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Freshwater mussels (order: Unionida) are highly imperiled globally and are increasingly the focus of captive propagation efforts to protect and restore wild populations. The Upper Tennessee River Basin (UTRB) in Virginia is a freshwater biodiversity hotspot hosting at least 45 of North America's ~300 species of freshwater mussels, including 21 threatened and endangered species listed under the U.S. Endangered Species Act. Recent studies have documented that viruses and other microbes have contributed to freshwater mussel population declines in the UTRB. We conducted a multi-year longitudinal study of captive-reared hatchery mussels released to restoration sites throughout the UTRB to evaluate their viromes and compare them to captive hatchery environments. We documented 681 viruses from 27 families. The hatchery mussels had significantly less viruses than those deployed to wild sites, with only 20 viruses unique to the hatchery mussels. After the hatchery mussels were released into the wild, their number of viruses initially spiked and then increased steadily over time, with 451 viruses in total unique to the mussels in the wild. We found Clinch densovirus 1 (CDNV-1), a virus previously associated with mass mortality events in the Clinch River, in all samples, but the wild site mussels consistently had significantly higher CDNV-1 levels than those held in the hatchery. Our data document substantial differences between the viruses in the mussels in the hatchery and wild environments and rapid virome shifts after the mussels are released to the wild sites. These findings indicate that mussel release programs might benefit from acclimatization periods or other measures to mitigate the potential negative effects of rapid exposure to infectious agents found in natural environments.

Carbon dioxide (CO 2 ) is gaining interest as a tool to combat aquatic invasive species, including zebra mussels (Dreissena polymorpha). However, the effects of water chemistry on CO 2 efficacy are not well described. We conducted five trials in which we exposed adult zebra mussels to a range of CO 2 in water with adjusted total hardness and specific conductance. We compared dose-responses and found differences in lethal concentration to 50% of organisms (LC50) estimates ranging from 108.3 to 179.3 mg/L CO 2 and lethal concentration to 90% of organisms (LC90) estimates ranging from 163.7 to 216.6 mg/L CO 2 . We modeled LC50 and LC90 estimates with measured water chemistry variables from the trials. We found sodium (Na + ) concentration to have the strongest correlation to changes in the LC50 and specific conductance to have the strongest correlation to changes in the LC90. Our results identify water chemistry as an important factor in considering efficacious CO 2 concentrations for zebra mussel control. Additional research into the physiological responses of zebra mussels exposed to CO 2 may be warranted to further explain mode of action and reported selectivity. Further study could likely develop a robust and relevant model to refine CO 2 applications for a wider range of water chemistries. Environ Toxicol Chem 2024;43:1312-1319. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Invasive freshwater mussels, such as the zebra ( Dreissena polymorpha ), quagga ( Dreissena rostriformis bugensis ), and golden ( Limnoperna fortunei ) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels., Competing Interests: S.T.S and M.C.S. are co-founders of Biomilab LLC and are members of its scientific advisory board and have management/advisory roles and a financial interest. Biomilab LLC has patents related to this work. Bio Bureau Biotecnologia is a private, for-profit company with commercial interests in invasive mussel biocontrol. J.A. A. is a shareholder of Bio Bureau Biotecnologia and its Scientific Director. M.F.R. is a shareholder, founder, and member of the scientific advisory board of Bio Bureau Biotecnologia. The remaining authors have no competing interests to declare., (© 2023 The Author(s).)

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund ("sick") or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell ( Ortmanniana pectorosa; synonym Actinonaias pectorosa ) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N -methyl-l-alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health., Competing Interests: The authors declare no conflict of interest. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the US Government or the authors. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the US Fish and Wildlife Service., (© The Author(s) 2023. Published by Oxford University Press and the Society for Experimental Biology.)

Dreissenid mussels are one of the most problematic aquatic invasive species (AIS) in North America, causing substantial ecological and economic effects. To date, dreissenid mussel control efforts in open water have included physical, biological, and chemical methods. The feasibility of successful dreissenid mussel management or eradication in lakes is relatively undocumented in the freshwater management literature. This review presents information on 33 open water dreissenid mussel control projects in 23 North America lakes. We reviewed data from past dreissenid mussel control projects and identified patterns and knowledge gaps to help inform adaptive management strategies. The three key lessons learned include (1) pre- and post-treatment survey methods that are designed to meet management objectives are beneficial, e.g., by sampling for all life stages and taking into account that no survey method is completely comprehensive; (2) defining the treatment area-particularly ensuring it is sufficiently large to capture all life stages present-is critical to meeting management objectives; and (3) control projects provide an opportunity to collect water chemistry, effects on non-target organisms, and other efficacy-related data that can inform safe and effective adaptive management., (© 2023. The Author(s).)

