Genomic profiling of climate adaptation in Aedes aegypti along an altitudinal gradient in Nepal indicates nongradual expansion of the disease vector.

Driven by globalization, urbanization and climate change, the distribution range of invasive vector species has expanded to previously colder ecoregions. To reduce health-threatening impacts on humans, insect vectors are extensively studied. Population genomics can reveal the genomic basis of adaptation and help to identify emerging trends of vector expansion. By applying whole genome analyses and genotype-environment associations to populations of the main dengue vector Aedes aegypti, sampled along an altitudinal gradient in Nepal (200-1300 m), we identify putatively adaptive traits and describe the species' genomic footprint of climate adaptation to colder ecoregions. We found two differentiated clusters with significantly different allele frequencies in genes associated to climate adaptation between the highland population (1300 m) and all other lowland populations (≤800 m). We revealed nonsynonymous mutations in 13 of the candidate genes associated to either altitude, precipitation or cold tolerance and identified an isolation-by-environment differentiation pattern. Other than the expected gradual differentiation along the altitudinal gradient, our results reveal a distinct genomic differentiation of the highland population. Local high-altitude adaptation could be one explanation of the population's phenotypic cold tolerance. Carrying alleles relevant for survival under colder climate increases the likelihood of this highland population to a worldwide expansion into other colder ecoregions.

The knowledge, attitude and practice of community people on dengue fever in Central Nepal: a cross-sectional study.

BACKGROUND: Since 2006, Nepal has experienced frequent Dengue fever (DF) outbreaks. Up to now, there have been no knowledge, attitude and practice (KAP) studies carried out on DF in Nepal that have included qualitative in-depth and quantitative data. Thus, we aimed to explore and compare the KAP of people residing in the lowland (< 1500 m) and highland (> 1500 m) areas of Nepal. METHODS: A cross-sectional mixed-method study was conducted in six districts of central Nepal in September-October 2018 including both quantitative (660 household surveys) and qualitative data (12 focus group discussions and 27 in-depth interviews). The KAP assessment was executed using a scoring system and defined as high or low based on 80% cut-off point. Logistic regression was used to investigate the associated factors, in quantitative analysis. The deductive followed by inductive approach was adopted to identify the themes in the qualitative data. RESULTS: The study revealed that both the awareness about DF and prevention measures were low. Among the surveyed participants, 40.6% had previously heard about DF with a significantly higher number in the lowland areas. Similarly, IDI and FGD participants from the lowland areas were aware about DF, and it's associated symptoms, hence they were adopting better preventive practices against DF. The findings of both the qualitative and quantitative data indicate that people residing in the lowland areas had better knowledge on DF compared to people in highland areas. All IDI participants perceived a higher chance of increasing future dengue outbreaks due to increasing temperature and the mobility of infected people from endemic to non-endemic areas. The most quoted sources of information were the television (71.8%) and radio (51.5%). Overall, only 2.3% of the HHS participants obtained high knowledge scores, 74.1% obtained high attitude scores and 21.2% obtained high preventive practice scores on DF. Among the socio-demographic variables, the area of residence, educational level, age, monthly income, SES and occupation were independent predictors of knowledge level, while the education level of the participants was an independent predictor of the attitude level. CONCLUSIONS: Our study found a very low level of knowledge and insufficient preventive practices. This highlights an urgent need for extensive dengue prevention programs in both highland and lowland communities of Nepal.

A barcoding pipeline for mosquito surveillance in Nepal, a biodiverse dengue-endemic country.

