Transcriptome analysis of the spider Phonotimpus pennimani reveals novel toxin transcripts

Background: Phonotimpus pennimani (Araneae, Phrurolithidae) is a small-sized (3-5 mm) spider endemic to the Tacaná volcano in Chiapas, Mexico, where it is found in soil litter of cloud forests and coffee plantations. Its venom composition has so far not been investigated, partly because it is not a species of medical significance. However, it does have an important impact on the arthropod populations of its natural habitat. Methods: Specimens were collected in Southeastern Mexico (Chiapas) and identified taxonomically by morphological characteristics. A partial sequence from the mitochondrial gene coxI was amplified. Sequencing on the Illumina platform of a transcriptome library constructed from 12 adult specimens revealed 25 toxin or toxinlike genes. Transcripts were validated (RT-qPCR) by assessing the differential expression of the toxin-like PpenTox1 transcript and normalising with housekeeping genes. Results: Analysis of the coxI-gene revealed a similarity to other species of the family Phrurolithidae. Transcriptome analysis also revealed similarity with venom components of species from the families Ctenidae, Lycosidae, and Sicariidae. Expression of the toxinlike PpenTox1 gene was different for each developmental stage (juvenile or adult) and also for both sexes (female or male). Additionally, a partial sequence was obtained for the toxin-like PpenTox1 from DNA. Conclusion: Data from the amplification of the mitochondrial coxI gene confirmed that P. pennimani belongs to the family Phrurolithidae. New genes and transcripts coding for venom components were identified.(AU)

Effects of dietary lysozyme on immune response and fecal microflora in both sows and their offspring

Two studies were conducted to investigate the effects of dietary lysozyme on immune response, fecal microflora in sows and their offspring fed lysozyme from late gestation to the onset of lactation, and growth performance in weaned piglets. Four antibiotic-based treatments (chlortetracycline, colistin, and lysozyme) were applied in experiment 1. Lysozyme addition significantly increased final body weight, average daily gain, and average daily feed intake, improved feed:gain ratio (F:G), and decreased diarrhea rate in weaned piglets. In experiment 2, postpartum sows were fed diets either with amoxicillin and cephalosporin (SC) or lysozyme (SE). Piglets from SC sows were administered enrofloxacin and those from SE sows were administered lysozyme. Lysozyme treatment decreased serum IL-1, IL-6, and IL-10, but did not influence IL-8, TNF-α, or IFN-γ in weaned piglets. Sequencing revealed that lysozyme significantly decreased Chao-1 index in sows and weaned piglets, increased Bifidobacterium longum in sows, and Lactobacillus coleohominis, L. mucosae, L. amylovorus, and L. hamsteri in weaned piglets. The results suggest that dietary supplementation of lysozyme improved the growth performance of weaned piglets, and dietary supplementation of lysozyme for sows increased immune function and modulated the intestinal flora structure in sows and their offspring.

Development of microsatellite loci and population genetics of the catfish Pimelodus yuma (Siluriformes: Pimelodidae)

Pimelodus yuma (formerly Pimelodus blochii) is a freshwater fish, endemic to the Colombian Magdalena-Cauca and Caribbean basins that experiences habitat disturbances resulting from anthropogenic activities. Due to the lack of information about the population genetics of this species, this study developed 14 species-specific microsatellite loci to assess the genetic diversity and population structure of samples from the lower section of the Cauca River. The studied species showed genetic diversity levels higher than the average values reported for Neotropical Siluriformes and significant inbreeding levels as was described for some congeners. Furthermore, P. yuma comprises two coexisting genetic groups that exhibit gene flow along the lower section of the Cauca River. This information constitutes a baseline for future monitoring of the genetic diversity and population structure in an anthropic influenced sector of the Magdalena-Cauca basin.(AU)

Pimelodus yuma (anteriormente Pimelodus blochii) es un pez dulceacuícola endémico de las cuencas colombianas Magdalena-Cauca y Caribe que experimenta alteraciones del hábitat como resultado de actividades antropogénicas. Debido a la falta de información sobre la genética poblacional de esta especie, este estudio desarrolló 14 loci microsatélites especie-específicos para evaluar la diversidad genética y la estructura poblacional de muestras de la sección baja del río Cauca. La especie estudiada mostró niveles de diversidad genética más altos que los valores promedio reportados para Siluriformes neotropicales y niveles de endogamia significativos como se describió para algunos congéneres. Además, P. yuma comprende dos grupos genéticos coexistentes que exhiben flujo de genes a lo largo de la sección baja del río Cauca. Esta información constituye una línea base para futuros monitoreos de la diversidad genética y la estructura poblacional en un sector de influencia antrópica de la cuenca Magdalena-Cauca.(AU)

