ITS Fragments Research Articles

Updates on Ecology and Life Cycle of Sulcascaris sulcata (Nematoda: Anisakidae) in Mediterranean Grounds: Molecular Identification of Larvae Infecting Edible Scallops.

Sulcascaris sulcata is a nematode parasite of sea turtles, widespread in neritic foraging grounds with variable prevalence, reaching 30% in loggerhead turtles Caretta caretta feeding in northern Adriatic Sea. Ulcerative gastritis associated to high intensity of infection is reported in this host species. The life cycle of S. sulcata has been elucidated in Australian and American waters, demonstrating the ability of the species of infecting a wide range of intermediate hosts, represented by bivalve and gastropod molluscs. During regular sanitary inspections, nematode larvae were found within the adductor muscle of Pecten jacobeus and Aequipecten opercularis collected from the Northern Adriatic Sea. Morphological and molecular analyses were performed for the identification of larvae, and molecular data were obtained from adult S. sulcata as well. Analysis of the sequences of ITS fragment, and mitochondrial genes cox1 and cox2, revealed high genetic similarity among all the samples, and no geographical clustering was observed between adult parasites collected in Adriatic and Tyrrhenian Seas. A common allele pool was detected also between the two developmental stages, included larvae from Pecten and Aequipecten. The comparison with other members of the family Anisakidae demonstrated that S. sulcata formed a clear monophyletic cluster. This study reports the first identification of intermediate hosts for S. sulcata within the Mediterranean Sea. Infection in edible scallops justifies the exclusion of the product from the market and zoonotic potential of larvae of this anisakid nematode are yet to be completely excluded. Fidelity of sea turtles to selected foraging grounds, such as the Northern Adriatic shelf, warrants the life cycle of S. sulcata to perpetrate in the area; at the same time, long distance migrations of individuals justify the dispersal of infecting elements over the Mediterranean basin, regardless of turtles' origin.

Genotyping the causative agents of cutaneous leishmaniasis in Ahvaz southwest of Iran.

With sand flies as the main vectors, Leishmania species cause ancient zoonotic diseases called leishmaniasis. Iran is an endemic country regarding cutaneous leishmaniasis. A number of 100 smear slides were collected from cutaneous lesions referred to Ahvaz health centers. The DNA was extracted and ITS1-PCR using LITSR and L5.8S primer pair was performed to detect the genus Leishmania. Then, enzymatic digestion of PCR products was done by HaeIII (species detection), TaqI (strain detection), DpnI and HpaII (mutation assessment). Furthermore, 50 samples were sent for sequencing. Microscopic examination showed amastigote form in all 100 slides. Also, molecular identification confirmed the infection of all cases to Leishmania genus, representing a 350bp band. HaeIII digestion yielded 150 and 200bp bands, indicating Leishmania major, while 130 and 200bp fragments following TaqI digestion suggested A1 strain of the parasite. Moreover, no likely mutations was detected in ITS1 fragment of obtained parasites using DpnI (140 and 200bp digestion) and HpaII (without digestion). The sequencing result also was consistent with our findings, having 100% homology to A1 strain sequence (AY550178). Leishmania major A1 strain was the predominant species in clinical samples of Ahvaz. Nevertheless, future researches should address the parasite strains in other foci and hosts of epidemiological significance.

Morphological and molecular characterization of the Colomerus vitis erineum strain (Trombidiformes: Eriophyidae) from grapevine erinea and buds.

The grapevine erineum mite strain (GEM) of Colomerus vitis (Pagenstecher) has spread throughout the main viticultural areas worldwide and was recently demonstrated to be a vector of Grapevine pinot gris virus (GPGV) and Grapevine inner necrosis virus (GINV). Its females mainly overwinter under the outer bud scales as winter morphs (deutogynes). Goals of this study were to characterize the morphology of protogynes (spring-summer morphs) and deutogynes (winter morphs), to confirm their genetic similarity, and to establish the seasonal period of the deutogyne occurrence. Buds or leaves from a single vineyard (cv. Luisa), Bari area, Apulia, Italy, infested with GEM were sampled 6 × from December 2015 to January 2017. Sixty-six traits commonly used for taxonomic identification were analysed on females. The length of the tibial setae l' on leg I and the tarsal setae ft' on leg II, as well as the number of smooth dorsal semiannuli differed significantly between protogynes and deutogynes, and were easier to detect than other significantly distinctive traits. ITS1 was investigated in individuals collected from buds and erinea, and the sequences confirmed that these two morphs have identical ITS1 fragments. The 1-year study demonstrated the simultaneous presence of protogynes and deutogynes in July and September 2016, whereas only protogynes were found in April and May 2016, and only deutogynes in December 2015 and January 2017.