BACKGROUND: Vector-borne diseases are on the rise on a global scale, which is anticipated to further accelerate because of anthropogenic climate change. Resource-limited regions are especially hard hit by this increment with the currently implemented surveillance programs being inadequate for the observed expansion of potential vector species. Cost-effective methods that can be easily implemented in resource-limited settings, e.g. under field conditions, are thus urgently needed to function as an early warning system for vector-borne disease epidemics. Our aim was to enhance entomological capacity in Nepal, a country with endemicity of numerous vector-borne diseases and with frequent outbreaks of dengue fever. METHODS: We used a field barcoding pipeline based on DNA nanopore sequencing (Oxford Nanopore Technologies) and verified its use for different mosquito life stages and storage methods. We furthermore hosted an online workshop to facilitate knowledge transfer to Nepalese scientific experts from different disciplines. RESULTS: The use of the barcoding pipeline could be verified for adult mosquitos and eggs, as well as for homogenized samples, dried specimens, samples that were stored in ethanol and frozen tissue. The transfer of knowledge was successful, as reflected by feedback from the participants and their wish to implement the method. CONCLUSIONS: Cost effective strategies are urgently needed to assess the likelihood of disease outbreaks. We were able to show that field sequencing provides a solution that is cost-effective, undemanding in its implementation and easy to learn. The knowledge transfer to Nepalese scientific experts from different disciplines provides an opportunity for sustainable implementation of low-cost portable sequencing solutions in Nepal.

The ecophysiological plasticity of Aedes aegypti and Aedes albopictus concerning overwintering in cooler ecoregions is driven by local climate and acclimation capacity.

Aedes aegypti and Aedes albopictus transmit diseases such as dengue, and are of major public health concern. Driven by climate change and global trade/travel both species have recently spread to new tropic/subtropic regions and Ae. albopictus also to temperate ecoregions. The capacity of both species to adapt to new environments depends on their ecophysiological plasticity, which is the width of functional niches where a species can survive. Mechanistic distribution models often neglect to incorporate ecophysiological plasticity especially in regards to overwintering capacity in cooler habitats. To portray the ecophysiological plasticity concerning overwintering capability, we conducted temperature experiments with multiple populations of both species originating from an altitudinal gradient in South Asia and tested as follows: the cold tolerance of eggs (-2 °C- 8 days and - 6 °C- 2 days) without and with an experimental winter onset (acclimation: 10 °C- 60 days), differences between a South Asian and a European Ae. albopictus population and the temperature response in life cycles (13 °C, 18 °C, 23 °C, 28 °C). Ecophysiological plasticity in overwintering capacity in Ae. aegypti is high in populations originating from low altitude and in Ae. albopictus populations from high altitude. Overall, ecophysiological plasticity is higher in Ae. albopictus compared to Ae. aegypti. In both species acclimation and in Ae. albopictus temperate continental origin had a huge positive effect on survival. Our results indicate that future mechanistic prediction models can include data on winter survivorship of both, tropic and subtropic Ae. aegypti, whereas for Ae. albopictus this depends on the respective temperate, tropical region the model is focusing on. Future research should address cold tolerance in multiple populations worldwide to evaluate the full potential of the ecophysiological plasticity in the two species. Furthermore, we found that Ae. aegypti can survive winter cold especially when acclimated and will probably further spread to colder ecoregions driven by climate change.

Knowledge on exotic mosquitoes in Germany, and public acceptance and effectiveness of Bti and two self-prepared insecticides against Aedes japonicus japonicus.

Mosquito-borne diseases are a continuous challenge to public health. To prevent transmission, Integrated Vector Management (IVM) applies preventive, control, and communicational strategies that should be feasible, environmentally benign, and sustainable. IVM shows higher efficiency when being supported by local communities. Accordingly, we applied a social-ecological approach to identify the public acceptance of control measures and effectiveness of Eurocent coins containing copper, clove essential oil (EO) and Bacillus thuringiensis israelensis (Bti). We performed field and laboratory experiments to demonstrate the toxicity of alternative substances against Aedes japonicus japonicus. In expert interviews, we asked for (1) knowledge on exotic mosquitoes in Germany, (2) potential chances of alternative substances in future mosquito control, and (3) their needs for further clarification before application. We assessed potential users' (4) awareness of exotic mosquitoes and (5) willingness to apply the substances. Self-prepared copper coins and EO were clearly preferred by potential users over Bti. However, 100% mortality of the sensitive first stage could not be reached with the number of ten 5-Eurocent coins showing limited toxicity. Clove EO was shown to work as oviposition deterrent and larvicide with a LC50 of 17 mg l-1 (95% CI: 15-19 mg l-1). This study shows the importance of potential users' perspectives in IVM and the need for authorised insecticides.

