Impact of environmental variables on the isolation,diversity and antibacterial activity of endophytic fungal communities from Madhuca indica Gmel. at different locations in India

A total of 1,897 isolates of endophytic fungi from Madhuca indica Gmel., representing 40 morphologically distinct fungal taxa were obtained from 2,700 segments of stem, bark and leaf from three different locations (Loc 1, Loc 2 and Loc 3) in Uttar Pradesh, India. Out of 40 taxa, 28 were identified microscopically and the remaining 12 by molecular methods. Coelomycetes (62.41 %) were the most prevalent fungal group followed by hyphomycetes (28.89 %) and ascomycetes (8.70 %). Colonisation frequency (CF) was greater in stem (82.55 %) than in leaf (65.00 %) and bark (63.22 %). Due to the dominance of a few taxa, species richness and Shannon and Simpson diversity indices were lower in stem than in leaf and bark at each location. Interestingly, less rainfall and lower temperatures disfavoured the overall colonisation of fungal endophytes at Loc 2. The stem samples from all locations were very similar in their endophytic composition, whereas bark and leaf samples showed differences. The dominant endophytic fungi isolated were Phomopsis sp. 1 (9.185 %), and Colletotrichum gloeosporioides (7.00 %). Principal component analysis showed 55 % tissue specificity with 51.08 % maximum variance. Antibacterial activity revealed that 58.33 % endophytic fungi were active against at least one or more bacterial pathogens, whereas the crude extract of five endophytic fungi inhibited the growth of five or more than five (50 %) of the pathogens tested. This report illustrates the value of having an adequate sample size from different tissues and different locations for species and chemical diversity in search of novel natural products.     

Use of cereals as basal medium for the formulation of alternative culture media for fungi

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliformehad an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.     

Endophytic Fungal Assemblages from Inner Bark and Twig of Terminalia arjuna W. & A. (Combretaceae)

Summary Fungal endophytes reside in healthy tissues of all terrestrial plant taxa studied to date and are diverse and abundant in tropical woody angiosperms. Endophytic fungi were isolated from Terminalia arjuna, an important ethno pharmacological plant extensively used in ayurvedic medicines to treat heart ailments. Isolations were made from symptomless fresh inner bark as well as twig samples of five plants collected from three locations of riparian vegetation during two seasons (monsoon and winter) of 2003 and 2004. Two hundred and seventy eight isolates, representing 22 genera, were obtained from both seasons. Monsoon seasonal isolations representing 22 genera showed greater diversity. Coelomycetes were more numerous during the winter season than hyphomycetes and ascomycetes. Among the endophytes, the genus Pestalotiopsis dominated the endophyte assemblage of T. arjuna collected from different locations, dominance was greater during the winter season than the monsoon season. Endophytic colonization frequency was greater in inner bark (18.5%) than twigs (4.6%). The genera Pestalotiopsis (54.5%), Chaetomium (10.5%) and Myrothecium (9%) were the most predominant endophytes. Rarefaction indices indicated the highest expected number of species for bark samples, monsoon isolations and location 1 (Mysore).     

Social media user’s knowledge on soil plant relationship in Saudi Arabia

The study aimed to investigate the soil-plant relationship by looking at knowledge levels of social media users. The study examined the relationship between the users’ characteristics and their knowledge on soil-plant relationship. Online survey was designed and distributed to gather the data. The number of response was received from 383 respondents where 375 participants provided completed information and the remaining eight responses were eliminated due to quality standards. The results show most of the participants (68.8%) are moderately depend on social media to acquire knowledge on soil-plant relationship and indicated “Twitter” as the highly utilized platform followed by the “Youtube”. Meanwhile, 48.8% of the participants indicated that social media have a high impact on their knowledge-based information on soil-plant relationship and agricultural contents. Inferential analysis shows there was a significant positive relationship (p < 0.01) between independent variables; Twitter, Youtube, Instagram, and Snapchat and the degree of trust on these platforms, and the level’s knowledge on soil-plant relationship (dependent variable). Only 13% of the variance accounted for the impact of social media on participants’ agricultural knowledge-base can be predicted from the combination of participants’ reliance on a set of social media platforms (Twitter, Instagram, Youtube, and Snapchat). The study revealed the trust and confidence of the users using social media on agricultural information/soil plant relationship had a medium effect in social and educational research.     