Strongyloidiasis in northern Vietnam: epidemiology, clinical characteristics and molecular diagnosis of the causal agent

BackgroundStrongyloidiasis is a health problem in Vietnam, but appropriate information is still limited. The aim of this study was to determine the prevalence, geographical distribution, epidemiological aspects, symptoms and other health indicators of Strongyloides stercoralis infections in patients from 27 provinces of northern Vietnam attending the Hanoi Medical University Hospital during 2016 and 2017.MethodsBlood samples of 2000 patients were analyzed for S. stercoralis infection with an IgG ELISA test. Seroprevalence was analyzed by gender, age group, locality of origin (rural or urban areas) and symptoms. Stools from the seropositive patients were examined for the detection of worms which were subsequently used for species identification by morphology and rDNA ITS1 sequencing.ResultsA seroprevalence of 20% was detected, showing an increasing prevalence from young to older age groups but without significant gender difference. Seroprevalence was higher in rural areas than in urban areas, both in general and individually in all provinces without exception, and lower in the mountainous areas than in the large valley lowlands. The follow-up of the 400 patients showed eosinophilia in 100% of cases, diarrhoea in 64.5%, digestion difficulties in 58.0%, stomachache in 45.5%, stomach and duodenal ulcers in 44.5%, itching in 28.0% and fever in 9.5%. The prevalence of symptoms and signs were also higher in older age groups than in younger age groups. Worms were detected in stools of 10.5% of the patients. Sequencing of a 501-bp nuclear ribosomal DNA ITS1 fragment allowed for the verification of infection by Strongyloides stercoralis.ConclusionsTo our knowledge, this study is the largest survey of human strongyloidiasis in Vietnam so far and the first molecular identification of this nematode species in this country. Long-term chronicity may probably be usual in infected subjects, mainly in the older age groups.

Application of ITS2 secondary structure phylogenetic information in DNA barcode identification of Chrysanthemum indicum and its related plants

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.

Gut microbiome composition in the Hispanic Community Health Study/Study of Latinos is shaped by geographic relocation, environmental factors, and obesity

BackgroundHispanics living in the USA may have unrecognized potential birthplace and lifestyle influences on the gut microbiome. We report a cross-sectional analysis of 1674 participants from four centers of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), aged 18 to 74 years old at recruitment.ResultsAmplicon sequencing of 16S rRNA gene V4 and fungal ITS1 fragments from self-collected stool samples indicate that the host microbiome is determined by sociodemographic and migration-related variables. Those who relocate from Latin America to the USA at an early age have reductions in Prevotella to Bacteroides ratios that persist across the life course. Shannon index of alpha diversity in fungi and bacteria is low in those who relocate to the USA in early life. In contrast, those who relocate to the USA during adulthood, over 45 years old, have high bacterial and fungal diversity and high Prevotella to Bacteroides ratios, compared to USA-born and childhood arrivals. Low bacterial diversity is associated in turn with obesity. Contrasting with prior studies, our study of the Latino population shows increasing Prevotella to Bacteroides ratio with greater obesity. Taxa within Acidaminococcus, Megasphaera, Ruminococcaceae, Coriobacteriaceae, Clostridiales, Christensenellaceae, YS2 (Cyanobacteria), and Victivallaceae are significantly associated with both obesity and earlier exposure to the USA, while Oscillospira and Anaerotruncus show paradoxical associations with both obesity and late-life introduction to the USA.ConclusionsOur analysis of the gut microbiome of Latinos demonstrates unique features that might be responsible for health disparities affecting Hispanics living in the USA.