Evaluating the efficiency of advanced wastewater treatment: target analysis of organic contaminants and (geno-)toxicity assessment tell a different story.

At a pilot scale wastewater treatment plant ozonation and powdered activated carbon filtration were assessed for their efficacy to remove trace organic contaminants from secondary treated effluents. A chemical analysis of 16 organic compounds was accompanied by a comprehensive suite of in vitro and in vivo bioassays with the focus on genotoxicity to account for the potential formation of reactive oxidation products. In vitro experiments were performed with solid phase extracted water samples, in vivo experiments with native wastewater in a flow through test system on site at the treatment plant. The chemical evaluation revealed an efficient oxidation of about half of the selected compounds by more than 90% at an ozone dose of 0.7 g/g DOC. A lower oxidizing efficiency was observed for the iodinated X-ray contrast media (49-55%). Activated carbon treatment (20 mg/L) was less effective for the removal of most pharmaceuticals monitored. The umuC assay on genotoxicity delivered results with about 90% decrease of the effects by ozonation and slightly lower efficiency for PAC treatment. However, the Ames test on mutagenicity with the strain YG7108 revealed a consistent and ozone-dose dependent increase of mutagenicity after wastewater ozonation compared to secondary treatment. Sand filtration as post treatment step reduced the ozone induced mutagenicity only partly. Also the fish early life stage toxicity test revealed an increase in mortality after ozonation and a reduced effect after sand filtration. Only activated carbon treatment reduced the fish mortality compared to conventional treatment on control level. Likewise the in vivo genotoxicity detected with the comet assay using fish erythrocytes confirmed an increased (geno-)toxicity after ozonation, an effect decrease after sand-filtration and no toxic effects after activated carbon treatment. This study demonstrates the need for a cautious selection of methods for the evaluation of advanced (oxidative) treatment technologies and of the effectiveness of post-treatments for elimination of adverse effects caused by oxidative treatments case by case.

Do contaminants originating from state-of-the-art treated wastewater impact the ecological quality of surface waters?

Since the 1980s, advances in wastewater treatment technology have led to considerably improved surface water quality in the urban areas of many high income countries. However, trace concentrations of organic wastewater-associated contaminants may still pose a key environmental hazard impairing the ecological quality of surface waters. To identify key impact factors, we analyzed the effects of a wide range of anthropogenic and environmental variables on the aquatic macroinvertebrate community. We assessed ecological water quality at 26 sampling sites in four urban German lowland river systems with a 0-100% load of state-of-the-art biological activated sludge treated wastewater. The chemical analysis suite comprised 12 organic contaminants (five phosphor organic flame retardants, two musk fragrances, bisphenol A, nonylphenol, octylphenol, diethyltoluamide, terbutryn), 16 polycyclic aromatic hydrocarbons, and 12 heavy metals. Non-metric multidimensional scaling identified organic contaminants that are mainly wastewater-associated (i.e., phosphor organic flame retardants, musk fragrances, and diethyltoluamide) as a major impact variable on macroinvertebrate species composition. The structural degradation of streams was also identified as a significant factor. Multiple linear regression models revealed a significant impact of organic contaminants on invertebrate populations, in particular on Ephemeroptera, Plecoptera, and Trichoptera species. Spearman rank correlation analyses confirmed wastewater-associated organic contaminants as the most significant variable negatively impacting the biodiversity of sensitive macroinvertebrate species. In addition to increased aquatic pollution with organic contaminants, a greater wastewater fraction was accompanied by a slight decrease in oxygen concentration and an increase in salinity. This study highlights the importance of reducing the wastewater-associated impact on surface waters. For aquatic ecosystems in urban areas this would lead to: (i) improvement of the ecological integrity, (ii) reduction of biodiversity loss, and (iii) faster achievement of objectives of legislative requirements, e.g., the European Water Framework Directive.

Deriving bio-equivalents from in vitro bioassays: assessment of existing uncertainties and strategies to improve accuracy and reporting.