Development of microsatellite loci and population genetics of the catfish Pimelodus yuma (Siluriformes: Pimelodidae)

Pimelodus yuma (formerly Pimelodus blochii) is a freshwater fish, endemic to the Colombian Magdalena-Cauca and Caribbean basins that experiences habitat disturbances resulting from anthropogenic activities. Due to the lack of information about the population genetics of this species, this study developed 14 species-specific microsatellite loci to assess the genetic diversity and population structure of samples from the lower section of the Cauca River. The studied species showed genetic diversity levels higher than the average values reported for Neotropical Siluriformes and significant inbreeding levels as was described for some congeners. Furthermore, P. yuma comprises two coexisting genetic groups that exhibit gene flow along the lower section of the Cauca River. This information constitutes a baseline for future monitoring of the genetic diversity and population structure in an anthropic influenced sector of the Magdalena-Cauca basin.(AU)

Pimelodus yuma (anteriormente Pimelodus blochii) es un pez dulceacuícola endémico de las cuencas colombianas Magdalena-Cauca y Caribe que experimenta alteraciones del hábitat como resultado de actividades antropogénicas. Debido a la falta de información sobre la genética poblacional de esta especie, este estudio desarrolló 14 loci microsatélites especie-específicos para evaluar la diversidad genética y la estructura poblacional de muestras de la sección baja del río Cauca. La especie estudiada mostró niveles de diversidad genética más altos que los valores promedio reportados para Siluriformes neotropicales y niveles de endogamia significativos como se describió para algunos congéneres. Además, P. yuma comprende dos grupos genéticos coexistentes que exhiben flujo de genes a lo largo de la sección baja del río Cauca. Esta información constituye una línea base para futuros monitoreos de la diversidad genética y la estructura poblacional en un sector de influencia antrópica de la cuenca Magdalena-Cauca.(AU)

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil / Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Abstract Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.

Resumo Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados.

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil / Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.(AU)

Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados.(AU)

Beneficial effects of a calf starter versus forage on rumen development and bacteria populations in beef calves / Efeitos benéficos de um concentrado de bezerro versus forragem no desenvolvimento ruminal e na população de bactérias em bezerros

Rumen development depends on the intake of solid food that is fermented into volatile fatty acids that stimulate the development of the rumen papillae in calves. The starter feeding can promote the growth of papillae in the rumen and as a consequence an earlier weaning. We evaluated the effects of calf starter on ruminal development, and productive response of lactating bull calves raised for meat in the tropics. Twelve male Brahman × Swiss American cross beef calves from a dual-purpose system were randomly assigned two treatments with six animals per treatment: milk-fed calves + Taiwan grass (Pennisetum purpureum, MT) and MT + calf starter, (MTS). Feed intake and growth were measured at 7-day intervals throughout until 210 d of age. At 90 days old, three calves from each treatment were harvested, and fluid and ruminal tissues were collected from the cranial, ventral, dorsal, and dorsal blind ruminal sacs for measurements of many papillaes per cm2 (NP), papillae length (LP) and papillae width (WP). Ruminal bacterial genotype identification was determined by amplicon generation with the Illumina platform. Calf starter-improved weight (Live weight, LW) and average weight gain (ADG) and NP, but, LP and WP was similar in both treatments (p 0.05). In calves with starter feed treatment, we observed the bacteria Desulfonauticus autotrophicus sp. nov.that was not previously reported in ruminants. Use of calf starter showed benefit for calves with improved feed intake and rumen development because promoted a greater number of rumen papillae.(AU)

