Sequence analysis of the internal transcribed spacer 2 (ITS2) region of rDNA for identifying Trichogramma species and evaluating genetic diversity / Análise de sequência do rDNA da região espaçadora do transcrito interno 2 (ITS2) para identificar espécies de Trichogramma e avaliar a diversidade genética

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.

Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.

Morphology and molecular taxonomy of the tongue worm, genus Raillietiella (Pentastomida) from the " lungs of berber skinks Eumeces schneideri (Scincidae): First report / Morfología y taxonomía molecular de un pentastómido del género Raillietiella obtenido del pulmón del eslizón bereber Eumeces schneideri: Primer informe

Abstract In the present study, pentastomids belonging to the order Cephalobaenida were isolated from the lungs of Berber skinks Eumeces schneideri (Famiy: Scincidae), which were morphologically described by light and scanning electron microscopy and taxonomically justi- fied by 18s rDNA molecular analyses of the parasites. Seventeen host specimens were collected from well-vegetated wadis at high altitudes, Jizan, Saudi Arabia as new type locality; twelve specimens (70.59%) were infected. All of the recovered parasites were adults, possessed small broadly triangular cephalothorax flattened on the ventral surface and merged smoothly with a uniformly thick and squat abdomen and terminated in a pair of divergent lobes. The results obtained indicated that the parasites belong to the sharp-tipped posterior-hook Raillietiella spp. distinguished from other raillietiedids of the same group some important characteristic fea- tures including annulus number, shape and dimensions of the buccal cadre, copulatory spicules, and anterior and posterior hooks. The anterior hook of the female specimens (n = 5) had a blade length (AB) of 135±5 (110-146) ^m and shank length (BC) 158±5 (150-169) ^m while the posterior hook was much larger with AB measuring 221 ±5 (200-236) m and BC 286 ±6 (280-289) -o.m. For the male specimens (n = 5), the anterior hook had an AB of 73 ±3 (72-75) -j.m and a BC 102±5 (100-103) ^m. The posterior hook was much larger with AB 190.6±5 (190-191).

Resumen En el presente estudio se aisló un pentastómido perteneciente al orden Cephalobaenida del pulmón de un eslizón bereber (Eumeces schneideri, Familia: Scincidae). Se efectuó su descripción morfológica basada en observación por microscopía óptica y de bar rido y se justificó su ubicación taxonómica mediante análisis molecular del gen 18S del ADNr. Se recolectaron 17 especímenes del citado huésped en valles ubicados a elevadas altitudes, en la región de Jizan (Arabia Saudí); 12 de ellos (70,59%) estaban infectados. Todos los parásitos recuperados eran adultos y poseían un pequeño cefalotórax triangular, aplanado en la super ficie ventral, que se fusionaba con un abdomen abultado y terminado en un par de lóbulos divergentes. Los resultados indicaron que este parásito pertenece a Raillietiella spp., que agrupa especies con gancho posterior puntiagudo; estas se distinguen de otros miembros de la Familia Raillietiella por algunos rasgos característicos, como el número de anillos y la forma y dimensiones del cuadro bucal, las espículas copulatorias y los ganchos anterior y posterior. La caracterización morfológica demostró que el parásito recuperado era muy similar a R. aegypti, previamente aislada del mismo huésped. El alineamiento de secuencias mediante el método de probabilidad máxima basado en el análisis del gen 18s del ADNr detectó identidades del 88-95% con los géneros de pentastómidos disponibles en GenBank. Dentro del árbol filogenético se pudo incluir este parásito dentro del clado monofilético pentastómido con máxima identidad con las especies de Raillietiella. Las secuencias obtenidas fueron depositadas en GenBank, con número de acceso MK970649.1. El presente análisis molecular confirma por primera vez la posición taxonómica de Raillietiella aegypti, anteriormente aislado del mismo huésped.

Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) from the blood of Echis pyramidum: morphology and SSU rDNA sequence / Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) do sangue de Echis pyramidum: morfologia e sequência de SSU rDNA

Abstract Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 μm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 μm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.

