Biological-Activity-Based Prioritization of Antidepressants in Wastewater in England and Japan.

Antidepressants are one of the most commonly prescribed pharmaceuticals. Although they have been frequently detected in aquatic environments around the globe, little is known regarding their adverse effects on humans and aquatic organisms. Recently, an in vitro monoamine transporter inhibition assay was developed to detect transporter-inhibitory activities of antidepressants in wastewater in Japan. However, it was unclear which antidepressants were responsible for transporter-inhibitory activities in wastewater. Herein, the per capita consumption of 32 antidepressants, their excretion of unchanged parent compounds, per capita water consumption, removal rate during wastewater treatment processes, and potency values from the monoamine transporter inhibition assay were used to prioritize antidepressants of concern in effluent wastewater in England and Japan. In both countries, sertraline and O-desmethylvenlafaxine had the highest contribution to inhibitory activities against the human serotonin transporter (hSERT) and zebrafish SERT (zSERT), respectively. It was found that the antidepressants inhibited the zSERT more strongly than the hSERT. The inhibitory activities found against the zSERT in wastewater in England and Japan were higher than thresholds for abnormal behavior in fish. The antidepressants prioritized in this study provide insight into launching environmental monitoring and ecotoxicological studies of antidepressants.

Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and ß-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC and SFH-1) in Enterobacterales isolated from surface waters in Japan.

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan. METHODS: Eighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21). RESULTS: Various rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were co-located on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. CONCLUSIONS: This study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

Characterization of G protein-coupled estrogen receptors in Japanese medaka, Oryzias latipes.

The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17ß-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

Biological Activity-Based Prioritization of Pharmaceuticals in Wastewater for Environmental Monitoring: G Protein-Coupled Receptor Inhibitors.

Pharmaceuticals raise concerns for aquatic species owing to their biological activities. It is estimated that nearly 40% of marketed pharmaceuticals target G protein-coupled receptors (GPCRs). Using an in vitro transforming growth factor-α (TGFα) shedding assay, we previously detected antagonistic activities of GPCR-acting pharmaceuticals against angiotensin (AT1), dopamine (D2), acetylcholine (M1), adrenergic family members (ß1), and histamine (H1) receptors at up to µg-antagonist-equivalent quantities/L in wastewater in England and Japan. However, which pharmaceuticals were responsible for biological activities in wastewater remained unclear. Here, we used (1) the consumption of GPCR-acting pharmaceuticals, particularly antagonists, as calculated from prescriptions, (2) their urinary excretion, and (3) their potency measured by the TGFα shedding assay to prioritize them for analysis in wastewater in England and Japan. We calculated predicted activities of 48 GPCR-acting pharmaceuticals in influents in England and Japan and identified which were mainly responsible for antagonistic activities in wastewater against each GPCR. Mixtures of pharmaceuticals tested in this study were confirmed to behave additively. The combination of consumption and potency is useful in prioritizing pharmaceuticals for environmental monitoring and toxicity testing.

Wastewater-derived antagonistic activities of G protein-coupled receptor-acting pharmaceuticals in river water.

Pharmaceuticals are widely detected in aquatic environments, and their potential risks to aquatic species are of concern because they are designed to be biologically active. Here, we used an in vitro assay, called the transforming growth factor α shedding assay, to measure the biological activities of G protein-coupled receptor (GPCR)-acting pharmaceuticals present in river water and effluents from municipal wastewater treatment plants (WWTPs) in Japan from 2014 to 2016. Antagonistic activities against angiotensin (AT1), dopamine (D2), adrenergic (ß1), acetylcholine (M1) and histamine (H1) receptors were detected in river water, and were stronger downstream than upstream owing to effluent from WWTPs along the river. Ozonation at one WWTP reduced these activities. Concentrations of sulpiride (D2 antagonist) could explain 73% of antagonistic activities against the D2 receptor; those of metoprolol, atenolol and propranolol (ß1 antagonists) could explain 16% of activities against the ß1 receptor; and those of pirenzepine (M1 antagonist) could explain 15% of activities against the M1 receptor. Therefore, other receptor antagonists also occur. GPCR-acting pharmaceuticals should be given more attention in environmental monitoring and toxicity testing.