O desenvolvimento do rumen depende da ingestão de alimentos sólidos fermentados em ácidos graxos voláteis que estimulam o desenvolvimento das papilas de rúmen em bezerros. A alimentação inicial pode promover o crescimento das papilas no rúmen e, como consequência, um desmame mais cedo. Avaliamos os efeitos da concentrado de bezerros no desenvolvimento ruminal, e a resposta produtiva dos bezerros lactantes criados para carne nos trópicos. Doze bezerros de carne bovina cruza Brahman × suíço-americano machos de de um sistema de dupla finalidade foram aleatoriamente atribuídos a dois tratamentos, com seis animais por tratamento: bezerros alimentados com leite + grama taiwanesa (Pennisetumpurpureum, MT) e MT+ concentrado de bezerro, (MTS). A ingestão de ração e o crescimento foram medidos. Aos 90 dias de idade, três bezerros de cada tratamento foi amostrado, e foram coletados tecidos fluidos dos sacos ruminais cranianos, ventral, dorsal e dorsal cegos para medições do número de papilas por cm2 (NP), comprimento das papilas (LP) e largura da papila (PM). A identificação do genótipo das bactérias ruminais foi determinada pela geração de amplicon com a plataforma de ilumine. O concentrado de bezerro melhorou o peso (LW) e o ganho diário médio (AGD) e NP, mas, LP e WP foram semelhantes nos dois tratamentos. Bezerros em MTS uma bactéria não encontrada anteriormente em ruminantes Desulfonauticus autotrophicus sp. nov. foi detectado. O uso de concentrado de bezerro mostrou benefício para bezerros com melhor ingestão de ração e desenvolvimento de rúmen, pois promove um maior número de papilas de rúmen.(AU)

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

Identification of candidate genes associated with milk yield trait in buffaloes ( Bubalus bubalis ) by restriction-site-associated DNA sequencing

The objectives of our present study included the screening of single nucleotide polymorphisms (SNP) that show significant differences in allelic frequencies between two buffalo populations (Egyptian and Chinese buffaloes), categorization of functional genes associated with these SNP by gene ontology, and pathway analyses to further understand their potential values as candidate genes closely associated with milk yield trait in buffaloes. In this study, double digest restrictionsite associated DNA sequencing was performed on Illumina HiSeq 2500 platform for 20 and 25 female buffaloes from Egypt and China, respectively. Approximately 118 Gb of sequencing data were obtained, and a total of 110,129 and 150,535 putative SNP were detected in Egyptian and Chinese buffaloes, respectively. Focused only on those SNP that differed significantly in allelic frequencies between the two populations, we found that genes associated with these SNP were significantly over-represented in the ionotropic glutamate receptor pathway, the endothelin signaling pathway, and the gonadotropin-releasing hormone receptor pathway, which contained a total of 29 genes. Of these, nine genes (ADCY5, CACNA1A, CREB1, INHBA, INHBB, PIK3R1, PLCB1, PRKCE, and SMAD2) participating in the hormonal regulation of lactation, were considered to be promising candidate genes worthy of further investigations for favorable alleles associated with milk yield. Our results provide useful information about genetic variations in Egyptian and Chinese buffaloes. The potential influences of nine candidate genes and their associated SNP on milk yield need to be validated in more buffalo populations.(AU)

In-depth genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain contaminating chicken carcass

Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil. Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means...

Full-Length Genomic Characterization of Chicken Anemia Virus in Turkey

Chicken infectious anemia (CIA) is an immune-suppressive disease caused by chicken anemia virus (CAV). It is characterized by lymphoid atrophy, aplastic anemia, especially in chicks. In this study, full-length genomic characterization of CAV DNA from the broiler flocks in Turkey and phylogenetic analysis were aimed. In the study, CAV DNA were found positive for 37 (53%) flocks with PCR studies from thymus tissues of each 70 broiler flocks. And 17 purified CAV DNA PCR products from these 37 CAV isolates were full length sequenced with the NGS method (Illumina MiSeq). Also with the phylogenetic analyses, full length PCR products of 17 purified CAV isolates have been determined as 2298bp genome size and 99% similarity with each other. The highest similarity (99%) has been detected with the isolates from China and Taiwan. Furthermore, a 97-98% similarity has been detected with vaccine strains (Cux-1, 26P4 and Del Ros) and also 88-90 % similarity has been detected with GyV4 and GyV3 isolates. As a result, in the study full length genomic characterization of CAV DNA from the 7 regions of Turkey were determined. And also all Turkish CAV isolates and vaccine strains were in group 2 according to the phylogenetic tree were obtained. But these isolates and vaccine strains were not found in the same group with GyV3 and GyV4 strains. Besides, these CAV isolates were showed more similarity to the isolates reported from Taiwan and China than the vaccine strains.(AU)

