Insights into the panorama of multiple DNA viruses in municipal wastewater and recycled sludge in Tianjin, China.

Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, and little is known about the diversity of DNA viral communities and their driving factors in municipal wastewater treatment environments. Herein, we conducted a pilot study to explore DNA virus profiles in municipal wastewater and recycled sludge by metagenomics method, and track their temporal changes in northern China. Results showed that 467 viral species were co-shared among all the samples. We identified six families of human viruses with a prevalence of 0.1%, which were rare but relatively stable in wastewater and sludge for six months. Adenoviridae, Parvoviridae, and Herpersviridae were the most dominant human viral families in municipal wastewater and recycled sludge. A time series of samples revealed that the dynamic changes of human DNA viruses were stable based on qPCR results, particularly for high-risk fecal-oral transmission viruses of adenovirus, bocavirus, polyomavirus, human gamma herpesvirus, human papillomavirus, and hepatitis B virus. Concentrations of Adenovirus (5.39-7.48 log10 copies/L) and bocavirus (4.36-7.48 log10 copies/L) were observed to be the highest in these samples compared to other viruses. Our findings demonstrated the DNA viruses' high prevalence and persistence in municipal wastewater treatment environments, highlighting the value of enhancing public health responses based on wastewater-based epidemiology.

Early larval exposure to flumethrin induces long-term impacts on survival and memory behaviors of adult worker bees Apis mellifera.

Flumethrin has been supplied as an acaricide for Varroa mite control in world-wide apiculture due to its low lethal effects on honey bees. However, little is known about the effects of short-term flumethrin exposure in the larval stage on adult life stage of bees involving survival status, foraging and memory-related behaviors. Here, we found that exposure to flumethrin at 1 mg/L during larval stage reduced survival and altered foraging activities including induced precocious foraging activity, decreased foraging trips and time, and altered rotating day-off status of adult worker bees using the radio frequency identification system. Furthermore, larval exposure at 1 mg/L flumethrin influenced the correct proboscis extension responses of 7-day-old worker bees and decreased homing rates of 20-day-old worker bees, suggesting that 1 mg/L flumethrin exposure at larval stage could affect memory-related behaviors of adult bees; meanwhile, three genes related to memory (GluRA, Nmdar1 and Tyr1) were certainly down-regulated varying different flumethrin concentrations (0.01, 0.1, and 1 mg/L). Combined with transcriptomic sequencing, differentially expressed genes involved in olfactory memory of adult bees were completely down-regulated under flumethrin exposure. Our findings highlight the unprecedented impact of short-term exposure of insecticides on honey bees in long-term health monitoring under field conditions.

Dissemination of antibiotic resistance genes from aboveground sources to groundwater in livestock farms.

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are prevalent in various environments on livestock farms, including livestock waste, soil, and groundwater. Contamination of groundwater by ARB and ARGs in livestock farms is a growing concern as it may have potentially huge risks to human health. However, the source of groundwater-borne ARB and ARGs in animal farms remains largely unknown. In this study, different types of samples including groundwater and its potential contamination sources from aboveground (pig feces, wastewater, and soil) from both working and abandoned swine feedlots in southern China were collected and subjected to metagenomic sequencing and ARB isolation. The source tracking based on metagenomic analysis revealed that 56-95 % of ARGs in groundwater was attributable to aboveground sources. Using metagenomic assembly, we found that 45 ARGs predominantly conferring resistance to aminoglycosides, sulfonamides, and tetracyclines could be transferred from the aboveground sources to groundwater, mostly through plasmid-mediated horizontal gene transfer. Furthermore, the full-length nucleotide sequences of sul1, tetA, and TEM-1 detected in ARB isolates exhibited the close evolutionary relationships between aboveground sources and groundwater. Some isolated strains of antibiotic-resistant Pseudomonas spp. from aboveground sources and groundwater had the high similarity (average nucleotide identity > 99 %). Notably, the groundwater-borne ARGs were identified as mainly carried by bacterial pathogens, potentially posing risks to human and animal health. Overall, this study underscores the dissemination of ARGs from aboveground sources to groundwater in animal farms and associated risks.

Spatial distribution of two acaricides and five neonicotinoids in beehives and surrounding environments in China.

