two rats), Rattus argentiventer (one rat), and Rattus tiomanicus (two rats) in Indonesia; Rattus rattus (one rat) in the Philippines; and Niviventer confucianus (four rats) in mainland China. Furthermore, large- or medium-sized non-T. lewisi-like trypanosomes were detected in two soricids, Crocidura dracula in Vietnam and Anourosorex yamashinai in Taiwan, respectively. Molecular genetic characterization of the small subunit (SSU) ribosomal RNA gene (rDNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene indicated that the trypanosomes from all the murid hosts had identical SSU rDNA or gGAPDH gene nucleotide sequences except for those in N. confucianus in mainland China. These N. confucianus-infecting trypanosomes also showed several unique morphological features such as smaller bodies, anteriorly positioned nuclei, and larger rod-shaped kinetoplasts when compared with T. lewisi trypomastigotes. Trypanosoma (Herpetosoma) niviventerae n. sp. is erected for this new species. Similarly, based on morphological and molecular genetic characterization, Trypanosoma sapaensis n. sp. and Trypanosoma anourosoricis n. sp. are proposed for the trypanosomes in C. dracula in Vietnam and A. yamashinai in Taiwan, respectively. More effort directed toward the morphological and molecular genetic characterization of the trypanosomes of rodents and soricids is required to fully understand the real biodiversity of their hemoflagellates.

Prevalence of Cryptosporidium spp. infections in large - scale pig farms in north of Yangtze River in Anhui Province

To characterize the prevalence and species/genotypes of Cryptosporidium spp. in farmed pigs in the north of the Yangtze River in Anhui Province. A total of 500 samples of pig feces were obtained from seven largescale pig farms in the north of the Yangtze River in Anhui Province. PCR and sequences analysis of the small subunit rDNA gene were used to detect and identify the Cryptosporidium species/genotypes. The overall prevalence of Cryptosporidium was 4.8% (24/500). Additionally, Cryptosporidium prevalence was 40.0% in Qianshan and 6.3% in Chuzhou, respectively. No Cryptosporidium infection was found in other sampling areas. The DNA sequence analysis of the SSUrDNA gene revealed that all of the isolates represented C. scrofarum. The Cryptosporidium infection rate (9.1%) of pigs (> 60 days) was significantly higher than the rates of both pigs (< 30 days) and pigs (30-60 days) (both P < 0.01). C. scrofarum in the farmed pigs in the north of the Yangtze River in Anhui Province may be a source of Cryptosporidium infection and pose a potential public health threat to humans and other animals, and therefore, the status should be paid more attention to.

A new species of entomopathogenic nematode Oscheius safricana n. sp. (Nematoda: Rhabditidae) from South Africa

In a recent entomopathogenic nematode (EPN) survey in the North West province of South Africa, Oscheius safricana was isolated from soil samples using the Galleria mellonella bait method. Morphological studies using light microscopy and scanning electron microscopy, molecular analysis of the internal transcribed spacer region (ITS), D2\\D3 expansion segments of the large subunit rDNA gene (LSU) and concise small subunit rDNA gene (SSU), revealed that it was a new species, described herein as Oscheius safricana n. sp. Oscheius safricana n. sp. was characterised by unique ribosomal DNA sequences, amphidelphic reproduction, six separate lips each two bristle-like sensillae, narrow pharynx, valvated basal bulb, lateral field with four lines, leptoderan and closed bursa and fused spicules. This EPN belongs to the group Insectivorus and is morphologically closest to O. necromenus, O. chongmingensis and O. carolinensis. Oscheius safricana n. sp. is symbiotically associated with Serratia marcescens strain MCB.http://www.zoobank.org/urn:lsid:zoobank.org:pub:642E1B7E-B88F-4CE0-9D06-4FA9ECA48847

Molecular identification of Blastocystis sp. subtypes in water samples collected from Black sea, Turkey

The aim of this study was to identify the subtypes of Blastocystis sp. and complete a phylogenetic analysis of 268 water samples that were collected from the Samsun, Amasya and Sinop Provinces of the Black Sea in Turkey, between the years 2011 and 2014. Blastocystis sp. was investigated in 48 uncultured sea water samples that were collected from 4 sites within the Sinop Province. A total of 100 river water samples were collected from 37 sites in the Samsun Province and 120 river water samples were collected from 10 sampling sites within the Amasya Province. The small subunit (SSU) rDNA gene Polymerase chain reaction (PCR) were performed for the detection of Blastocytis sp. and the PCR-positive samples were sequenced. Subsequently, the (SSU) rDNA sequences were aligned by Bioedit and phylogenetic trees were constructed for Blastocystis with reference to the genotypes from GenBank. Blastocystis sp. were found in 3 out of the 75 (4%) river water samples that were collected from the Samsun Province. Six of the 120 (5%) river water samples and 1 out of the 48 (2%) seawater samples were positive for Blastocystis in the Amasya and Sinop Provinces. There were two different subtypes (ST; 1 and 3) found from sequencing all of the samples from the investigated sites. Two and one PCR products were found to be positive for ST1 and ST3 from the different samples collected within the Samsun Province. Two and 4 PCR products from the Amasya Province were ST1 and ST3, respectively and only one sample from the Sinop Province was found to be positive for ST1. This is the first report to identify and report the occurrence of Blastocystis subtypes within the Black Sea of Turkey.

Incidence of Oscheius onirici (Nematoda: Rhabditidae), a potentially entomopathogenic nematode from the marshlands of Wisconsin, USA.

In a search for an entomopathogenic nematode to control cranberry insect pests, three Oscheius populations (Rhabditidae) were recovered through the Galleria-bait method from one sample taken in a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit rDNA gene, D2/D3 expansion segments of the large subunit rDNA gene, internal transcribed spacer, and mitochondrial cytochrome oxidase subunit 1 (CoxI) genes revealed this as Oscheius onirici, a species recently described from a karst cave soil of central Italy. The species belongs to the dolichura-group and is characterized by its DNA sequences; hermaphroditic reproduction; and males not found. A Bacillus-like bacterium appears to be associated with this nematode based on our microscopic and SEM observations; however its identity and persistent association with the nematode has not been confirmed. Nonetheless, this nematode is capable of infecting and killing the sparganothis fruitworm Sparganothis sulfureana Clemens (Lepidoptera: Tortricidae), the brown-banded cockroach Supella longipalpa Fabricius (Blattodea: Ectobiidae), and the cranberry fruitworm Acrobasis vaccinii Riley (Lepidoptera: Pyralidae), under laboratory conditions, and each in less than 72 hr. The mealworm Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) and the greater wax moth Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), are also susceptible, but take 3.5 and 5.2 days to die, respectively. This species is a new potential bio-control agent on insects.