The Iranian Hyalomma (Acari: Ixodidae) with molecular evidences to understand taxonomic status of species complexes

The identification of Hyalomma is a challenging issue in the systematics of ixodid ticks. Here, we examined 960 adult males of Hyalomma tick from 10 provinces of Iran using morphological and molecular methods. PCR was carried out on 60 samples to amplify an ITS2 fragment of nuclear and a COI fragment of mitochondrial genomes. Nine species, namely H. aegyptium, H. anatolicum, H. asiaticum, H. scupense, H. dromedarii, H. excavatum, H. marginatum, H. rufipes and H. schulzei were identified. The validity of H. rufipes and H. excavatum can be challenged. We concluded that these species should be regarded as H. marginatum and H. anatolicum complexes, respectively. Furthermore, the taxonomic status of the two closely related H. anatolicum and H. asiaticum was confirmed as distinct species.

Genotyping of Causative Agents of Cutaneous Leishmaniasis in Patients Using PCR-RFLP Method in Dasht-e-Azadegan County, Southwest of Iran, in 2016

Background: As a neglected disease, cutaneous leishmaniasis renders considerable incidence rates. Eastern Mediterranean countries such as Iran are endemic for this infection. Objectives: Herein, we determined the species/strains of the causative agents of cutaneous leishmaniasis in Dasht-e-Azadegan, Khuzestan, Iran, using PCR-RFLP in 2016. Methods: We microscopically examined 80 smear slides from suspected patients referring to health centers of the city. After DNA extraction from slide materials, the amplification of the ITS1 fragment was done using the LITSR and L5.8S primer pair for Leishmania molecular detection. Subsequently, HaeIII (species level), TaqI (strain level), DpnI, and HpaII (mutation analysis) digestion was exerted based on the RFLP method. Results: Microscopic examination revealed the amastigote forms of Leishmania in all 80 samples. A 350 bp band was amplified by ITS1-PCR, which confirmed the infection at the molecular level. Following HaeIII digestion, 150 and 200 bp fragments were produced indicating Leishmania major. Also, TaqI digestion rendered 130 and 200 bp bands suggesting the A1 strain. Moreover, no mutations were detected in the genome of the identified L. major A1 strains by DpnI (140 and 200 bp bands) and HpaII (no digestion) digestion. Conclusions: The genotypic heterogeneity of Leishmania species is of utmost importance for better treatment choices, appropriate diagnosis, and preventive measures. Future work in the area should address the parasite strains in alternative vector/reservoir hosts.

Correlates of Gut Function in Children Hospitalized for Severe Acute Malnutrition, a Cross-sectional Study in Uganda.

Children with severe acute malnutrition (SAM) may have impaired intestinal function, which can result in malabsorption, diarrhoea, and poor growth. This study evaluated the gut function of children with SAM using fecal and blood biomarkers and assessed their correlates. A cross-sectional study, nested in a randomized trial (www.isrctn.com, ISRCTN 16454889), was conducted at Mulago hospital, Uganda among subgroups of 400 children with complicated SAM and 30 community controls. Gut function was evaluated by 5 biomarkers: plasma citrulline, fecal myeloperoxidase and fecal neopterin, bacterially derived 16S rRNA gene and internal transcribed Spacer region (ITS) specific for Candida spp. in blood. Compared with controls, children with SAM had lower median plasma citrulline (5.14 vs 27.4 μmol/L, P < 0.001), higher median fecal myeloperoxidase (18083 vs 7482 ng/mL, P = 0.001), and fecal neopterin (541 vs 210 nmol/L, P < 0.001). A higher blood concentration of 16S rRNA gene copy numbers was observed among children with SAM (95 vs 28 copies/μl, P = 0.05), whereas there was no difference in the blood concentration of Candida-specific ITS fragment.Among those with SAM, plasma citrulline was lower in children with edema, diarrhoea, dermatosis, and plasma C-reactive protein (CRP) >10 mg/L. Fecal neopterin was positively correlated with symptoms of fever and cough whereas it was negatively correlated with mid-upper arm circumference (MUAC), weight-for-height z score (WHZ), edema, and dermatosis. Children with complicated SAM seem to have impaired gut function characterized by reduced enterocyte mass, intestinal inflammation, and increased bacterial translocation.