Resumo Hepatozoon pyramidumi sp. n. é descrito a partir do sangue da víbora em escamas e quilhas serrilhadas, Echis pyramidum, capturada na Arábia Saudita. Cinco de dez espécimes de víbora examinadas (50%) foram encontradas infectadas com Hepatozoon pyramidumi sp. n. com nível de parasitemia de 20% a 30%. A infecção foi restrita apenas aos eritrócitos. Foram observadas duas formas morfologicamente diferentes de estágios intra-eritrocíticos: gamontes de tamanho pequeno e madura. As formas menores de gamontes apresentaram média de 10,7 × 3,5 μm. Os gamontes maduros apresentaram forma de salsicha, com pequenos polos recurvados, medindo 16,3 × 4,2 μm, em média. Os eritrócitos infectados estavam aumentados de tamanho; seus núcleos encontravam-se deformados e, algumas vezes, deslocados de sua posição central, quando comparados às células normais não-infectadas. Foram observados estágios merogônicos em células endoteliais pulmonares e nas células do parênquima hepático. Os merontes maduros apresentavam 17,8 × 13,6 µm e continham merozoítos em forma de banana com tamanho médio de ~ 15 × 2 µm. A análise filogenética baseada nas sequências SSU rDNA agrupou Hepatozoon pyramidumi sp. n com Hepatozoon spp. detectados em répteis de várias regiões geográficas. Por meio de análises morfológicas e moleculares com espécies intimamente relacionadas, demonstrou-se a singularidade dessa nova espécie de Hepatozoon.

COⅠ and 16S rDNA Sequence Identification of Common Necrophagous Flies in Fujian Province / 法医学杂志

Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） and 16S ribosomal deoxyribonucleic acid （16S rDNA）, and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means （UPGMA）, respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.

Phyllodistomum vaili(Plagiorchiida: Gorgoderidae) infecting Parupeneus rubescens (Perciformes: Mullidae): morphology and phylogeny / Phyllodistomum vaili (Plagiorchiida: Gorgoderidae) infectando Parupeneus rubescens (Perciformes: Mullidae): morfologia e filogenia

Abstract Knowledge of the Arabian Gulf fish's parasite fauna is very poor. Until recently, only scattered reports from different locations are known for ecto- and endoparasites. Therefore, the present study aimed to investigate the digenean species that infects one of the most economically fish species in the Arabian Gulf, the rosy goatfish Parupeneus rubescens . One plagiorchiid species has been described, belonging to the Gorgoderidae family, and has been named as Phyllodistomum vaili Ho, Bray, Cutmore, Ward & Cribb, 2014 based on its morphological and morphometric characteristics. In order to accurately classify and characterize this plagiorchiid species, molecular analysis was carried out using both nuclear 18S and 28S rRNA gene regions and revealed that the present plagiorchiid species was associated with other species belonging to the Gorgoderidae family and deeply embedded in the Phyllodistomum genus, closely related to the previously described P. vaili (gb- KF013187.1, KF013173.1). The present study therefore revealed that the species Phyllodistomum is the first account as endoparasites from the rosy goatfish inhabiting the Arabian Gulf.

Resumo O conhecimento da fauna de parasitas dos peixes do Golfo Árabe é escasso. Atualmente, apenas relatórios dispersos de diferentes locais são conhecidos para ecto e endoparasitas. Portanto, o presente estudo teve como objetivo investigar as especies digenéticas que infectam uma das espécies economicamente mais importantes do Golfo Arábico, o peixe-cabra rosado Parupeneus rubescens . Uma espécie de plagiorquídeo foi descrita, pertencente à família Gorgoderidae e foi denominada Phyllodistomum vaili Ho, Bray, Cutmore, Ward & Cribb, 2014, com base em suas propriedades morfológicas e morfométricas. A fim de classificar e caracterizar com precisão essa espécie de plagiorquídeo, a análise molecular foi realizada usando as regiões nucleares do gene 18S e 28S rRNA, revelando que a atual espécie de plagiorchídeo estava associada a outras espécies pertencentes à família Gorgoderidae e, profundamente incorporada ao gênero Phyllodistomum , intimamente relacionado ao P. vaili descrito anteriormente (gb - KF013187.1, KF013173.1). O presente estudo revelou, portanto, que a espécie Phyllodistomum vailli é o primeiro relato como endoparasita do peixe-cabra rosado que habita o Golfo Arábico.