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.     

Litter fall of the paperbark tree (Melaleuca cuticularis) in the marshes of the Blackwood River Estuary,Western Australia

Paperbark low closed forest, dominated by Melaleuca cuticularis, produced 430 g (dry weight), m^?2 of litter over a year, containing some 3.4 g.m^?2 of nitrogen and 77 mg.m^?2 of phosphorus. Twigs and bark made up more than 50% of the total annual litter fall. The twigs and bark contribute most of the nitrogen (54 %) and phosphorus (56%), compared with leaf fall (37% and 35%) and flower and fruit fall (8% and 9%). The fall of leaves, twigs and bark was primarily related to wind, and flower and fruit fall was greatest after flowering. The litter must make a significant contribution to the accretion of peat. Since the forest covers some 200 ha of the lower Blackwood River estuary, it may contribute some 8001 of litter to the ecosystem each year, containing some 6600 kg of nitrogen and 154 kg of phosphorus.     

Antimicrobial endophytic fungal assemblages inhabiting bark of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. of Indo-Burma mega biodiversity hotspot

Fungal endophytes were isolated from inner bark of Taxus baccata L., an important source of potent anticancer drug taxol. Bark samples were collected from two locations of Arunachal Pradesh, India, part of the Indo-Burma mega biodiversity hotspot, during two seasons i.e. monsoon and winter. Altogether 77 fungal strains representing 18 genera were isolated from T. baccata bark during the present investigation. The colonizing frequency was recorded as 38.5% and the fungal community comprised of 78% of Hyphomycetes, 5.2% of Coelomycetes, 2.6% of Zygomycetes and Ascomycetes and 9.1% of sterile mycelia. Most common and frequently isolated genera were Fusarium, Penicillium and Aspergillus. Simpson and Shannon diversity indices indicated higher species diversity during monsoon than during winter seasons irrespective of the locations. The two locations harbored 5 to 37 endophyte species and the similarity index was low during winter and high during monsoon. Ethyl acetate extract of fermentation cultures of these fungi were tested for their antimicrobial activity against a panel of human pathogenic Gram-positive and Gram-negative bacteria and fungi. Fifteen fungal isolates out of the isolated strains displayed antimicrobial activity. An endophytic fungus, identified as Fusarium sp. displayed significant antimicrobial activity against all the test pathogens.     

Historical and current climate drive spatial and temporal patterns in fungal endophyte diversity

Horizontally-transmitted foliar endophytic fungi can moderate plant tolerance to abiotic and biotic stress. Previous studies have found correlations between climate and endophyte beta diversity, but were unable to clearly separate drivers related to long-term climate, annual weather, and host plants. To address this, we characterized endophyte communities in the perennial C₄ grass, Panicum hallii, across a precipitation gradient in central Texas over 3 years. A total of 65 unique leaf endophytes were isolated and identified based on ITS and LSU regions of rDNA. Mean annual rainfall and temperature were the primary drivers of endophyte richness and community composition, followed by annual weather conditions. In contrast, little explanatory value was provided by plant host traits, vegetation structure, or spatial factors. The importance of historical climate and annual weather in endophyte distributions suggests that species sort by environment and are likely to be affected by future climate change.     

Toward Objective,Morphology-Based Taxonomy: A Case Study on the Malagasy Nesomyrmex sikorai Species Group (Hymenoptera: Formicidae)

Madagascar is one of the world’s greatest biodiversity hotspots, meriting special attention from biodiversity scientists. It is an excellent testing ground for novel techniques in taxonomy that aim to increase classification objectivity and yield greater taxonomic resolving power. Here we reveal the diversity of a unique and largely unexplored fragment of the Malagasy ant fauna using an advanced combination of exploratory analyses on quantitative morphological data allowing for increased objectivity in taxonomic workflow. The diversity of the Nesomyrmex sikorai species-group was assessed via hypothesis-free nest-centroid-clustering combined with recursive partitioning to estimate the number of morphological clusters and determine the most probable boundaries between them. This combination of methods provides a highly automated and objective species delineation protocol based on continuous morphometric data. Delimitations of clusters recognized by these exploratory analyses were tested via confirmatory Linear Discriminant Analysis (LDA) and Multivariate Ratio Analysis (MRA). The final species hypotheses are corroborated by many qualitative characters, and the recognized species exhibit different spatial distributions and occupy different ecological regions. We describe and redescribe eight morphologically distinct species including six new species: Nesomyrmex excelsior sp. n., N. modestus sp. n., N. reticulatus sp. n., N. retusispinosus (Forel, 1892), N. rugosus sp. n., N. sikorai (Emery, 1896), N. striatus sp. n., and N. tamatavensis sp. n. An identification key for their worker castes using morphometric data is provided.     