Freshwater mussels (Unionida) are among the world's most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina ; syn: Ortmanniana ligamentina ) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019-2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell ( Actinonaias pectorosa ; syn: Ortmanniana pectorosa ) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.

Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells ( Actinonaias pectorosa ) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

Researchers most often focus on individual toxicants when identifying effective chemical control agents for aquatic invasive species; however, toxicant mixtures may elicit synergistic effects. Synergistic effects may decrease required concentrations and shorten exposure durations for treatments. We investigated four toxicants (EarthTec QZ, Clam-Trol CT-2, niclosamide, and potassium chloride) that have been considered to control invasive zebra mussels (Dreissena polymorpha Pallas, 1771). We determined the toxicity of binary mixtures for five different mixture ratios to adult mussels. We compared our observations to predictions made with concentration addition and independent action paradigms, as based on the dose-response relationships of each individual toxicant. We calculated the model deviation ratio for each combination at the LC 50 and LC 90 and identified three possible interactions: synergy, antagonism, and additivity. We found that mixtures of niclosamide and Clam-Trol CT-2 were the most synergistic while mixtures that included potassium chloride were largely additive to antagonistic. The use of synergistic combinations has potential to decrease the overall volume and concentration of individual toxicants required for dreissenid mussel treatments, thereby decreasing cost., (Copyright © 2021. Published by Elsevier B.V.)

Carbon dioxide (CO 2 ) has been approved by the US Environmental Protection Agency as a new aquatic pesticide to control invasive Asian carps and other aquatic nuisance species in the United States. However, limited CO 2 toxicity data could make it challenging for resource managers to characterize the potential risk to nontarget species during CO 2 applications. The present study quantified the toxicity of CO 2 to 2 native riverine fishes, bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas), using 12-h continuous flow-through CO 2 exposure at 5, 15, and 25 °C water temperatures. Resulting survival indicated that bluegill (median lethal concentration [LC50] range 91-140 mg/L CO 2 ) were more sensitive to CO 2 than fathead minnow (LC50 range 235-306 mg/L CO 2 ) across all water temperatures. Bluegill were also more sensitive to CO 2 at 5 °C (LC50 91 mg/L CO 2 , 95% CI 85-96 mg/L CO 2 ) than at 25 °C (LC50 140 mg/L CO 2 , 95% CI 135-146 mg/L CO 2 ). Fathead minnow showed an opposite response and were less sensitive at 5 °C (LC50 306 mg/L CO 2 , 95% CI 286-327 mg/L CO 2 ) relative to 25 °C (LC50 235 mg/L CO 2 , 95% CI 224-246 mg/L CO 2 ). Our results show that CO 2 toxicity can differ by species and water temperature. Data from the present study may inform decisions related to the use of CO 2 as a control tool. Environ Toxicol Chem 2020;39:2247-2255. Published 2020. This article is a U.S. government work and is in the public domain in the USA., (Published 2020. This article is a U.S. government work and is in the public domain in the USA.)

Zebra mussels (Dreissena polymorpha) have exacerbated the decline of native freshwater mussels (order Unionida) in North America since their arrival in the 1980s. Options for controlling invasive mussels, particularly in unionid mussel habitats, are limited. Previously, carbon dioxide (CO 2 ) showed selective toxicity for zebra mussels, relative to unionids, when applied in cool water (12 °C). We first determined 96-h lethal concentrations of CO 2 at 5 and 20 °C to zebra mussels and responses of juvenile plain pocketbook (Lampsilis cardium). Next, we compared the time to lethality for zebra mussels at 5, 12, and 20 °C during exposure to partial pressure of CO 2 (PCO 2 ) values of 110 to 120 atm (1 atm = 101.325 kPa) and responses of juvenile plain pocketbook and fragile papershell (Leptodea fragilis). We found efficacious CO 2 treatment regimens at each temperature that were minimally lethal to unionids. At 5 °C, plain pocketbook survived 96-h exposure to the highest PCO 2 treatment (139 atm). At 20 °C, the 96-h lethal concentration to 10% of animals (LC10) for plain pocketbook (173 atm PCO 2 , 95% CI 147-198 atm) was higher than the LC99 for zebra mussels (118 atm PCO 2 , 95% CI 109-127 atm). Lethal time to 99% mortality (LT99) of zebra mussels in 110 to 120 atm PCO 2 ranged from 100 h at 20 °C to 300 h at 5 °C. Mean survival of both plain pocketbook and fragile papershell juveniles exceeded 85% in LT99 CO 2 treatments at all temperatures. Short-term infusion of 100 to 200 atm PCO 2 at a range of water temperatures could reduce biofouling by zebra mussels with limited adverse effects on unionid mussels. Environ Toxicol Chem 2020;39:1546-1557. Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