Effect of depleted uranium on a soil microcosm fungal community and influence of a plant-ectomycorrhizal association

Fungi are one of the most biogeochemically active components of the soil microbiome, becoming particularly important in metal polluted terrestrial environments. There is scant information on the mycobiota of uranium (U) polluted sites and the effect of metallic depleted uranium (DU) stress on fungal communities in soil has not been reported. The present study aimed to establish the effect of DU contamination on a fungal community in soil using a culture-independent approach, fungal ribosomal intergenic spacer analysis (F-RISA). Experimental soil microcosms also included variants with plants (Pinus silvestris) and P. silvestris/Rhizopogon rubescens ectomycorrhizal associations. Soil contamination with DU resulted in the appearance of RISA bands of the ITS fragments of fungal metagenomic DNA that were characteristic of the genus Mortierella (Mortierellomycotina: Mucoromycota) in pine-free microcosms and for ectomycorrhizal fungi of the genus Scleroderma (Basidiomycota) in microcosms with mycorrhizal pines. The precise taxonomic affinity of the ITS fragments from the band appearing for non-mycorrhizal pines combined with DU remained uncertain, the most likely being related to the subphylum Zoopagomycotina. Thus, soil contamination by thermodynamically unstable metallic depleted uranium can cause a significant change in a soil fungal community under experimental conditions. These changes were also strongly affected by the presence of pine seedlings and their mycorrhizal status which impacted on DU biocorrosion and the release of bioavailable uranium species.

Determination of Echinococcus Granulosus Genotypes in Livestock Slaughtered in Shush County, Southwest Iran Using PCR-RFLP.

SummaryEchinococosis is a zoonotic disease caused by the larval stages of Echinococcus spp. that occurs in most parts of the world. Herein, we aimed to evaluate the genotypes of isolated hydatid cysts from slaughtered animals in Shush county, southwestern Iran. Totally, 96 hydatid cysts were collected, including 11 buffaloes, 13 cattle, 12 goat and 60 sheep. The PCR was done by a primer pair (BDI and 4s) to amplify ITS1 fragment. Four restriction endonucleases including AluI, HpaII, RsaI, and TaqI were used for RFLP products and enzymatic reactions were electrophoresed. Finally, twenty PCR products were sent for sequencing and phylogenetic tree was drawn with MEGA6. Molecular identification of 96 hydatid cysts demonstrated a distinctive 1000 bp fragment in all samples from four animal hosts. RFLP analysis showed similar digestion patterns in all samples. AluI digestion yielded 800 bp and 200 bp fragments, HpaII digestion made 700 bp and 300 bp fragments and RsaI digestion entailed 655 and 345segments. Moreover, TaqI rendered no digestion pattern on rDNA-ITS1 region. Additionally, E. granulosus sensu stricto (G1-3 complex) was the prevailing genotype in all livestock samples, according to PCR-RFLP and sequencing analyses.

&lt;p class="Body"&gt;Comparison of five DNA extracting protocols from Typhlodromus (Anthoseius) bagdasarjani Wainsten and Arutunjan (Acari: Phytoseiidae)

Obtaining clean DNA from mites to be used in molecular studies has always been a challenging task. In this study, five protocols (CTAB-based handmade kit, salting out protocol, STE protocol, modified CTAB-based protocol and wizard Genomic DNA purification kit) were evaluated to extract DNA from Typhlodromus (Anthoseius) bagdasarjani. Quantitative and qualitative analyses of the DNA concentrations and a comparison between the ratio of absorption of wavelength from 260 to 280 nm in a completely randomized split plot design for five extraction protocols and three levels of 1, 5, 10 mites were performed. PCR protocol was used to examine the amplification of the ITS1 fragment from the specimens. The results showed that wizard kit and CTAB modified protocol were the bests. However, economically, modified CTAB-based protocol is the best choice for extensive experiments, since this protocol allows extracting DNA from an individual mite and provides the highest concentration DNA (143.35±0.85ng) and the best OD (1.80±0.03) for molecular studies.