First record of Pseudoterranova decipiens (Nematoda, Anisakidae) infecting the Red spot emperor Lethrinus lentjan in the Red Sea / Primeiro registro de Pseudoterranova decipiens (Nematoda, Anisakidae) infectando o imperador da mancha vermelha Lethrinus lentjan no Mar Vermelho

Abstract The current parasitological study was carried out to investigate helminth parasites infecting the Red spot emperor Lethrinus lentjan inhabiting Hurghada City at the Gulf of Suez, Red Sea, Egypt. Third-stage larvae of nematode parasite was isolated from the intestine as well as body cavity of the examined fish. Light and scanning electron microscopy revealed that this parasite belonged to Anisakidae family within the genus Pseudoterranova. The present species is named Pseudoterranova decipiens based on the presence of triangular mouth aperture with prominent boring teeth and soft swellings of the cuticle, long muscular esophagus, ventrally excretory pore, and narrow transverse slit of anal opening followed by a short mucron. The morphological characteristics of this species were confirmed by molecular analysis of 18S rDNA gene region of the present parasite. It demonstrated a close identity ≥89% with taxa under family Anisakidae, 85% with Raphidascarididae, and 79-84% with Toxocaridae. A preliminary genetic comparison between gene sequence of the present parasite and other oxyurid species placeed it as a putative sister taxon to other Pseudoterranova decipiens described previously. This study demonstrated that the 18S rDNA gene region of Pseudoterranova decipiens yielded a unique sequence that confirmed its taxonomic position in Anisakidae.

Resumo O presente estudo parasitológico foi realizado para investigar os helmintos parasitos que infectam o peixe imperador Lethrinus lentjan, que habita a cidade de Hurghada no Golfo de Suez, Mar Vermelho, no Egito. Larvas de terceiro estágio de parasitos nematoides foram isoladas do intestino e da cavidade do corpo do peixe examinado. Microscopia eletrônica de luz e de varredura revelou que este parasita pertence à família Anisakidae dentro do gênero Pseudoterranova. A espécie atual é denominada Pseudoterranova decipiens baseada na presença de abertura triangular da boca com dentes proeminentes chatos e inchaços moles da cutícula, esôfago muscular longo, poro ventralmente excretor e fenda transversal estreita da abertura anal seguida por um mucron curto. As características morfológicas desta espécie foram confirmadas pela análise molecular da região do gene 18S rDNA do presente parasito. Demonstrou uma identidade próxima ≥89% com taxa sob família Anisakidae, 85% com Raphidascarididae, e 79-84% com Toxocaridae. Uma comparação genética preliminar entre a sequência genética do presente parasito e outras espécies de oxiurídeos coloca-o como um taxon irmão putativo para outros Pseudoterranova descritos anteriormente. Este estudo demonstra que a região do gene 18S rDNA de Pseudoterranova decipiens produz uma sequência única que confirma sua posição taxonômica em Anisakidae.

Molecular identification and histological aspects of Renicola sloanei (Digenea: Renicolidae) in Puffinus puffinus (Procellariiformes): a first record / Identificação molecular e aspectos histológicos de Renicola sloanei (Digenea: Renicolidae) em Puffinus puffinus (Procellariiformes): primeiros registros

Abstract Renicolids are parasites that inhabit the renal tubules and ureters of molluscivorous and piscivorous birds. Puffinus puffinus is a migratory seabird that was identified as the definitive host of Renicola spp. Studies focusing on the renicolid species and the resulting renal lesions are valuable for their association with causes of stranding in seabirds. The aim of this study was to identify the renicolid trematodes and evaluate the histological findings in two P. puffinus stranded on the coast of Paraná state, Brazil. The parasites were evaluated by histologic, ultrastructural and molecular assays, while tissue changes were analyzed by histologic methods. The morphological and morphometrical characteristics of the parasites, along with polymerase chain reaction and sequencing assays (ribosomal and mitochondrial regions), identified the species as Renicola sloanei. The results also suggest that this helminth can be the adult form of Cercaria pythionike. The dilation of collecting ducts was the main histological finding in the kidneys. In conclusion, R. sloanei was identified, and for the first time, P. puffinus was described as a host of this digenean inducing mild renal changes.