Managed bees commonly suffer from cross-contamination with acaricides and neonicotinoids, posing robust threats to bee population health. However, their residual characteristics and spatial distribution in beehives and surrounding environments are poorly understood. This study detected two common acaricides and five neonicotinoids in 240 beehive samples and 44 surrounding environmental samples collected from 25 Chinese provinces. The results showed that 40.0% of the honey samples contained acaricides and 83.1% contained neonicotinoids. Neonicotinoid concentrations in honey were geographically distinguished by the "Hu Huanyong line", and concentrations of neonicotinoids in honey from eastern areas were 2.65-fold higher than those in honey from western areas. Compared to the approved acaricide amitraz, the banned acaricide coumaphos was detected more frequently in honey and was positively correlated with that quantified in the paired pollen samples. Although coumaphos was identified in only three soil samples, lower coumaphos residues in honey might be associated with persistent pollution in the surrounding environment. Conversely, neonicotinoids were detected at higher levels in honey than in the pollen and soil, demonstrating that the neonicotinoid residues in honey have a cumulative effect. This study contributes to a better understanding of the pesticide contamination scenarios that underlie the exposure risks of bees.

Molecular characterization of human bocavirus in municipal wastewaters using amplicon target sequencing.

Human bocavirus (HBoV) is an emerging health concern worldwide, associated with range of clinical manifestations, including gastroenteritis and respiratory infections. Therefore, it is crucial to comprehend and minimize their prevalence in different systems. In this study, we conducted regular sampling throughout the year in two different sizes and work processes of wastewater treatment plants (WWTPs) in Tianjin, China. Our objective was to investigate the occurrence, prevalence, and endurance of HBoV in wastewater, while also evaluating the efficacy of amplicon target sequencing in directly detecting HBoV in wastewater. At two WWTPs, HBoV2 (45.51 %-45.67 %) and HBoV3 (38.30 %-40.25 %) were the most common genotypes identified, and the mean concentration range of HBoV was 2.54-7.40 log10 equivalent copies/l as determined by multiplex real-time quantitative PCR assay. A positive rate of HBoV was found in 96.6 % (29/30) samples of A-WWTP, and 96.6 % (26/27) samples of B-WWTP. The phylogenetic analysis indicated that the nucleotide similarity between the HBoV DNA sequences to the reference HBoV sequences published globally ranged from 90.14 %-100 %. A significant variation in the read abundance of HBoV2 and HBoV3 in two wastewater treatment plants (p < 0.05) was detected, specifically in the Winter and Summer seasons. The findings revealed a strong correlation between the genotypes detected in wastewater and the clinical data across various regions in China. In addition, it is worth mentioning that HBoV4 was exclusively detected in wastewater and not found in the clinical samples from patients. This study highlights the high prevalence of human bocavirus in municipal wastewater. This finding illustrates that amplicon target sequencing can amplify a wide variety of viruses, enabling the identification of newly discovered viruses.

Metabolic profiling of Apis mellifera larvae treated with sublethal acetamiprid doses.

Acetamiprid is a neonicotinoid insecticide used in crop protection worldwide. Such widespread application can pose risks to pollinator insects, particularly to honeybees (Apis mellifera); therefore, the evaluation of the harmful effects of acetamiprid is necessary. Recent studies report behavior and gene expression dysfunction in honeybees, related to acetamiprid contamination. However, most studies do not consider potential metabolism disorders. To examine the effects of sublethal acetamiprid doses on the hemolymph metabolism of honeybees, worker bee larvae(2 days old) were fed with sucrose water containing different concentrations of acetamiprid (0, 5, and 25 mg/L) until capped (6 days old). The hemolymph (200 µL) of freshly capped larvae was collected for liquid chromatography-mass spectrometry (LC-MS). Overall, increasing acetamiprid exposure induced greater metabolic variations in worker bee larvae(treated groups compared to untreated). In the positive ion mode, 36 common differential metabolites in the acetamiprid-treated groups were screened from the identified differential metabolites. Of these, 19 metabolites were upregulated, and 17 were downregulated. 10 common differential metabolites were screened in the negative ion mode. 3 metabolites were upregulated, and 7 metabolites were downregulated. These common metabolites included traumatic acid, indole etc. These commonly differentiated metabolites were classified as compounds with biological roles, lipids, and phytochemical compounds, and others. The metabolic pathways of common differentiated metabolites with significant differences (P < 0.05) included the metabolism of tryptophan, purines, phenylalanine, etc. As the concentration of acetamiprid increased, the content of traumatic acid increased, the content of tryptophan metabolite l-kynurenine and indole decreased, and the content of lipids also decreased. Our results revealed that the damage to honeybee larvae increased when the acetamiprid solution formulations residue in their food had a concentration higher than 5 mg/L, causing metabolic disorders in various substances in larvae. Analysis of these metabolic processes can provide a theoretical basis for further research on the metabolism of acetamiprid-treated honeybees and elucidate the detoxification mechanisms.

Exposure to acetamiprid influences the development and survival ability of worker bees (Apis mellifera L.) from larvae to adults.