Du’an Karst of Guangxi: a kingdom of the cavernicolous genus Dongodytes Deuve (Coleoptera,Carabidae, Trechinae)

Recent cave biodiversity surveys carried out in Du’an County and its adjacent areas of northwestern Guangxi, China, have revealed some exciting scientific findings. In a very limited area seven new species of the cavernicolous trechine genus Dongodytes Deuve, 1993 were found and are described: Dongodytes (s. str.) elongatus sp. n., Dongodytes (s. str.) troglodytes sp. n., Dongodytes (s. str.) lani sp. n., Dongodytes (Dongodytodes) brevipenis sp. n., Dongodytes (Dongodytodes) jinzhuensis sp. n., Dongodytes (Dongodytodes) inexpectatus sp. n. and Dongodytes (Dongodytodes) yaophilus sp. n. Diagnoses and notes on the genus, subgenera, and two known species in Du’an Karst, Dongodytes (s. str.) baxian Tian, 2011 and Dongodytes (Dongodytodes) deharvengi Tian, 2011, are also given. A key to subgenera and all species of Dongodytes is provided. To date, Dongodytes becomes one of the richest in species genera of subterranean carabid trechines in China with 12 species which are arranged into two subgenera. Dongodytes (s. str.) Deuve is composed of seven species, four of which from Du’an County, each of other three from Bama, Fengshan and Tian’e Counties, respectively. All species of the subgenus Dongodytodes Tian, 2011 are recorded from Du’an Karst. By having 10 species (nine Dongodytes and one Libotrechus Uéno, 1998), Du’an Karst holds the richest specific diversity of cavernicolous Trechinae in China. Dongodytes species are distributed in a very limited area of the river Hongshui He drainages in northwestern Guangxi, and the river acts as a natural barrier of Dongodytes dispersal at only a specific level. However, all members of Dongodytodes are recorded from the eastern or northern bank of Hongshui He.     

Diatoms and Other Epibionts Associated with Olive Ridley (Lepidochelys olivacea) Sea Turtles from the Pacific Coast of Costa Rica

Although the sea turtles have long been familiar and even iconic to marine biologists, many aspects of their ecology remain unaddressed. The present study is the first of the epizoic diatom community covering the olive ridley turtle’s (Lepidochelys olivacea) carapace and the first describing diatoms living on sea turtles in general, with the primary objective of providing detailed information on turtle epibiotic associations. Samples of turtle carapace including the associated diatom biofilm and epizoic macro-fauna were collected from Ostional beach (9° 59´ 23.7´´ N 85° 41´ 52.6´´ W), Costa Rica, during the arribada event in October 2013. A complex diatom community was present in every sample. In total, 11 macro-faunal and 21 diatom taxa were recorded. Amongst diatoms, the most numerous were erect (Achnanthes spp., Tripterion spp.) and motile (Haslea sp., Navicula spp., Nitzschia spp., Proschkinia sp.) forms, followed by adnate Amphora spp., while the most common macro-faunal species was Stomatolepas elegans (Cirripedia). Diatom densities ranged from 8179 ± 750 to 27685 ± 4885 cells mm^-2. Epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. The relatively low diatom species number, stable species composition and low inter-sample dissimilarities (14.4% on average) may indicate a mutualistic relationship between the epibiont and the basibiont. Dispersal of sea turtle diatoms is probably highly restricted and similar studies will help to understand both diatom diversity, evolution and biogeography, and sea turtle ecology and foraging strategies.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

New records of the genus Iporhogas Granger (Hymenoptera,Braconidae, Rogadinae) from Vietnam,with description of four new species

The genus Iporhogas Granger, 1949 (Braconidae: Rogadinae) is recorded for the first time for Vietnam. Four new species of the genus Iporhogas, viz. Iporhogas albilateralis sp. n., I. contrastus sp. n., I. simulatus sp. n. and I. tricoloratus sp. n., from Vietnam are described and illustrated, and additionally, one species, Iporhogas guangxiensis Chen & He, 1997, is newly recorded for Vietnam’s fauna of the family Braconidae. A key to the five Vietnamese species of the genus Iporhogas and a checklist with distributions of the ten species are provided.     