Resumo Renicolídeos são parasitos que habitam túbulos renais e ureteres de aves que se alimentam de moluscos e peixes. Puffinus puffinus, ave marinha migratória, foi registrada como hospedeiro definitivo de Renicola spp. Estudos relacionados com as espécies de renicolídeos e as lesões renais resultantes são importantes para o entendimento das causas de óbito de aves marinhas. O objetivo deste estudo foi identificar os trematódeos renicolídeos e avaliar os achados histológicos em dois P. puffinus encalhados no litoral do Estado do Paraná, Brasil. Os parasitos foram avaliados por ensaios histológicos, ultraestruturais e moleculares, enquanto as alterações teciduais foram analisadas por métodos histológicos. As características morfológicas e morfométricas dos parasitos, juntamente com a reação em cadeia da polimerase e sequenciamento (regiões ribossomal e mitocondrial), identificaram a espécie como Renicola sloanei. Os resultados também sugerem que este helminto pode ser a forma adulta de Cercaria pythionike. A dilatação dos ductos coletores foi o principal achado histológico renal. Em conclusão, R. sloanei foi identificado, e pela primeira vez P. puffinus foi descrito como hospedeiro deste digenético induzindo alterações renais discretas.

First molecular detection of Mycoplasma ovis (Hemotropic mycoplasmas) from Sheep in Brazil / Primeira detecção molecular de Mycoplasma ovis (Micoplasmas Hemotrópicos) em Ovinos no Brasil

Abstract Mycoplasma ovis is an emerging zoonotic pathogen with a worldwide distribution and can cause mild to severe hemolytic anemia, icterus, and poor weight gain in animals. Although M. ovis has been described in small ruminants worldwide, data on M. ovis in sheep in Brazil is unknown. The objective of the present study was to present the first report of hemotropic mycoplasma (HM) in sheep from Brazil. We evaluated factors associated with this infection, such age group, tick presence, and anemia. Blood samples were collected from 33 sheep from a farm in southern Brazil and screened for hemoplasmas using PCR. Out of 33 samples, 26 (78.8%) tested positive for M. ovis. The sequencing of positive samples showed 100% identity with multiple M. ovis 16S rDNA sequences. No association was observed between the presence of M. ovis and the FAMACHA© score (p = 0.620). Rhipicephalus (Boophilus) microplus (15/33, 45.4%) was the tick species found on the animals. No significant association between M. ovis infection and presence of ticks (p = 0.4134) and age group (p = 0.4221) was observed. This is the first report of M. ovis infection in sheep from Brazil and only the second report of this pathogen in sheep in Latin America.

Resumo Mycoplasma ovis é um patógeno zoonótico emergente com distribuição mundial e pode causar anemia hemolítica de leve a grave, icterícia e baixo ganho de peso em animais. Embora M. ovis tenha sido descrito em pequenos ruminantes em todo o mundo, os dados sobre M. ovis em ovinos no Brasil são desconhecidos. O objetivo deste estudo foi apresentar o primeiro relato de micoplasmas hemotrópicos em ovinos no Brasil. Avaliamos os fatores associados a essa infecção, como faixa etária, presença de carrapatos e anemia. Amostras de sangue foram coletadas de 33 ovelhas de uma fazenda no sul do Brasil e testadas para hemoplasmas usando a PCR. Das 33 amostras, 26 (78,8%) apresentaram resultado positivo. O sequenciamento das amostras positivas mostrou 100% de identidade com múltiplas sequências de M. ovis 16S rDNA. Não foi observada associação entre a presença de M. ovis e o escore FAMACHA© (p = 0,620). Rhipicephalus (Boophilus) microplus (15/33, 45,4%) foi a espécie de carrapato encontrada nos animais. Não houve associação significativa entre infecção por M. ovis e presença de carrapatos (p = 0,4134) e faixa etária (p = 0,4221). Este é o primeiro relato de infecção por M. ovis em ovinos no Brasil e o segundo relato deste patógeno em ovinos na América Latina.