A general and efficient catalytic approach to synthesis of the furo[2,3-c]pyridazine ring system is reported. Building on the easily accessible 2-bromo-3-aminopyridizinone skeleton, a tandem Sonogashira coupling-cycloisomerization provides ready access to functionalized furopyridazines. A wide functional group tolerance was observed in the tandem reaction, which proceeds in high yield in 1-3 h. The structure of the heterocyclic ring system was confirmed through single-crystal X-ray crystallography.

Carbon dioxide (CO 2 ) has shown promise as a tool to control movements of invasive Asian carp, but its effects on native freshwater biota have not been well studied. The authors evaluated lethal and sublethal responses of juvenile fatmucket (Lampsilis siliquoidea) mussels to CO 2 at levels (43-269 mg/L, mean concentration) that bracket concentrations effective for deterring carp movement. The 28-d lethal concentration to 50% of the mussels was 87.0 mg/L (95% confidence interval [CI] 78.4-95.9) and at 16-d postexposure, 76.0 mg/L (95% CI 62.9-90.3). A proportional hazards regression model predicted that juveniles could not survive CO 2 concentrations >160 mg/L for more than 2 wk or >100 mg/L CO 2 for more than 30 d. Mean shell growth was significantly lower for mussels that survived CO 2 treatments. Growth during the postexposure period did not differ among treatments, indicating recovery of the mussels. Also, CO 2 caused shell pitting and erosion. Behavioral effects of CO 2 included movement of mussels to the substrate surface and narcotization at the highest concentrations. Mussels in the 110 mg/L mean CO 2 treatment had the most movements in the first 3 d of exposure. If CO 2 is infused continuously as a fish deterrent, concentrations <76 mg/L are recommended to prevent juvenile mussel mortality and shell damage. Mussels may survive and recover from brief exposure to higher concentrations. Environ Toxicol Chem 2017;36:671-681. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Natural products have been widely used to dissect the basic mechanisms of fundamental life science and as clinical therapeutics. Recently, there has been significant interest in discovering new chemical pharmacophores in natural products to fulfil the vast demand for novel kinase inhibitors and address critical unmet medical needs with respect to signal transduction pathways. In this review, we summarize the history of several different classes of natural product-derived kinase inhibitors, discuss their kinome-wide target profiles and examine their structural binding modes based on available 3D X-ray structures. In particular, their origin, target activity, selectivity, scope and potential therapeutic development are highlighted against the backdrop of medicinal chemistry.

Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Addition of in situ generated di- or trisubstituted alkenylalanes to N-acyliminium ions provides rapid access to functionalized isoindolinones. Subsequent ring closing metathesis leads to tricyclic products. These transformations proceed under mild conditions and allow for the convergent synthesis of biologically significant scaffolds from readily available starting materials.

Oxidative rearrangement of cyclic enol ethers leads to alpha-alkoxyesters. In the presence of a neighboring spiroether, this approach provides a stereoselective access to spiroketals. A modified proposal for the biosynthesis of acutumine is presented.

[Chemical reaction: See text] A three-step cascade reaction involving a water-accelerated catalytic carboalumination, a Claisen rearrangement, and a nucleophilic carbonyl addition converts terminal alkynes and allyl vinyl ethers into allylic alcohols containing up to three contiguous asymmetric carbon centers. Stoichiometric quantities of water as an additive increase the rate of the [3,3] sigmatropic rearrangement as well as the diastereoselectivity of the carbonyl addition process. Reaction products contain 1,6-diene functionalities that are readily cyclized to substituted cyclopentenes. An extension of this methodology to a sequence involving a [1,3] sigmatropic shift was feasible with a cyclopropylmethyl vinyl ether substrate.