TEKNIK PCR ITS-RFLP UNTUK SELEKSI ISOLAT JAMUR PADA PENGUJIAN AGEN PENGENDALI HAYATI PADA SERANGAN GANODERMA TANAMAN Acacia mangium

In vivo control activities test of BCA candidate is required to evaluate its effectiveness. This test involved identification of the fungal species that inhabited the experimental plant after inoculation of BCA and pathogen. Identification of inhabitant fungi in the BCA control activity test can be conducted by culturing the fungi on artificial media. Fungal species can be identified based on morphological characters or genetic characters of fungal culture. This study was conducted to determine the potential of PCR ITS-RFLP molecular markers in the initial selection process of fungal isolates before identification based on DNA sequences and to select the enzyme that produce polymorphic PCR ITS RFLP pattern. Three enzymes (DraI, EcoRI and HinfI) were successfully separated 8 fungal cultures used in this study into three groups based on the pattern of PCR ITS DNA, while five other enzymes (BamHI, BclI HaeII, HpaI, and HindIII) were failed to cut the DNA ITS fragments, except for one isolate.

Effects of dietary supplementation with lysozyme on the structure and function of the cecal microbiota in broiler chickens.

Lysozyme is known to eliminate intestinal pathogens in poultry and improve their growth performance. However, whether it can replace antibiotic growth promoters without the associated risk of the emergence of antibiotic-resistant bacterial strains is not known, and the effects of lysozyme supplementation on the composition, biodiversity, and function of the chicken gut microbiota remain unclear. Here, we used the 16S rRNA gene and ITS fragment Illumina sequencing combined with transcriptomic analysis to address this issue. A total of 400 1-d-old Di Gao chicks were allocated randomly to five groups, each consisting of four replicates (20 birds/group). The chicks were fed a starter (1–21 d) and a grower (22–42 d) diet supplemented with 0 (control), 40 (LYS40), 100 (LYS100), or 200 ppm (LYS200) lysozyme, or 400 ppm flavomycin as an antibiotic control for 6 weeks. Lysozyme administration did not contribute significantly (P > 0.05) to the growth of the broiler chickens. No significant (P > 0.05) differences in the diversity and composition of the bacterial and fungal communities in the cecal microbiota of chickens in the different diet groups were found. However, lysozyme supplementation led to a significant (P < 0.05) enrichment of genes involved in the synthesis/degradation of bacterial outer membranes and cell walls, cross-cell substrate transport, and carbohydrate metabolic processes, thus possibly promoting the cecal microbiota carbon and energy metabolism. Bacteroides contributed 31.9% of glycoside hydrolase genes (17,681–24,590), 26.1% of polysaccharide lyase genes (479–675), 20.7% of carbohydrate esterase genes (3,509–4,101), 8.8% of auxiliary activity genes (705–1,000), 16.2% of glycosyltransferase genes (5,301–6,844), and 13.9% of carbohydrate-binding module genes (8838–15,172) identified in the cecal samples. Thus, they were the main players in the breakdown of non-starch polysaccharides in the cecum, although Parabacteroides, Alistipes, Prevotella, Clostridium, Blastocystis, Barnesiella, Blautia, Faecalibacterium, Subdoligranulum, Megamonas, Eubacterium, Ruminococcus, Paenibacillus, Bifidobacterium, Akkermansia, and other bacteria also participated.

Molecular characterization of Hymenolepis nana based on nuclear rDNA ITS2 gene marker.

Hymenolepis nana is a zoonotic tapeworm with widespread distribution. The goal of the present study was to identify the parasite in the specimens collected from NorthWestern regions of Iran using PCR-sequencing method. A total of 1521 stool samples were collected from the study individuals. Initially, the identification of hymenolepis nana was confirmed by parasitological method including direct wet-mount and formalin-ethyl acetate concentration methods. Afterward, PCR-sequencing analysis of ribosomal ITS2 fragment was targeted to investigate the molecular identification of the parasite. Overall, 0.65% (10/1521) of the isolates were contaminated with H. nana in formalin-ethyl acetate concentration. All ten isolates were succefully amplified by PCR and further sequenced. The determined sequences were deposited in GenBank under the accession numbers MH337810 -MH337819. Our results clarified the presence of H. nana among the patients in the study areas. In addition, the molecular technique could be accessible when the human eggs are the only sources available to identify and diagnose the parasite.

