Background: Metagenomics approaches are increasingly being utilized as “dipstick” for microbial carriage. In this study, 16S rRNA metagenomics was used to probe for microbial community that resides in the ticks, tho...Background: Metagenomics approaches are increasingly being utilized as “dipstick” for microbial carriage. In this study, 16S rRNA metagenomics was used to probe for microbial community that resides in the ticks, those they pick from the environment, wildlife and livestock and to identify potential tick borne zoonoses. Methods: Tick DNA from 463 tick pools collected from domestic animals between 2007 and 2008 were amplified with primers that target the 16S rRNA V3-V4 domain and then sequenced on Illumina Miseq platform using 300 cycles version 3 kits. Ticks were pooled according to species and animal from which they were collected. A non-target control was used to track laboratory contaminants. Sequence data were analyzed using Mothur v1.3 pipeline and R v3.3.1 software and taxonomy determined using SILVA rRNA database. Shannon diversity index was used to compute bacterial diversity in each tick species before computing the means. Results: A total of 645 bacteria genera grouped into 27 phyla were identified. Four phyla contributed 97.4% of the 36,973,934 total sequences. Proteobacteria contributed 61.2% of these sequences that tarried to 33.8% genera, compared to 15.9% (23.4% genera) for Firmicutes, 15.6% (20% genera) for Actinobacteria and 4.7% (11.6% genera) for Bacteroidetes. The remaining 23 phyla only contributed 2.6% of the sequence reads (11.2% genera). Amongst the 645 genera, three groups were discernible, with the biggest group comprised commensals/symbionts that contributed 93.6% of the genera, but their individual sequence contribution was very low. Group two comprised genera that are known to contain pathogenic species, with Coxiella contributing 15,445,204 (41.8%) sequences, Corynebacterium (13.6%), Acinetobacter (4.3%), Staphylococcus (3.9%), Bacillus (2.7%) and Porphyromonas (1.6%), Ralstonia (1.5%), Streptococcus (1.3%), Moraxella (1.3%), amongst others. Group three comprised genera known to contain tick borne zoonotic pathogens (TBZ): Rickettsiae, Anaplasma, Francisella, Ehrlichia, Bartonella and Borrelia. Individually the TBZ contributed Amblyomma variegatum carried the least diverse bacteria (mean Shannon diversity index of 2.69 ± 0.92) compared to 3.79 ± 1.10 for A. gemma, 3.71 ± 1.32 for A. hebraeum, 4.15 ± 1.08 for other Amblyomma spp, 3.79 ± 1.37 for Hyalomma truncatum, 3.67 ± 1.38 for other Hyalomma spp, 3.86 ± 1.27 for Rhipicephalus annulatus, 3.56 ± 1.21 for Rh. appendiculatus, and 3.65 ± 1.30 for Rh. Pulchellus, but the difference was not significant (p = 0.443). Conclusion: This study illustrates the utility of 16S rRNA metagenomics in revealing the complexity of bacteria communities that reside and/or transit through the tick having been picked from the environment, livestock and/or wild animals, some with potential to cause zoonoses.展开更多
Anastrepha fraterculus is a significant fruit fly pest in Argentina and other South American countries. Previous studies showed the key role of gut bacteria in the protection and nutrient assimilation of fruit flies, ...Anastrepha fraterculus is a significant fruit fly pest in Argentina and other South American countries. Previous studies showed the key role of gut bacteria in the protection and nutrient assimilation of fruit flies, particularly the importance of the biological fixation of nitrogen (diazotrophy). The presence of diazotrophic bacteria in A. fraterculus sp. 1 has been demonstrated through molecular, culture-independent methods. This study is aimed to characterize the composition and diversity of culturable gut bacteria of A. fraterculus sp. 1 males from different origins, and explore their metabolic roles, focusing on diazotrophic bacteria. Three male groups were studied: wild-caught (WW), lab-reared from wild larvae (WL), and lab-colony raised (LL). Gut bacteria were collected and characterized via 16S rRNA gene sequencing, with potential diazotrophs screened using selective media (SIL and NFb). Phylogenetic analysis of 16S rRNA gene mapped potential diazotrophs across the bacterial collection, while biochemical profiling and ARDRA (Amplified rDNA Restriction Analysis) were used to quickly differentiate diazotrophic bacteria. PCR testing for the nifH gene, associated with nitrogen fixation, was also performed. Bacterial diversity was highest in WW, followed by WL, and lowest in LL. In LL and WL, Enterobacter was the most frequent genus, while Klebsiella dominated in WW. Among the 20 SIL+ isolates identified, 10 came from WW, 9 from WL, and 1 from LL. One of these isolates (Enterobacter sp.) was tested as a supplement to the adult diet, without showing a beneficial effect on males pheromone calling behavior. Three isolates were also NFb+;two had the nifH gene. ARDRA was effective for rapid diazotroph discrimination. These findings highlight the potential of gut symbiotic bacteria in eco-friendly pest management strategies like the sterile insect technique (SIT). By using diazotrophic bacteria, protein requirements in artificial diets could be reduced, cutting costs and improving the affordability of SIT programs.展开更多