New occurrence of Kudoa orbicularis parasitizing the freshwater catfish Trachelyopterus galeatus (Siluriformes: Auchenipteridae) in the Brazlian Amazon region / Nova ocorrência de Kudoa orbicularis parasitando peixe de água doce Trachelyopterus galeatus (Siluriformes: Auchenipteridae) na região Amazônica brasileira

Abstract The aim of this was describe an infection by Kudoa orbicularis in freshwater catfish Trachelyopterus galeatus. A sample of 80 specimens of T. galeatus was collected in the municipality of Cachoeira do Arari, Marajó Island, in the state of Pará, Brazil. Pseudocysts were found in the muscle fibers of the epaxial and hypaxial regions of 85.0% of the specimens analyzed, reflecting a high infection rate. The pseudocysts contained spores that were pseudo-square in shape, with a mean length of 4.65 µm (range: 4.04-5.54) and mean width of 1.53 µm (1.56-1.74). Analyses on the morphology of the spores and a partial 934-bp sequence of the SSU rDNA gene confirmed that the microparasite was Kudoa orbicularis. This is the second record of this microparasite in a siluriform host in the Brazilian Amazon region.

Resumo O objetivo deste estudo foi descrever a infecção por Kudoa orbicularis em Trachelyopterus galeatus. Foram analisados 80 espécimes de T. galeatus capturados no município de Cachoeira do Arari, ilha de Marajó, estado do Pará, Brasil. A presença de pseudocistos nas fibras musculares das regiões epiaxial e hipoaxial em 85,0% dos exemplares analisados, mostra alto grau de infecção. Os pseudocistos continham esporos de formato pseudoquadrado, medindo 4,65 (4,04-5,54) µm de comprimento e 5,25 (4,78-5,98) µm de largura, com quatro cápsulas polares de tamanho iguais medindo 2,22 (2,05-2,32) µm de comprimento e 1,53 (1,56-1,74) µm de largura. Através das análises morfológicas dos esporos e molecular de uma sequência parcial de 934bps do gene SSU rDNA, confirma que o microparasito é Kudoa orbicularis, sendo este o segundo registro desse microparasito em hospedeiro da ordem Siluriformes da Amazônia brasileira.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Application of 18S rDNA Clone Library to Detect Diatom Population Diversity in Dianchi / 法医学杂志

Objective To detect the diatom population diversity in Dianchi by constructing a 18S rDNA clone library. Methods DNA from diatoms in 6 water samples of Dianchi was amplified with diatom 18S rDNA specific primer.The 18S rDNA clone library was constructed, and clones were randomly selected for sequence. Sequence alignment was performed by BLAST. The diatom population distribution in Dianchi was analyzed and the phylogenetic tree of diatom 18S rDNA in Dianchi waters was established with the MEGA v7.0.14 software. Results Two hundred and forty clones were sequenced, with 167 diatom sequences obtained, including 11 diatom species such as Stephanodiscus, Diatoma, and Melosira. There were certain differences in diatom population distribution among the 6 samples. Conclusion The population distribution of diatom species in Dianchi shows unique features and the sequence analysis of diatom 18S rDNA has a certain reference value to the inference of forensic drowning sites.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica

ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Variations in culturable bacterial communities and biochemical properties in the foreland of the retreating Tianshan No. 1 glacier

Abstract As a glacier retreats, barren areas are exposed, and these barren areas are ideal sites to study microbial succession. In this study, we characterized the soil culturable bacterial communities and biochemical parameters of early successional soils from a receding glacier in the Tianshan Mountains. The total number of culturable bacteria ranged from 2.19 × 105 to 1.30 × 106 CFU g-1 dw and from 9.33 × 105 to 2.53 × 106 CFU g-1 dw at 4 °C and 25 °C, respectively. The number of culturable bacteria in the soil increased at 25 °C but decreased at 4 °C along the chronosequence. The total organic carbon, total nitrogen content, and enzymatic activity were relatively low in the glacier foreland. The number of culturable bacteria isolated at 25 °C was significantly positively correlated with the TOC and TN as well as the soil urease, protease, polyphenoloxidase, sucrase, catalase, and dehydrogenase activities. We obtained 358 isolates from the glacier foreland soils that clustered into 35 groups using amplified ribosomal DNA restriction analysis. These groups are affiliated with 20 genera that belong to six taxa, namely, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroides, and Deinococcus-Thermus, with a predominance of members of Actinobacteria and Proteobacteria in all of the samples. A redundancy analysis showed that the bacterial succession was divided into three periods, an early stage (10a), a middle stage (25-74a), and a late stage (100-130a), with the total number of culturable bacteria mainly being affected by the soil enzymatic activity, suggesting that the microbial succession correlated with the soil age along the foreland.

Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L. ) and plant growth-promoting properties

Abstract A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacteria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with more than 63% inhibitory activities were further characterized through optical epifluorescence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacillus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regression analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings.

Analysis of bacterial communities and characterization of antimicrobial strains from cave microbiota

Abstract In this study for the first-time microbial communities in the caves located in the mountain range of Hindu Kush were evaluated. The samples were analyzed using culture-independent (16S rRNA gene amplicon sequencing) and culture-dependent methods. The amplicon sequencing results revealed a broad taxonomic diversity, including 21 phyla and 20 candidate phyla. Proteobacteria were dominant in both caves, followed by Bacteroidetes, Actinobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, and the archaeal phylum Euryarchaeota. Representative operational taxonomic units from Koat Maqbari Ghaar and Smasse-Rawo Ghaar were grouped into 235 and 445 different genera, respectively. Comparative analysis of the cultured bacterial isolates revealed distinct bacterial taxonomic profiles in the studied caves dominated by Proteobacteria in Koat Maqbari Ghaar and Firmicutes in Smasse-Rawo Ghaar. Majority of those isolates were associated with the genera Pseudomonas and Bacillus. Thirty strains among the identified isolates from both caves showed antimicrobial activity. Overall, the present study gave insight into the great bacterial taxonomic diversity and antimicrobial potential of the isolates from the previously uncharacterized caves located in the world's highest mountains range in the Indian sub-continent.

Characterization of multidrug-resistant Acinetobacter ssp. strains isolated from medical intensive care units in Cali - Colombia / Caracterización de cepas de Acinetobacter spp. resistentes a múltiples fármacos aisladas de unidades de cuidados intensivos en Cali - Colombia

Abstract Introduction: The extensive use of antibiotics has led to the emergence of multi-resistant strains in some species of the genus Acinetobacter. Objective: To investigate the molecular characteristics of multidrug-resistant of Acinetobacter ssp. strains isolated from 52 patients collected between March 2009 and July 2010 in medical intensive care units in Cali - Colombia. Methods: The susceptibility to various classes of antibiotics was determined by disc diffusion method, and the determination of the genomic species was carried out using amplified ribosomal DNA restriction analysis (ARDRA) and by sequencing of the 16s rDNA gene. Also, the genes of beta-lactamases as well as, integrases IntI1 and IntI2 were analyzed by PCR method. Results: The phenotypic identification showed that the isolates belong mainly to A. calcoaceticus- A. baumannii complex. All of them were multi-resistant to almost the whole antibiotics except to tigecycline and sulperazon, and they were grouped into five (I to V) different antibiotypes, being the antibiotype I the most common (50.0%). The percent of beta-lactamases detected was: blaTEM (17.3%), blaCTX-M (9.6%), blaVIM (21.2%), blaIMP (7.7%), blaOXA-58 (21.2%), and blaOXA-51 (21.2%). The phylogenetic tree analysis showed that the isolates were clustering to A. baumannii (74.1%), A. nosocomialis (11.1%) and A. calcoaceticus (7.4 %). Besides, the integron class 1 and class 2 were detected in 23.1% and 17.3% respectively. Conclusion: The isolates were identified to species A. baumanii mainly, and they were multiresistant. The resistance to beta-lactams may be by for presence of beta-lactamases in the majority of the isolates.