Genetic Analysis of Toxocara Spp. in Stray Cats and Dogs in Van Province, Eastern Turkey.

IntroductionToxocara canis and Toxocara cati are roundworms of dogs and cats. The purpose of this study was to investigate the infection caused by these ascarids in cats and dogs, using microscopic and molecular analysis methods.Material and MethodsAdult ascarids were gathered from the faeces of dogs and cats in Van province, in 2015–2016. Existing keys and PCR sequencing of the ITS-2 fragment were used to identify the morphological features of the parasite species.ResultsIt was observed that out of 20 adult ascarids, 17 and 3 were found to be Toxocara canis and Toxocara cati, respectively. The ITS-2 gene region was amplified by PCR to perform molecular analysis. Genotyping indicated that the dogs and cats were infected with T. canis and T. cati, respectively, and none had Toxascaris leonina.ConclusionTo the best of our knowledge, this is the first report on the molecular characteristics of adult ascaridoid nematodes from cats and dogs in Turkey. The molecular approaches established in this study enable molecular identification and genetic structure studies of the ascaridoids.

Root growth, function and rhizosphere microbiome analyses show local rather than systemic effects in apple plant response to replant disease soil.

Apple replant disease (ARD) is the phenomenon of soil decline occurring after repeated planting of apple trees at the same site. This study aimed to elucidate whether ARD is systemic, i.e. whether the contact of parts of the root system with ARD soil causes the whole plant to show poor shoot and root growth. A split-root experiment was conducted with seedlings of ‘M26’, offering the same plant for its root system the choice between the substrates ARD soil (+ARD), γ-sterilized ARD soil (-ARD) or soil from a grass parcel (Control) with the following combinations: +ARD/+ARD, -ARD/-ARD; +ARD/-ARD; +ARD/Control. Root growth was analysed throughout the 34-day growing period. Samples from bulk, rhizosphere and rhizoplane soil were collected separately for each compartment, and analysed by fingerprints of 16S rRNA gene or ITS fragments amplified from total community (TC) DNA. The response of the plant to +ARD was not systemic as root growth in -ARD compartment was always superior to root growth in +ARD soil. Crosswise 15N-labelling of the N-fertilizer applied to the split-root compartments showed that nitrate-N uptake efficiency was higher for roots in -ARD soil compared to those in +ARD. Bacterial and fungal community composition in the rhizoplane and rhizosphere of the same plants differed significantly between the compartments containing +ARD/-ARD or +ARD/Control. The strongest differences between the bacterial fingerprints were observed in the rhizoplane and rhizosphere. Bacterial genera with increased abundance in response to ARD were mainly Streptomyces but also Sphingobium, Novosphingobium, Rhizobium, Lysobacter and Variovorax. The strongest differences between the fungal fingerprints were observed in bulk soil. Our data showed that the response of the apple plant to ARD soil is local and not systemic.

Dynamics of soil respiration and microbial communities: Interactive controls of temperature and substrate quality