Full-Length Genomic Characterization of Chicken Anemia Virus in Turkey

Chicken infectious anemia (CIA) is an immune-suppressive disease caused by chicken anemia virus (CAV). It is characterized by lymphoid atrophy, aplastic anemia, especially in chicks. In this study, full-length genomic characterization of CAV DNA from the broiler flocks in Turkey and phylogenetic analysis were aimed. In the study, CAV DNA were found positive for 37 (53%) flocks with PCR studies from thymus tissues of each 70 broiler flocks. And 17 purified CAV DNA PCR products from these 37 CAV isolates were full length sequenced with the NGS method (Illumina MiSeq). Also with the phylogenetic analyses, full length PCR products of 17 purified CAV isolates have been determined as 2298bp genome size and 99% similarity with each other. The highest similarity (99%) has been detected with the isolates from China and Taiwan. Furthermore, a 97-98% similarity has been detected with vaccine strains (Cux-1, 26P4 and Del Ros) and also 88-90 % similarity has been detected with GyV4 and GyV3 isolates. As a result, in the study full length genomic characterization of CAV DNA from the 7 regions of Turkey were determined. And also all Turkish CAV isolates and vaccine strains were in group 2 according to the phylogenetic tree were obtained. But these isolates and vaccine strains were not found in the same group with GyV3 and GyV4 strains. Besides, these CAV isolates were showed more similarity to the isolates reported from Taiwan and China than the vaccine strains.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.(AU)

Next-generation sequencing analysis reveals high bacterial diversity in wild venomous and non-venomous snakes from India

The oral cavities of snakes are replete with various types of bacterial flora. Culture-dependent studies suggest that some of the bacterial species are responsible for secondary bacterial infection associated with snakebite. A complete profile of the ophidian oral bacterial community has been unreported until now. Therefore, in the present study, we determined the complete bacterial compositions in the oral cavity of some snakes from India. Methods: Total DNA was isolated from oral swabs collected from three wild snake species (Indian Cobra, King Cobra and Indian Python). Next, the DNA was subjected to PCR amplification of microbial 16S rRNA gene using V3-region-specific primers. The amplicons were used for preparation of DNA libraries that were sequenced on an Illumina MiSeq platform. Results: The cluster-based taxonomy analysis revealed that Proteobacteria and Actinobacteria were the most predominant phyla present in the oral cavities of snakes. This result indicates that snakes show more similarities to birds than mammals as to their oral bacterial communities. Furthermore, our study reports all the unique and common bacterial species (total: 147) found among the oral microbes of snakes studied, while the majority of commonly abundant species were pathogens or opportunistic pathogens to humans. A wide difference in ophidian oral bacterial flora suggests variation by individual, species and geographical region. Conclusion: The present study would provide a foundation for further research on snakes to recognize the potential drugs/antibiotics for the different infectious diseases.(AU)

Genome-wide association study using the Bayes C method for important traits in dairy yield in Colombian Holstein Cattle / Associação genômica usando o método Bayes C para características importantes na produção leiteira em Gado Holandês na Colômbia

The objective of this study was to determine the genome association between markers of bovine LD BeadChip with dairy important traits. Information of breeding program of the Universidad Nacional de Colombia was used. BLUP-EBVs were used for dairy yield (DY), fat percentage (FP), proteinpercentage (PP) and somatic cell score (SCS). 150 animals were selected for blood or semen DNA extraction and genotyping with BovineLD BeadChip (Illumina). Autosomal information was retained and the editing information was performed using Plink v1.07 software. The effects of SNPs were determined by Bayes C with GS3 software. The minor allele frequency for most of the markers on the bead chip was high, which increases the probability of finding important loci segregating in the population. Estimations of fraction markers with an effect were close to zero in almost all cases. The most important markers were mapped by trait using ENSEMBL. A total of 6,510 autosomal SNPs were retained, out of which only a proportion with effect was taken from the mixed function of Bayes C. Important genes as ANKS1B, CLCN1, NMBR and CTSD, were found for each trait for AL, FP, PP and SCS respectively. Finally, Bayes C estimation allowed to identify specific SNPs possibly associated with QTLs.(AU)