Quantification of Pharmaceutical Related Biological Activity in Effluents from Wastewater Treatment Plants in UK and Japan.

While pharmaceuticals are now routinely detected in aquatic environments, we know little of the biological activity their presence might provoke. It is estimated that nearly 40% of all marketed pharmaceuticals are G protein-coupled receptors (GPCRs) acting pharmaceuticals. Here, we applied an in-vitro assay, called the TGFα shedding assay, to measure the biological activities of GPCRs-acting pharmaceuticals present in effluents from municipal wastewater treatment plants in the United Kingdom (UK) and Japan from 2014 to 2016. The results indicated that compounds were present in the wastewater with antagonistic activities against angiotensin (AT1), dopamine (D2), adrenergic (ß1), acetylcholine (M1), and histamine (H1) receptors in both countries. The most consistent and powerful antagonistic activity was against the H1, D2, and AT1 receptors at up to microgram-antagonist-equivalent quantity/L. Chemical analysis of the same UK samples was also conducted in parallel. Comparing the results of the bioassay with the chemical analysis indicated (1) the existence of other D2 or M1 receptor antagonists besides sulpiride (D2 antagonist) or pirenzepine (M1 antagonist) in wastewater and (2) that there might be a mixture effect between agonist and antagonistic activities against ß1 receptor. GPCR-acting pharmaceuticals should be paid more attention in the environmental monitoring and toxicity testing in future studies.

Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

Detection of physiological activities of G protein-coupled receptor-acting pharmaceuticals in wastewater.

Although pharmaceuticals are generally found at very low levels in aquatic environments, concern about their potential risks to humans and aquatic species has been raised because they are designed to be biologically active. To resolve this concern, we must know whether the biological activity of pharmaceuticals can be detected in waters. Nearly half of all marketed pharmaceuticals act by binding to the G protein-coupled receptors (GPCRs). In this study, we measured the physiological activity of pharmaceuticals in wastewater. We applied the in vitro transforming growth factor-α (TGFα) shedding assay, which accurately and sensitively detect GPCR activation, to investigate the agonistic/antagonistic activities of wastewater extracts against receptors for angiotensin (AT1), dopamine (D2, D4), adrenergic family members (α1B, α2A, ß1, ß3), acetylcholine (M1, M3), cannabinoid (CB1), vasopressin (V1A, V2), histamine (H1, H2, H3), 5-hydroxytryptamine (5-HT1A, 5-HT2C), prostanoid (EP3), and leukotriene (BLT1). As a result, antagonistic activity against AT1, D2, α1B, ß1, M1, M3, H1, and V2 receptors was detected at up to several µg/L for the first time. Agonistic activity against α2A receptor was also detected. The TGFα shedding assay is useful for measuring the physiological activity of GPCR-acting pharmaceuticals in the aquatic environment.

The challenge presented by progestins in ecotoxicological research: a critical review.

Around 20 progestins (also called gestagens, progestogens, or progestagens) are used today in assisting a range of medical conditions from endometrial cancer to uterine bleeding and as an important component of oral contraception. These progestins can bind to a wide range of receptors including progestin, estrogen, androgen, glucocorticoid, and mineralocorticoid receptor, as well as sex hormone and corticosteroid binding globulins. It appears that only five of these (four synthetic and one natural) progestins have so far been studied in sewage effluent and surface waters. Analysis has reported values as either nondetects or low nanograms per liter in rivers. Seven of the progestins have been examined for their effects on aquatic vertebrates (fish and frogs). The greatest concern is associated with levonorgestrel, norethisterone, and gestodene and their ability to reduce egg production in fish at levels of 0.8-1.0 ng/L. The lack of environmental measurements, and some of the contradictions in existing values, however, hampers our ability to make a risk assessment. Only a few nanograms per liter of ethynodiol diacetate and desogestrel in water would be needed for fish to receive a human therapeutic dose for these progestins according to modeled bioconcentration factors. But for the other synthetic progestins levels would need to reach tens or hundreds of nanograms per liter to achieve a therapeutic dose. Nevertheless, the wide range of compounds, diverse receptor targets, and the effect on fish reproduction at sub-nanogram-per-liter levels should prompt further research. The ability to impair female reproduction at very low concentrations makes the progestins arguably the most important pharmaceutical group of concern after ethinylestradiol.

Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis.

The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17ß-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.

Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Co-occurrence of estrogenic and antiestrogenic activities in wastewater: quantitative evaluation of balance by in vitro ERα reporter gene assay and chemical analysis.

Endocrine-disrupting chemicals are exogenous substances that alter the function of the endocrine system, with adverse health effects on organisms or their progeny. In vitro estrogen receptor (ER) reporter gene assays have long been used to measure estrogenic activity in wastewater. Nevertheless, there is still uncertainty about their usefulness in environmental monitoring on account of a discrepancy between the estrogenic response of the in vitro assay and concentrations of estrogenic compounds determined by chemical analysis. Here, we measured estrogenic and antiestrogenic activities in wastewater by ERα reporter gene assay. All samples were simultaneously analyzed for estrone, 17ß-estradiol, estriol, and 17α-ethynylestradiol, and the concentrations were used to predict estrogenic activity. All samples in which measured estrogenic activity was significantly lower than predicted showed strong antiestrogenic activity. In addition, we confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. These results indicate that antiestrogenic compounds in wastewater suppress the activity of natural estrogens, and the reporter gene assay represents the net activity.

Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17ß (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

Comparison on removal performance of virus, antibiotic-resistant bacteria, cell-associated and cell-free antibiotic resistance genes, and indicator chemicals by ozone in the filtrated secondary effluent of a sewage treatment plant.

Due to the widespread appearance of viruses, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) in the aquatic environment, more powerful oxidation processes such as ozonation are needed to enhance the efficiency of their inactivation and removal during wastewater treatment. However, information is lacking on the elimination rates of viruses, ARBs, cell-associated ARGs (ca-ARGs), and cell-free ARGs (cf-ARGs) during ozonation. This study examined the kinetics and dose-dependent inactivation of a virus (MS2 coliphage) and an ARB (Ampicillin-resistant [AmpR] E. coli) and the removal of ca- and cf-ARGs (plasmid-encoded blaTEM) by ozonation in a filtered secondary effluent (SE) of a municipal sewage treatment plant (STP). In addition, the ozonation kinetics of carbamazepine (CBZ) and metoprolol (MTP)-ubiquitous organic micropollutants with different removal rate constants-were also investigated in order to monitor their effectiveness as indicators for the abovementioned biological risk factors. Our results showed that ozonation was an efficient way to remove MS2, AmpRE. coli, ARGs, CBZ, and MTP. We investigated the kinetics of their inactivation/removal with respect to exposure in terms of CT (dissolved ozone concentration C and contact time T) value, and found their inactivation/removal constants were in the following order: MS2 (8.66 ×103 M-1s-1) ≈ AmpRE. coli (8.19 ×103 M-1s-1) > cf-ARG (3.95 ×103 M-1s-1) > CBZ (3.21 ×103 M-1s-1) > ca-ARG (2.48×103 M-1s-1) > MTP (8.35 ×102 M-1s-1). In terms of specific ozone dose, > 5-log inactivation of MS2 was observed at > 0.30 mg O3/mg DOC, while > 5-log inactivation of AmpRE. coli was confirmed at 1.61-2.35 mg O3/mg DOC. Moreover, there was almost no removal of ca-ARG when the specific ozone dose was < 0.68 mg O3/mg DOC. However, 2.86-3.42-log removal of ca-ARG was observed at 1.27-1.31 mg O3/mg DOC, while 1.14-1.36-log removal of cf-ARG was confirmed at 3.60-4.30 mg O3/mg DOC. As alternative indicators, > 4-log removal of CBZ was observed at > 1.00 mg O3/mg DOC, while > 2-log removal of MTP was confirmed at > 2.00 mg O3/mg DOC. Thus, it was observed that inactivation of E. coli needs a greater ozone dose to achieve the same level of inactivation of AmpRE. coli; for ARGs, cf-ARG can persist longer than ca-ARG if low dosages of ozone are applied in the filtrated SE, CBZ might act as an indicator with which to monitor the inactivation of viruses and ARBs, while MTP might act as an indicator with which to monitor removal of ARGs. Moreover, cf-ARG cannot be neglected even after ozonation due to the possibility that ca-ARGs can become cf-ARGs during ozonation and be discharged with the final effluent, posing a potential risk to the receiving environment.