In most cases, honey bees experience pesticide pollution in a long-term period through direct or indirect exposure, such as the development process from larvae to the pre-harvest stage. At present, little is known about how honey bees respond to pesticide stresses during the continuous development period. This study aims to examine effects of long-term acetamiprid exposure on the development and survival of honey bees, and further present the expression profile in larvae, 1-day-old, and 7-day-old adult worker bees that related to immune, detoxification, acetylcholinesterase (AChE) and memory. Honey bees from 2-day-old larvae to 14-day-old adults except the pupal stage were continuously fed with different acetamiprid solutions (0, 5, and 25 mg/L). We found that acetamiprid over 5 mg/L disturbed the development involving birth weight and emergence rate of newly emerged bees, and reduced the proportion of capped cells of larvae at 25 mg/L; gene expression related to immune and detoxification of worker bees exposed to acetamiprid was roughly activated, returned and then inhibited from larval to emerged and to the late adult stage, respectively. Moreover, lifespans of bees treated with acetamiprid at 25 mg/L were significantly reduced. The present study reflects the potential risk for honey bees continuously exposed to acetamiprid in the development stage.

Sublethal acetamiprid doses negatively affect the lifespans and foraging behaviors of honey bee (Apis mellifera L.) workers.

The neonicotinoid insecticide acetamiprid is applied widely for pest control in agriculture production. However, little is known about the effects of acetamiprid on the foraging behavior of nontarget pollinators. This study aims to investigate effects of sublethal acetamiprid doses on lifespans and foraging behaviors of honey bees (Apis mellifera L.) under natural swarm conditions. Newly emerged worker bees of each treatment received a drop of 1.5 µL acetamiprid solution (containing 0, 0.5, 1, and 2 µg/bee acetamiprid, diluted by water) on the thorax respectively. Bees from 2-day-old to deadline were monitored on foraging behaviors involving the age of bee for first foraging flights, rotating day-off status and the number of foraging flights using the radio frequency identification (RFID) system. We found that acetamiprid at 2 µg/bee significantly reduced the lifespan, induced precocious foraging activity, influenced the rotating day-off status and decreased foraging flights of worker bees. The abnormal behaviors of worker bees may be associated with a decline in lifespan. This work may provide a new perspective into the neonicotinoids that accelerate the colony failure.

Vancomycin exposure caused opportunistic pathogens bloom in intestinal microbiome by simulator of the human intestinal microbial ecosystem (SHIME).

Antibiotics are emerging organic pollutants posing high health risks to humans by causing human intestinal microbial disorders with increasing abundances of opportunistic pathogens, and fecal microbiota transplantation (FMT) has been confirmed to restore the dysbiosis of gut flora in many kinds of intestinal disease. However, to date, few studies have focused on the bloomed opportunistic pathogens associated human disease-related pathways as well as antibiotic resistance genes (ARGs) after vancomycin exposure, and there is limited information on using FMT for restoration of intestinal microbiome affected by antibiotics. Therefore, this study investigated effects of vancomycin on the opportunistic pathogens, human disease-related pathways as well as ARGs in human gut, and the restoration of intestinal microbiome by FMT. Results indicated that vancomycin treatment substantially increased human disease-related pathways and decreased abundances of ARGs. Besides, the bloomed opportunistic pathogens including Achromobacter, Klebsiella, and Pseudomonas, caused by vancomycin exposure, were positively correlated with human disease-related pathways. The microbiota abundance and genes of human disease-related pathways and antibiotic resistance showed a remarkable return towards baseline after FMT, but not for natural recovery. These findings suggest that impacts of vancomycin on human gut are profound and FMT will be a promising strategy in clinical application that can restore the dysbiosis of gut microbiota, which may be valuable for directing future work.

Effect of Astragalus membranaceus Oral Solution on Lifespan and Learning and Memory Ability of Honey Bees.

In this study, the effects of Astragalus membranaceus oral solution on lifespan and learning and memory abilities of honey bees were evaluated. Two groups of bees were fed with sucrose syrup (50%) containing low dose (1.33%) and high dose (13.3%) of A. membranaceus oral solution, respectively. The proboscis extension response (PER) analysis was applied to examine the learning and memory capabilities of bees. Two genes related to memory formation in honey bees were determined by real-time PCR. High dose (13.3%) of A. membranaceus significantly decreased the mean lifespan of bees compared to the bees fed with low dose (1.33%) and control bees. No significant differences in lifespan of bees were found between low-dose-fed bees and control bees. The results of PER experiments showed apparent improvement in the memorizing ability of the high-dose group (in comparison with the control group). Moreover, the relative expression levels of Nmdar1 in the low-dose group and control group were significantly lower than those in the high-dose group. It is preliminarily concluded that A. membranaceus has an adverse effect on the mean lifespan of honey bees but might be helpful in strengthening memories.