O Objetivo deste estudo foi determinar a associação genômica entre os marcadores do chip bovino LD (LD BeadChip) com características importantes na produção de leite. Foram utilizadas informações do programa de melhoramento genético da Universidade Nacional de Colômbia. BLUPEBVs foram utilizados para a produção de leite (DY), porcentagem de gordura (FP), porcentagem deproteína (PP) e escore de células somáticas (SCS). 150 animais foram selecionados para extração de DNAdo sangue ou sêmen e genotipados com o chip BovineLD (Illumina). A informação autossômica foi mantidae a edição da informação foi executada usando o programa Plink v1.07. Os efeitos dos SNPs foram determinados por Bayes C com o programa GS3. A frequência do alelo menor para a maioria dos marcadores no chip foi alta, o que aumenta a probabilidade de encontrar locos importantes segregando na população. As estimativas da fração de marcadores, com efeito, foram próximas de zero em quase todas as situações. Os marcadores mais importantes foram mapeados com ENSEMBL. Um total de 6510 SNPs autossômicos foram preservados, dos quais apenas uma proporção foi tomada com efeito a partir da função mista de Bayes C. Para cada característica foram encontrados genes importantes, como ANKS1B, CLCN1, NMBR e CTSD, para AL, FP, PP e SCS, respectivamente. Finalmente, a estimativa de Bayes-C permitiu aidentificação de SNPs com possível associação com QTLs.(AU)

Draft genome sequence of Chryseobacterium limigenitum SUR2T (LMG 28734T) isolated from dehydrated sludge

The type strain SUR2 of the novel species Chryseobacterium limigenitum was isolated from a dehydrated sludge of the municipal sewage treatment plant in Dogoše near Maribor in Slovenia. The draft genome, with 60 contigs, 4,697,725 bp, 34.4% of G+C content, was obtained using the Illumina HiSeq 2500-1 platform. Joint Genome Institute Microbial Genome Annotation Pipeline (MGAP v.4) has identified 4322 protein-coding sequences including resistance genes against arsenic and other heavy metals. In addition, a subclass B3 metallo-β-lactamase, which confers resistance to penicillins, cephalosporins and carbapenems, was also present in the genome. The genome sequence provides important information regarding bioremediation potential and pathogenic properties of this newly identified species.(AU)

Genome-wide association study using the Bayes C method for important traits in dairy yield in Colombian Holstein Cattle / Associação genômica usando o método Bayes C para características importantes na produção leiteira em Gado Holandês na Colômbia

The objective of this study was to determine the genome association between markers of bovine LD BeadChip with dairy important traits. Information of breeding program of the Universidad Nacional de Colombia was used. BLUP-EBVs were used for dairy yield (DY), fat percentage (FP), proteinpercentage (PP) and somatic cell score (SCS). 150 animals were selected for blood or semen DNA extraction and genotyping with BovineLD BeadChip (Illumina). Autosomal information was retained and the editing information was performed using Plink v1.07 software. The effects of SNPs were determined by Bayes C with GS3 software. The minor allele frequency for most of the markers on the bead chip was high, which increases the probability of finding important loci segregating in the population. Estimations of fraction markers with an effect were close to zero in almost all cases. The most important markers were mapped by trait using ENSEMBL. A total of 6,510 autosomal SNPs were retained, out of which only a proportion with effect was taken from the mixed function of Bayes C. Important genes as ANKS1B, CLCN1, NMBR and CTSD, were found for each trait for AL, FP, PP and SCS respectively. Finally, Bayes C estimation allowed to identify specific SNPs possibly associated with QTLs.

O Objetivo deste estudo foi determinar a associação genômica entre os marcadores do chip bovino LD (LD BeadChip) com características importantes na produção de leite. Foram utilizadas informações do programa de melhoramento genético da Universidade Nacional de Colômbia. BLUPEBVs foram utilizados para a produção de leite (DY), porcentagem de gordura (FP), porcentagem deproteína (PP) e escore de células somáticas (SCS). 150 animais foram selecionados para extração de DNAdo sangue ou sêmen e genotipados com o chip BovineLD (Illumina). A informação autossômica foi mantidae a edição da informação foi executada usando o programa Plink v1.07. Os efeitos dos SNPs foram determinados por Bayes C com o programa GS3. A frequência do alelo menor para a maioria dos marcadores no chip foi alta, o que aumenta a probabilidade de encontrar locos importantes segregando na população. As estimativas da fração de marcadores, com efeito, foram próximas de zero em quase todas as situações. Os marcadores mais importantes foram mapeados com ENSEMBL. Um total de 6510 SNPs autossômicos foram preservados, dos quais apenas uma proporção foi tomada com efeito a partir da função mista de Bayes C. Para cada característica foram encontrados genes importantes, como ANKS1B, CLCN1, NMBR e CTSD, para AL, FP, PP e SCS, respectivamente. Finalmente, a estimativa de Bayes-C permitiu aidentificação de SNPs com possível associação com QTLs.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)