Soil microbial communities mediate soil feedbacks to climate; a thorough understanding of their response to increasing temperatures is therefore central to predict climate-induced changes in carbon (C) fluxes. However, it is unclear how microbial communities will change in structure and function in response to temperature change and to the availability of organic C which varies in complexity. Here we present results from a laboratory incubation study in which soil microbial communities were exposed to different temperatures and organic C complexity. Soil samples were collected from two land-use types differing in climatic and edaphic conditions and located in two regions in southwest Germany. Soils amended with cellobiose (CB), xylan, or coniferyl alcohol (CA, lignin precursor) were incubated at 5, 15 or 25 °C. We found that temperature predominantly controlled microbial respiration rates. Increasing temperature stimulated cumulative respiration rates but decreased total microbial biomass (total phospholipid fatty acids, PLFAs) in all substrate amendments. Temperature increase affected fungal biomass more adversely than bacterial biomass and the temperature response of fungal biomass (fungal PLFAs, ergosterol and ITS fragment) depended upon substrate quality. With the addition of CB, temperature response of fungal biomass did not differ from un-amended control soils, whereas addition of xylan and CA shifted the fungal temperature optima from 5 °C to 15 °C. These results provide first evidence that fungi which decompose complex C substrates (CA and xylan) may have different life strategies and temperature optima than fungal communities which decompose labile C substrate (CB). Gram-positive and gram-negative bacteria differed strongly in their capacity to decompose CB under different temperature regimes: gram-positive bacteria had highest PLFA abundance at 5 °C, while gram-negative bacteria were most abundant at 25 °C. Bacterial community composition, as measured by 16S rRNA gene abundance, and PLFAs showed opposite temperature and substrate decomposition trends. Using multivariate statistics, we found a general association of microbial life strategies and key members of the microbial community: oligotrophic Alphaproteobacteria and Acidobacteria were associated with complex substrates and copiotrophic Actinobacteria with labile substrates. Our study provides evidence that the response of C cycling to warming will be mediated by shifts in the structure and function of soil microbial communities.

Rabbit trypanosome detection in Phlebotomus perniciosus sand flies from the leishmaniasis outbreak in Madrid, Spain

Phlebotomine sand flies are known vectors of several pathogens. In Spain, Phlebotomus perniciosus and Phlebotomus ariasi are the proven vectors of Leishmania infantum. Since 2010 a human leishmaniasis outbreak has been notified in Madrid region, central Spain. Studies have shown that P. perniciosus is the only vector confirmed in the focus area and that rabbits and hares are the wild reservoirs incriminated in the cycle of the parasite in the outbreak. Trypanosoma nabiasi is a trypanosomatid found in wild rabbits and its presence has been reported in wild rabbits from southern Spain. Moreover, co-infection with L. infantum was found in some of these animals. However, in Madrid region, there is no information about the transmission of this trypanosome in rabbits. Hence, in this study we investigate if T. nabiasi could be circulating in the aforementioned leishmaniasis focus. Wild P. perniciosus female sand flies were captured in the affected area and analyzed using molecular methods. T. nabiasi DNA was detected in 20 out of 155 female sand flies fed on rabbits by amplification and subsequent sequencing of ITS1 and SSU rRNA fragments. Therefore, we describe for the first time the presence of T. nabiasi and its co-infection with L. infantum in P. perniciosus female sand flies. More investigation is needed in order to elucidate the role of P. perniciosus in the transmission of T. nabiasi among rabbits and its potential consequences.

Anaerobic Fungi in Gorilla (Gorilla gorilla gorilla) Feces: an Adaptation to a High-Fiber Diet?

Many studies have demonstrated the importance of symbiotic microbial communities for the host with beneficial effects for nutrition, development, and the immune system. The majority of these studies have focused on bacteria residing in the gastrointestinal tract, while the fungal community has often been neglected. Gut anaerobic fungi of the class Neocallimastigomycetes are a vital part of the intestinal microbiome in many herbivorous animals and their exceptional abilities to degrade indigestible plant material means that they contribute significantly to fermentative processes in the enteric tract. Gorillas rely on a highly fibrous diet and depend on fermentative microorganisms to meet their daily energetic demands. To assess whether Neocallimastigomycetes occur in gorillas we analyzed 12 fecal samples from wild Western lowland gorillas (Gorilla gorilla gorilla) from Dzanga–Sangha Protected Areas, Central African Republic, and subjected potential anaerobic fungi sequences to phylogenetic analysis. The clone library contained ITS1 fragments that we related to 45 different fungi clones. Of these, 12 gastrointestinal fungi in gorillas are related to anaerobic fungi and our phylogenetic analyses support their assignment to the class Neocallimastigomycetes. As anaerobic fungi play a pivotal role in plant fiber degradation in the herbivore gut, gorillas might benefit from harboring these particular fungi with regard to their nutritional status. Future studies should investigate whether Neocallimastigomycetes are also found in other nonhuman primates with high fiber intake, which would also benefit from having such highly efficient fermentative microbes.