Bio-equivalents (e.g., 17ß-estradiol or dioxin equivalents) are commonly employed to quantify the in vitro effects of complex human or environmental samples. However, there is no generally accepted data analysis strategy for estimating and reporting bio-equivalents. Therefore, the aims of the present study are to 1) identify common mathematical models for the derivation of bio-equivalents from the literature, 2) assess the ability of those models to correctly predict bio-equivalents, and 3) propose measures to reduce uncertainty in their calculation and reporting. We compiled a database of 234 publications that report bio-equivalents. From the database, we extracted 3 data analysis strategies commonly used to estimate bio-equivalents. These models are based on linear or nonlinear interpolation, and the comparison of effect concentrations (ECX ). To assess their accuracy, we employed simulated data sets in different scenarios. The results indicate that all models lead to a considerable misestimation of bio-equivalents if certain mathematical assumptions (e.g., goodness of fit, parallelism of dose-response curves) are violated. However, nonlinear interpolation is most suitable to predict bio-equivalents from single-point estimates. Regardless of the model, subsequent linear extrapolation of bio-equivalents generates additional inaccuracy if the prerequisite of parallel dose-response curves is not met. When all these factors are taken into consideration, it becomes clear that data analysis introduces considerable uncertainty in the derived bio-equivalents. To improve accuracy and transparency of bio-equivalents, we propose a novel data analysis strategy and a checklist for reporting Minimum Information about Bio-equivalent ESTimates (MIBEST).

Whole effluent toxicity assessment at a wastewater treatment plant upgraded with a full-scale post-ozonation using aquatic key species.

Ozonation as final wastewater (WW) polishing step, following conventional activated sludge treatment is increasingly implemented in sewage treatment for contaminant degradation to prevent surface water pollution. While the oxidative degradation of chemicals has been extensively investigated, the in vivo toxicological characteristics of ozonated whole effluents are rarely a matter of research. In the present study, whole effluents were toxicologically evaluated with an in vivo test battery before and after full-scale ozonation and subsequent sand filtration on site at a treatment plant. One aquatic plant (duckweed, Lemna minor) and five invertebrate species of different systematic groups (Lumbriculus variegatus, Chironomus riparius, Potamopyrgus antipodarum, Daphnia magna) were exposed to the effluents in a flow-through-designed test system with a test duration of 7-28 d. None of the considered toxicity endpoints correlated with the pollutant elimination. A tendency towards an increased toxicity after ozonation was apparent in three of the test systems showing [statistically] significant adverse effects in the L. variegatus toxicity test (decrease in reproduction and biomass). After sand filtration, adverse effects were reduced to a similar level like after conventional treatment. Solely the Daphnia reproduction test revealed beneficial effects after ozonation in combination with sand filtration. Results of the test battery indicate the formation of adverse oxidation products during WW ozonation. L. variegatus appeared to be the most sensitive of the five test species. Sand filtration effectively removes or detoxifies toxic oxidation products, as toxic effects were subsequently reduced to the level after conventional treatment.

Ozonation and activated carbon treatment of sewage effluents: removal of endocrine activity and cytotoxicity.