Resumen Introducción: El uso extensivo de antibióticos ha llevado a la emergencia de cepas multirresistentes en algunas especies del género Acinetobacter. Objetivo: Investigar las características moleculares de resistencia a múltiples fármacos de cepas aisladas de Acinetobacter spp. colectadas entre marzo de 2009 y julio de 2010 en 52 pacientes de unidades de cuidados intensivos en Cali - Colombia. Métodos: La susceptibilidad a diversas clases de antibióticos se determinó mediante el método de difusión de disco, y la determinación de la especie genómica se llevó a cabo usando un análisis de restricción de ADN ribosómico amplificado (ARDRA) y mediante la secuenciación del gen 16s de ADNr. Además, se analizaron por el método de PCR los genes de las beta-lactamasas, como también, las integrasas IntI1 e IntI2. Resultados: La identificación fenotípica mostró que los aislamientos pertenecen principalmente al complejo A. calcoaceticus - A. baumannii. Todos ellos eran multirresistentes a casi todos los antibióticos excepto tigeciclina y sulperazón, y se agruparon en cinco (I a V) antibitipos diferentes, siendo el antibiotipo I el más común (50%). El porcentaje de betalactamasas detectadas fue: blaTEM (17,3%), blaCTX-M (9,6%), blaVIM (21,2%), blaIMP (7,7%), blaOXA-58 (21,2%), blaOXA- 51 (21.2%). El análisis del árbol filogenético mostró que los aislados se agrupaban en A. baumannii (74.1%), A. nosocomialis (11.1%) y A. calcoaceticus (7.4%). Además, el integrón clase 1 y clase 2 se detectaron en 23.1% y 17.3% respectivamente. Conclusión: los aislamientos se identificaron principalmente como la especie A. baumanii, y fueron multirresistentes. La resistencia a los betalactámicos puede deberse a la presencia de betalactamasas en la mayoría de los aislamientos.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico / Caracterización de toxinas Cry de aislados de Bacillus thuringiensis autóctonos de México

Abstract: Background: Chemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. Methods: Eight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. Results: An increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. Conclusions: The observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization.

Resumen: Introducción: Los pesticidas químicos, ampliamente usados en agricultura y en el control de vectores transmisores de enfermedades, han mostrado efectos tóxicos sobre el medio ambiente y las personas expuestas a ellos. Bacillus thuringiensis es una bacteria ampliamente utilizada como una alternativa segura y eficaz en el control biológico de plagas agrícolas. Sus toxinas son específicas de insectos, pero inocuas para mamíferos, e incluso poseen gran potencial para ser usadas como adyuvantes en vacunas. El objetivo de este trabajo fue caracterizar cepas autóctonas de B. thuringiensis con efectividad contra el gusano barrenador (Diatraea considerata Heinrich) de la caña de azúcar en cultivos del estado de Sinaloa, México, y como fuente de proteínas Cry, con potencial de utilizarse como adyuvantes en vacunas. Métodos: Se lograron aislar ocho cepas a partir de insectos muertos en campos agrícolas, las cuales fueron identificadas microbiológicamente como B. thuringiensis, lo que se confirmó por amplificación y secuenciación del 16S rDNA. La efectividad de los aislados para el control del gusano barrenador fue evaluada mediante bioensayos y las toxinas Cry fueron identificadas por análisis proteómico. Resultados: Se observó una mortalidad elevada en las larvas infectadas con las cepas de estudio. Particularmente, la cepa Bt-D, de la cual el análisis molecular mostró que posee una toxina tipo Cry1Ac. Conclusiones: Los resultados mostraron que la cepa Bt-D posee un elevado potencial patogénico hacia D. considerata y produce la proteína Cry1Ac, de la cual existen reportes de su aplicación como adyuvante en diferentes formas de inmunización.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Effects of environment, dietary regime and ageing on the dengue vector microbiota: evidence of a core microbiota throughout Aedes aegypti lifespan

Abstract Mosquito midgut microbiota is a key component of vector competence, as gut bacteria can disturb pathogen development. In this study, we addressed the microbiota composition of Aedes aegypti during its lifespan, under field conditions. We also investigated the possible effects of environment, dietary regime and ageing on the gut community composition. We employed culture independent and dependent approaches to characterise vector microbiota. There was evidence of a lifelong stable core microbiota after mosquitoes were released into an urban settlement, where they presumably fed on a range of vertebrate hosts and carbohydrate sources. This core was formed mainly of bacteria belonging to the genera Pseudomonas, Acinetobacter, Aeromonas and Stenotrophomonas and to the families Oxalobacteraceae, Enterobacteriaceae and Comamonadaceae. We showed that both dietary regime and age were associated with the abundance of some bacterial groups in the Ae. aegypti microbiota. The majority of the bacterial groups we identified have been detected in the midgut of Ae. aegypti from laboratory and wild populations, indicating a possible core microbiota associated with this mosquito species. Our findings suggest that Ae. aegypti harbours a stable bacterial community during its adult life, similar to mosquito populations from distinct geographic areas, which may be further explored for arbovirus biocontrol strategies.