文摘Background: Metagenomics approaches are increasingly being utilized as “dipstick” for microbial carriage. In this study, 16S rRNA metagenomics was used to probe for microbial community that resides in the ticks, those they pick from the environment, wildlife and livestock and to identify potential tick borne zoonoses. Methods: Tick DNA from 463 tick pools collected from domestic animals between 2007 and 2008 were amplified with primers that target the 16S rRNA V3-V4 domain and then sequenced on Illumina Miseq platform using 300 cycles version 3 kits. Ticks were pooled according to species and animal from which they were collected. A non-target control was used to track laboratory contaminants. Sequence data were analyzed using Mothur v1.3 pipeline and R v3.3.1 software and taxonomy determined using SILVA rRNA database. Shannon diversity index was used to compute bacterial diversity in each tick species before computing the means. Results: A total of 645 bacteria genera grouped into 27 phyla were identified. Four phyla contributed 97.4% of the 36,973,934 total sequences. Proteobacteria contributed 61.2% of these sequences that tarried to 33.8% genera, compared to 15.9% (23.4% genera) for Firmicutes, 15.6% (20% genera) for Actinobacteria and 4.7% (11.6% genera) for Bacteroidetes. The remaining 23 phyla only contributed 2.6% of the sequence reads (11.2% genera). Amongst the 645 genera, three groups were discernible, with the biggest group comprised commensals/symbionts that contributed 93.6% of the genera, but their individual sequence contribution was very low. Group two comprised genera that are known to contain pathogenic species, with Coxiella contributing 15,445,204 (41.8%) sequences, Corynebacterium (13.6%), Acinetobacter (4.3%), Staphylococcus (3.9%), Bacillus (2.7%) and Porphyromonas (1.6%), Ralstonia (1.5%), Streptococcus (1.3%), Moraxella (1.3%), amongst others. Group three comprised genera known to contain tick borne zoonotic pathogens (TBZ): Rickettsiae, Anaplasma, Francisella, Ehrlichia, Bartonella and Borrelia. Individually the TBZ contributed Amblyomma variegatum carried the least diverse bacteria (mean Shannon diversity index of 2.69 ± 0.92) compared to 3.79 ± 1.10 for A. gemma, 3.71 ± 1.32 for A. hebraeum, 4.15 ± 1.08 for other Amblyomma spp, 3.79 ± 1.37 for Hyalomma truncatum, 3.67 ± 1.38 for other Hyalomma spp, 3.86 ± 1.27 for Rhipicephalus annulatus, 3.56 ± 1.21 for Rh. appendiculatus, and 3.65 ± 1.30 for Rh. Pulchellus, but the difference was not significant (p = 0.443). Conclusion: This study illustrates the utility of 16S rRNA metagenomics in revealing the complexity of bacteria communities that reside and/or transit through the tick having been picked from the environment, livestock and/or wild animals, some with potential to cause zoonoses.
基金Funding was provided by FAO/IAEA contract 22515(CRP D42017),PICT-2019-04141,PIP-CONICET 0039PI USAL 2022-80020210100018.
文摘Anastrepha fraterculus is a significant fruit fly pest in Argentina and other South American countries. Previous studies showed the key role of gut bacteria in the protection and nutrient assimilation of fruit flies, particularly the importance of the biological fixation of nitrogen (diazotrophy). The presence of diazotrophic bacteria in A. fraterculus sp. 1 has been demonstrated through molecular, culture-independent methods. This study is aimed to characterize the composition and diversity of culturable gut bacteria of A. fraterculus sp. 1 males from different origins, and explore their metabolic roles, focusing on diazotrophic bacteria. Three male groups were studied: wild-caught (WW), lab-reared from wild larvae (WL), and lab-colony raised (LL). Gut bacteria were collected and characterized via 16S rRNA gene sequencing, with potential diazotrophs screened using selective media (SIL and NFb). Phylogenetic analysis of 16S rRNA gene mapped potential diazotrophs across the bacterial collection, while biochemical profiling and ARDRA (Amplified rDNA Restriction Analysis) were used to quickly differentiate diazotrophic bacteria. PCR testing for the nifH gene, associated with nitrogen fixation, was also performed. Bacterial diversity was highest in WW, followed by WL, and lowest in LL. In LL and WL, Enterobacter was the most frequent genus, while Klebsiella dominated in WW. Among the 20 SIL+ isolates identified, 10 came from WW, 9 from WL, and 1 from LL. One of these isolates (Enterobacter sp.) was tested as a supplement to the adult diet, without showing a beneficial effect on males pheromone calling behavior. Three isolates were also NFb+;two had the nifH gene. ARDRA was effective for rapid diazotroph discrimination. These findings highlight the potential of gut symbiotic bacteria in eco-friendly pest management strategies like the sterile insect technique (SIT). By using diazotrophic bacteria, protein requirements in artificial diets could be reduced, cutting costs and improving the affordability of SIT programs.