Concerns about endocrine disrupting compounds in sewage treatment plant (STP) effluents give rise to the implementation of advanced treatment steps for the elimination of trace organic contaminants. The present study investigated the effects of ozonation (O(3)) and activated carbon treatment (AC) on endocrine activities [estrogenicity, anti-estrogenicity, androgenicity, anti-androgenicity, aryl-hydrocarbon receptor (AhR) agonistic activity] with yeast-based bioassays. To evaluate the removal of non-specific toxicity, a cytotoxicity assay using a rat cell line was applied. Wastewater (WW) was sampled at two STPs after conventional activated sludge treatment following the secondary clarifier (SC) and after subsequent advanced treatments: O(3), O(3) + sand filtration (O(3-SF)), and AC. Conventional treatment reduced estrogenicity, androgenicity, and AhR agonistic activity by 78-99% compared to the untreated influent WW. Anti-androgenicity and anti-estrogenicity were not detectable in the influent but appeared in SC, possibly due to the more effective removal of respective agonists during conventional treatment. Endocrine activities after SC ranged from 2.0 to 2.8 ng/L estradiol equivalents (estrogenicity), from 4 to 22 µg/L 4-hydroxytamoxifen equivalents (anti-estrogenicity), from 1.9 to 2.0 ng/L testosterone equivalents (androgenicity), from 302 to 614 µg/L flutamide equivalents (anti-androgenicity), and from 387 to 741 ng/L ß-naphthoflavone equivalents (AhR agonistic activity). In particular, estrogenicity and anti-androgenicity occurred in environmentally relevant concentrations. O(3) and AC further reduced endocrine activities effectively (estrogenicity: 77-99%, anti-androgenicity: 63-96%, AhR agonistic activity: 79-82%). The cytotoxicity assay exhibited a 32% removal of non-specific toxicity after O(3) compared to SC. O(3) and sand filtration reduced cytotoxic effects by 49%, indicating that sand filtration contributes to the removal of toxicants. AC was the most effective technology for cytotoxicity removal (61%). Sample evaporation reduced cytotoxic effects by 52 (AC) to 73% (O(3)), demonstrating that volatile substances contribute considerably to toxic effects, particularly after O(3). These results confirm an effective removal or transformation of toxicants with receptor-mediated mode of action and non-specific toxicants during O(3) and AC. However, due to the limited extractability, polar ozonation by-products were neglected for toxicity analysis, and hence non-specific toxicity after O(3) is underestimated.

Comparative toxicity assessment of ozone and activated carbon treated sewage effluents using an in vivo test battery.

Wastewater treatment plants do not eliminate micropollutants completely and are thus important point sources for these substances. Ozonation and activated carbon treatment might be beneficial for ecosystem health as these techniques provide effective barriers to organic contaminants. However, a toxicity evaluation is required to investigate toxicity reduction and to assess the potential formation of toxic oxidation byproducts during ozonation. Therefore a comparative toxicity evaluation of different treated wastewater effluents was performed on site at a half scale treatment plant equipped with an ozonation step and an activated carbon treatment step in parallel subsequent to conventional activated sludge treatment. For this purpose four invertebrate and one higher plant toxicity test were selected to assess potential biological effects on whole organisms. The reproduction test with the mudsnail Potamopyrgus antipodarum exhibited a decreased reproductive output after advanced treatment compared to conventional treatment. This indicates an effective estrogenicity removal by ozonation and activated carbon treatment and is confirmed by results of the yeast estrogen screen with a reduction of in vitro estrogenic activity by >75%. The Lumbriculus variegatus test revealed a significantly enhanced toxicity after ozonation compared to conventional treatment whereas this effect was reduced following subsequent sand filtration. When ozonation was applied, a significantly increased genotoxicity was observed, detected with the comet assay using haemolymph of the zebra mussel. Again, this effect was removed by subsequent sand filtration to the level of conventional treatment. Activated carbon treatment even resulted in a significant reduction of genotoxicity. Adverse effects after the ozone reactor are possibly a result of the formation of toxic oxidation byproducts. Biologically active sand filtration obviously is an effective barrier to such compounds.

Toxication or detoxication? In vivo toxicity assessment of ozonation as advanced wastewater treatment with the rainbow trout.

Ozonation as advanced wastewater treatment method is an effective technique for micropollutant removal. However, the application of this method carries the inherent danger to produce toxic oxidation byproducts. For an ecotoxicological assessment conventionally treated wastewater, wastewater after ozonation and ozonated wastewater after sand filtration were evaluated in parallel at an operating treatment plant via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). The FELST revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW and to the ozonated water after sand filtration. Hence sand filtration obviously prevents from adverse ecotoxicological effects of ozonation. An additional test with yolk-sac larvae resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treated wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. Adverse ozonation effects may have been a result of the conversion of chemicals into more toxic metabolites. However, sand filtration reduced toxication effects indicating that these oxidation byproducts are readily degradable or adsorbable. The results indicate that in any case ozonation should not be applied without subsequent post treatment appropriate for oxidation byproducts removal (e.g. sand filtration).