Yoanna Eissler

Bacterial Active Community Cycling in Response to Solar Radiation and Their Influence on Nutrient Changes in a High-Altitude Wetland

Microbial communities inhabiting high-altitude spring ecosystems are subjected to extreme changes in solar irradiance and temperature throughout the diel cycle. Here, using 16S rRNA gene tag pyrosequencing (cDNA) we determined the composition of actively transcribing bacteria from spring waters experimentally exposed through the day (morning, noon, and afternoon) to variable levels of solar radiation and light quality, and evaluated their influence on nutrient recycling. Solar irradiance, temperature, and changes in nutrient dynamics were associated with changes in the active bacterial community structure, predominantly by Cyanobacteria, Verrucomicrobia, Proteobacteria, and 35 other Phyla, including the recently described Candidate Phyla Radiation (e.g., Parcubacteria, Gracilibacteria, OP3, TM6, SR1). Diversity increased at noon, when the highest irradiances were measured (3.3–3.9 H′, 1125 W m-2) compared to morning and afternoon (0.6–2.8 H′). This shift was associated with a decrease in the contribution to pyrolibraries by Cyanobacteria and an increase of Proteobacteria and other initially low frequently and rare bacteria phyla (< 0.5%) in the pyrolibraries. A potential increase in the activity of Cyanobacteria and other phototrophic groups, e.g., Rhodobacterales, was observed and associated with UVR, suggesting the presence of photo-activated repair mechanisms to resist high levels of solar radiation. In addition, the percentage contribution of cyanobacterial sequences in the afternoon was similar to those recorded in the morning. The shifts in the contribution by Cyanobacteria also influenced the rate of change in nitrate, nitrite, and phosphate, highlighted by a high level of nitrate accumulation during hours of high radiation and temperature associated with nitrifying bacteria activity. We did not detect ammonia or nitrite oxidizing bacteria in situ, but both functional groups (Nitrosomona and Nitrospira) appeared mainly in pyrolibraries generated from dark incubations. In total, our results reveal that both the structure and the diversity of the active bacteria community was extremely dynamic through the day, and showed marked shifts in composition that influenced nutrient recycling, highlighting how abiotic variation affects potential ecosystem functioning.

Detection and quantification of Chilean strains of infectious pancreatic necrosis virus by real-time RT-PCR assays using segment B as a target

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly prevalent disease that affects salmonid fish, mostly during their fresh water life period. Like many other viruses, IPNV produces highly heterogeneous populations. Therefore, diagnostic methods need to be checked constantly so that no variants of the virus escape detection. The IPNV genome is composed of two double-stranded RNA segments: A and B, polymerase chain reaction (PCR) methods normally use segment A as a target. In order to develop an optimized protocol to diagnose IPNV, we present a real-time RT-PCR (reverse transcription) technique, using primers designed to recognize segment B of the virus. To validate the ubiquity of the primers used, the IPNV isolates tested were sequenced and compared with previously published cladograms, which include a wide spectrum of genogroups. These primers made it possible to detect viral isolates belonging to genogroups 1 and 5, which were obtained from different locations linked to fish farming. As expected, we were able to detect the virus from distant Aquabirnavirus genogroups.

Active microbiome structure and its association with environmental factors and viruses at different aquatic sites of a high-altitude wetland

Salar de Huasco is a high‐altitude wetland characterized by a highly diverse microbial life adapted to extreme climatic and environmental conditions. Our study aims to determine active microbial community structure changes within different aquatic sites and its relationship with environmental factors and viruses as potential drivers of diversification in different aquatic areas of this ecosystem. In this study, bacteria and archaea composition (16S rRNA subunit pyrolibraries) and picoplankton and viral abundance were determined at ponds, springs and lagoon sites of the wetland during wet and dry seasons (February and July 2012, respectively). In general, mixosaline waters (1,400–51,000 μS/cm) usually found in ponds and lagoon presented higher picoplanktonic abundances compared to freshwater (<800 μS/cm) spring sites, ranging from 1.07 × 105 to 1.83 × 107 cells/ml. Viral abundance and viral to picoplankton ratio (VPR) also presented greater values at ponds compared to spring sites, reaching up to 4.78 × 108 viruses‐like particles and up to 351 for VPR. In general, ponds hold a higher microbial diversity and complexity associated also with the presence of microbial mats compared with water sources or lagoon (Shannon index H′ 2.6–3.9 vs. <2.0). A greater richness of archaea was also detected in ponds characterized by functional groups such as known methanogens and ammonia oxidizers, and uncultured groups. In total, our results indicate that among the different aquatic sites of the wetland, ponds presented a great microbial community diversification associated to a higher top‐down control by viruses which may influence nutrient and greenhouse gases cycling.

The activity of nitrifying microorganisms in a high-altitude Andean wetland

High-altitude wetland holds freshwater springs, evaporitic ponds and lagoon with variable salinity and nutrients, potentially influencing the ecology of nitrifying communities. In this study, nitrifying microorganisms in Salar de Huasco (Chile) were surveyed to determine bacterial and archaeal contribution to ammonium (AO), nitrite oxidation (NO), ammonium uptake (AU) during wet and dry seasons. The activity signals from these groups were assessed by specific amoA-qPCR transcription, 15N tracer studies and addition of group specific inhibitor experiments for nitrifying microorganisms (N1-guanyl-1, 7-diaminoheptane [GC7]-archaeal specific and allylthiourea [ATU]-bacterial specific). Nitrifying communities, i.e. Nitrosopumilus, Nitrosospira, Nitrosomonas, Kuenenia and Nitrospira, were more frequent (∼0.25% of 16S rRNA sequences) at low salinity sites. Bacterial amoA-qPCR transcripts also increased at low salinity and along in situ ammonium increase observed between wet/dry seasons. Nutrient changes through time and 15N tracer experiments results showed that AO and NO were detected and peaked mainly at low salinity-high ammonium sites (<37 000 μS cm-1 and >0.3 μM), whereas AU was predominant at evaporitic sites. Our results indicate that salinity and ammonium affect the nitrifying communities that are potentially more active at low-salinity sites but persistent at saltier evaporitic areas of the wetland when ammonium is available.

Genotyping of Infectious Pancreatic Necrosis virus through nested PCR and possible host-specific link by genogroup

Samples of fish organs from three salmonid species present in Chile, were analyzed through the nested polymerase chain reaction (nested PCR) to detect the presence of Infectious Necrosis Pancreatic virus (IPNV) and leading to their phylogenetic classification. The technique proved to be efficient and sensitive for detection and genotypification of viral samples which could not even be isolated in cell culture. The phylogenetic analysis showed the two genogroups previously described in the country, ie., European (Genogroup 5) and American (Genogroup 1), being the IPNV that belong to Genogroup 5 the dominant one (78.8%). It is clear that the Chilean IPNV is clustered within the Genogroup 1 forming a Chilean genotype that is separated from the reference strains (e.g. WB, VR-299). It was determined that there is a statistically significant relationship between the genonogroup that a viral isolate belongs and a specific host. Most of the viruses from Genogroup 5 were detected in Salmo salar , while the ones from Genogroup 1 were detected mainly in Oncorhynchus mykiss and O. Kisutch (P