Evaluation of virus removal in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes based on long-term monitoring at two wastewater treatment plants.

The membrane bioreactor (MBR) process always offers better wastewater treatment than conventional activated sludge (CAS) treatment. However, the difference in their efficacy of virus reduction remains unknown. To investigate this, we monitored virus concentrations before and after MBR and CAS processes over 2 years. Concentrations of norovirus genotypes I and II (NoV GI and GII), aichivirus (AiV), F-specific RNA phage genotypes I, II, and III (GI-, GII-, and GIII-FRNAPHs), and pepper mild mottle virus (PMMoV) were measured by a quantitative polymerase chain reaction (qPCR) method at two municipal wastewater treatment plants (WWTPs A and B) in Japan. Virus concentration datasets containing left-censored data were estimated by using both maximum likelihood estimation (MLE) and robust regression on order statistics (rROS) approaches. PMMoV was the most prevalent at both WWTPs, with median concentrations of 7.5 to 8.8 log10 copies/L before treatment. Log10 removal values (LRVs) of all viruses based on means and standard deviations of concentrations before and after treatment were consistently higher following MBR than following CAS. We used NoV GII as a model pathogen in a quantitative microbial risk assessment of the treated water, and we estimated the additional reductions required following MBR and CAS processes to meet the guideline of 10-6 DALYs pppy for safe wastewater reuse.

Explaining the impact of mutations on quantification of SARS-CoV-2 in wastewater.

Wastewater surveillance is an effective tool for monitoring community spread of COVID-19 and other diseases. Quantitative PCR (qPCR) analysis for wastewater surveillance is more susceptible to mutations in target genome regions than binary PCR analysis for clinical surveillance. The SARS-CoV-2 concentrations in wastewater estimated by N1 and N2 qPCR assays started to diverge around July 2022 in data from different sampling sites, analytical methods, and analytical laboratories in Japan. On the basis of clinical genomic surveillance data and experimental data, we demonstrate that the divergence is due to two mutations in the N1 probe region, which can cause underestimation of viral concentrations. We further show that this inaccuracy can be alleviated if the qPCR data are analyzed with the second derivative method or the Cy0 method instead of the crossing point method.

Tracking community infection dynamics of COVID-19 by monitoring SARS-CoV-2 RNA in wastewater, counting positive reactions by qPCR.

Wastewater-based epidemiology has proved useful for monitoring the COVID-19 infection dynamics in communities. However, in regions of low prevalence, low concentrations of SARS-CoV-2 RNA in wastewater make this difficult. Here, we used real-time reverse-transcription PCR (RT-qPCR) to monitor SARS-CoV-2 RNA in wastewater from October 2020 to December 2022 during the third, fourth, fifth, sixth, seventh, and eighth waves of the COVID-19 outbreak in Japan. Viral RNA was below the limit of detection in all samples during the third and fourth waves. However, by counting the number of positive replicates in qPCR of each sample, we found that the positive ratio to all replicates in wastewater was significantly correlated with the number of clinically confirmed cases by the date of symptom onset during the third, fourth, and fifth waves. Time-step analysis indicated that, for 2 days either side of symptom onset, COVID-19 patients excreted in their feces large amounts of virus that wastewater surveillance could detect. We also demonstrated that the viral genome copy number in wastewater, as estimated from the positive ratio of SARSA-CoV-2 RNA, was correlated with the number of clinically confirmed cases. The positive count method is thus useful for tracing COVID-19 dynamics in regions of low prevalence.

Eyestalk transcriptome and methyl farnesoate titers provide insight into the physiological changes in the male snow crab, Chionoecetes opilio, after its terminal molt.

The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.