A new species of Pseudorobillarda, an endophyte from Thuja occidentalis in Canada, and a key to the species

Pseudorobillarda monica sp. nov. is described and illustrated. The endophyte was isolated from living leaves and bark of twigs of a Thuja occidentalis bonsai (>90 years old) at the Montréal Botanical Garden and ornamental trees in Montréal urban plantations. This pycnidial fungus is typical of the genus in morphology but clearly differs from other species in Pseudorobillarda by the distinct size of the conidiomata and the shape and size of conidia and paraphyses. Its taxonomic placement is discussed and a key to the species of Pseudorobillarda is provided.     

High Diversity in Cretaceous Ichthyosaurs from Europe Prior to Their Extinction

Background

Ichthyosaurs are reptiles that inhabited the marine realm during most of the Mesozoic. Their Cretaceous representatives have traditionally been considered as the last survivors of a group declining since the Jurassic. Recently, however, an unexpected diversity has been described in Upper Jurassic–Lower Cretaceous deposits, but is widely spread across time and space, giving small clues on the adaptive potential and ecosystem control of the last ichthyosaurs. The famous but little studied English Gault Formation and ‘greensands’ deposits (the Upper Greensand Formation and the Cambridge Greensand Member of the Lower Chalk Formation) offer an unprecedented opportunity to investigate this topic, containing thousands of ichthyosaur remains spanning the Early–Late Cretaceous boundary.

Methodology/Principal Findings

To assess the diversity of the ichthyosaur assemblage from these sedimentary bodies, we recognized morphotypes within each type of bones. We grouped these morphotypes together, when possible, by using articulated specimens from the same formations and from new localities in the Vocontian Basin (France); a revised taxonomic scheme is proposed. We recognize the following taxa in the ‘greensands’: the platypterygiines ‘Platypterygius’ sp. and Sisteronia seeleyi gen. et sp. nov., indeterminate ophthalmosaurines and the rare incertae sedis Cetarthrosaurus walkeri. The taxonomic diversity of late Albian ichthyosaurs now matches that of older, well-known intervals such as the Toarcian or the Tithonian. Contrasting tooth shapes and wear patterns suggest that these ichthyosaurs colonized three distinct feeding guilds, despite the presence of numerous plesiosaur taxa.

Conclusion/Significance

Western Europe was a diversity hot-spot for ichthyosaurs a few million years prior to their final extinction. By contrast, the low diversity in Australia and U.S.A. suggests strong geographical disparities in the diversity pattern of Albian–early Cenomanian ichthyosaurs. This provides a whole new context to investigate the extinction of these successful marine reptiles, at the end of the Cenomanian.     

A willow drawing from 1786: the earliest depiction of intraspecific trait variation in plants?

Background and AimsThe study of intraspecific trait variation (ITV) in plants has a long history, dating back to the fourth century BC. Its existence was widely acknowledged by the end of the 18th century, although systematic and experimental studies commenced only a century later. However, the historiography of ITV has many gaps, especially with regard to early observations and visual documents. This note identifies an early depiction of plant ITV.MethodsThe botanical works of Johann Wolfgang von Goethe (1749–1832), a German writer and naturalist, were subjected to close reading. This included all publications and unpublished sources related to botany between 1785 and 1832 (e.g. notes, drafts, diaries, letters, drawings). This material is accessible in the multi-volume historical-critical edition of Goethe’s studies in natural science (Leopoldina-Ausgabe).Key ResultsA diary entry from 9 September 1786 described changes in leaf morphology along an elevation gradient in the Bavarian Alps. The leaves of an unidentified species of willow (Spix sp.) and gentian (Gentiana sp.) were said to become narrower with increasing elevation; leaves also stood further apart on twigs, and the latter became thinner. A crude drawing of two willow twigs illustrated the differences. Goethe conjectured that the differences were due to environmental conditions.ConclusionsGoethe’s notes were anecdotal, and it is unclear whether the observed plant individuals actually belonged to the same species. Nevertheless, the notes represent an early and clear articulation of the hypothesis that changes in environmental conditions can cause ITV in a natural plant population. The drawing may be the earliest visual record of environmentally caused plant ITV in the wild.     

Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria

The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis.     

Contributions to the knowledge of subterranean trechine beetles in southern China’s karsts: five new genera (Insecta,Coleoptera, Carabidae,Trechinae)

Recent discoveries reveal that southern China’s karsts hold the most diverse and morphologically modified subterranean trechine beetles in the world, albeit the first troglobitic blind beetle was only reported in the early 1990’s. In total, 110 species belonging to 43 genera of cavernicolous trechines have hitherto been recorded from the karsts of southern China, including the following five new genera proposed below: Shiqianaphaenops Tian, gen. n., to contain two species: Shiqianaphaenops majusculus (Uéno, 1999) (= Shenaphaenops majusculus Uéno, 1999, comb. n.), the type species from Cave Feng Dong, Shiqian, Guizhou, and Shiqianaphaenops cursor (Uéno, 1999) (= Shenaphaenops cursor Uéno, 1999, comb. n.), from Cave Shenxian Dong, Shiqian, Guizhou; and the monotypic Dianotrechus Tian, gen. n. (the type species: Dianotrechus gueorguievi Tian, sp. n., from Cave Dashi Dong, Kunming, Yunnan), Tianeotrechus Tian & Tang, gen. n. (the type species: Tianeotrechus trisetosus Tian & Tang, sp. n., from Cave Bahao Dong, Tian’e County, Guangxi), Huoyanodytes Tian & Huang, gen. n. (the type species: Huoyanodytes tujiaphilus Tian & Huang, sp. n., from Longshan, Hunan) and Wanhuaphaenops Tian & Wang, gen. n. (the type species: Wanhuaphaenops zhangi Tian & Wang, sp. n., from Cave Songjia Dong, Chenzhou, Hunan).     

Blastocystis Isolates from Patients with Irritable Bowel Syndrome and from Asymptomatic Carriers Exhibit Similar Parasitological Loads,but Significantly Different Generation Times and Genetic Variability across Multiple Subtypes

Blastocystis spp is a common intestinal parasite of humans and animals that has been associated to the etiology of irritable bowel syndrome (IBS); however, some studies have not found this association. Furthermore, many biological features of Blastocystis are little known. The objective of present study was to assess the generation times of Blastocystis cultures, from IBS patients and from asymptomatic carriers. A total of 100 isolates were obtained from 50 IBS patients and from 50 asymptomatic carriers. Up to 50 mg of feces from each participant were cultured in Barret’s and in Pavlova’s media during 48 h. Initial and final parasitological load were measured by microscopy and by quantitative PCR. Amplicons were purified, sequenced and submitted to GenBank; sequences were analysed for genetic diversity and a Bayesian inference allowed identifying genetic subtypes (ST). Generation times for Blastocystis isolates in both media, based on microscopic measures and molecular assays, were calculated. The clinical symptoms of IBS patients and distribution of Blastocystis ST 1, 2 and 3 in both groups was comparable to previous reports. Interestingly, the group of cases showed scarce mean nucleotide diversity (π) as compared to the control group (0.011±0.016 and 0.118±0.177, respectively), whilst high gene flow and small genetic differentiation indexes between different ST were found. Besides, Tajima’s D test showed negative values for ST1-ST3. No statistical differences regarding parasitological load between cases and controls in both media, as searched by microscopy and by qPCR, were detected except that parasites grew faster in Barret’s than in Pavlova’s medium. Interestingly, slow growth of isolates recovered from cases in comparison to those of controls was observed (p<0.05). We propose that generation times of Blastocystis might be easily affected by intestinal environmental changes due to IBS probably because virulent strains with slow growth may be selected, reducing their genetic variability.