Climate causes shifts in grey seal phenology by modifying age structure.

There are numerous examples of phenological shifts that are recognized both as indicators of climate change and drivers of ecosystem change. A pressing challenge is to understand the causal mechanisms by which climate affects phenology. We combined annual population census data and individual longitudinal data (1992-2018) on grey seals, Halicheorus grypus, to quantify the relationship between pupping season phenology and sea surface temperature. A temperature increase of 2°C was associated with a pupping season advance of approximately seven days at the population level. However, we found that maternal age, rather than sea temperature, accounted for changes in pupping date by individuals. Warmer years were associated with an older average age of mothers, allowing us to explain phenological observations in terms of a changing population age structure. Finally, we developed a matrix population model to test whether our observations were consistent with changes to the stable age distribution. This could not fully account for observed phenological shift, strongly suggesting transient modification of population age structure, for example owing to immigration. We demonstrate a novel mechanism for phenological shifts under climate change in long-lived, age- or stage-structured species with broad implications for dynamics and resilience, as well as population management.

Maze learning and memory in a decapod crustacean.

Spatial learning is an ecologically important trait well studied in vertebrates and a few invertebrates yet poorly understood in crustaceans. We investigated the ability of European shore crabs, Carcinus maenas, to learn a complex maze over four consecutive weeks using food as a motivator. Crabs showed steady improvement during this conditioning period in both the time taken to find the food and in the number of wrong turns taken. Crabs also clearly remembered the maze as when returned two weeks later but without any food, they all returned to the end of the maze in under 8 min. Crabs that had not been conditioned to the maze (naive animals) took far longer to reach the end, and many (42%) did not venture to the end of the maze at all during the 1 h study period. This study provides an initial description of spatial learning in a benthic decapod; a better appreciation of this adaptive trait in these animals will develop our understanding of resource exploitation by benthic crustaceans and their ecological roles.

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality.

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.

Combined use of the entomopathogenic fungus, Metarhizium brunneum, and the mosquito predator, Toxorhynchites brevipalpis, for control of mosquito larvae: Is this a risky biocontrol strategy?

Mosquitoes transmit several diseases, which are of global significance (malaria, dengue, yellow fever, Zika). The geographic range of mosquitoes is increasing due to climate change, tourism and trade. Both conidial and blastospore formulations of the entomopathogenic fungus, Metarhizium brunneum ARSEF 4556, are being investigated as mosquito larvicides. However, concerns have been raised over possible non-target impacts to arthropod mosquito predators such as larvae of Toxorhynchites brevipalpis which feed on larvae of mosquito vector species. Laboratory-based, small container bioassays showed, that T. bevipalpis larvae are susceptible to relatively high concentrations (i.e. ≥107 spores ml-1) of inoculum with blastospores being significantly more virulent than conidia. At lower concentrations (e.g. <107 spores ml-1), it appears that M. brunneum complements T. brevipalpis resulting in higher control than if either agent was used alone. At a concentration of 105 spores ml-1, the LT50 of for conidia and blastospores alone was 5.64 days (95% CI: 4.79-6.49 days) and 3.89 days (95% CI: 3.53-4.25 days), respectively. In combination with T. brevipalpis, this was reduced to 3.15 days (95% CI: 2.82-3.48 days) and 2.82 days (95% CI: 2.55-3.08 days). Here, combined treatment with the fungus and predator was beneficial but weaker than additive. At 107 and 108 blastospores ml-1, mosquito larval mortality was mostly due to the fungal pathogen when the predator was combined with blastospores. However, with conidia, the effects of combined treatment were additive/synergistic at these high concentrations. Optimisation of fungal concentration and formulation will reduce: (1) risk to the predator and (2) application rates and costs of M. brunneum for control of mosquito larvae.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens.

BACKGROUND: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. RESULTS: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. CONCLUSIONS: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

Zebrafish Rab5 proteins and a role for Rab5ab in nodal signalling.

The RAB5 gene family is the best characterised of all human RAB families and is essential for in vitro homotypic fusion of early endosomes. In recent years, the disruption or activation of Rab5 family proteins has been used as a tool to understand growth factor signal transduction in whole animal systems such as Drosophila melanogaster and zebrafish. In this study we have examined the functions for four rab5 genes in zebrafish. Disruption of rab5ab expression by antisense morpholino oligonucleotide (MO) knockdown abolishes nodal signalling in early zebrafish embryos, whereas overexpression of rab5ab mRNA leads to ectopic expression of markers that are normally downstream of nodal signalling. By contrast MO disruption of other zebrafish rab5 genes shows little or no effect on expression of markers of dorsal organiser development. We conclude that rab5ab is essential for nodal signalling and organizer specification in the developing zebrafish embryo.

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish. RESULTS: flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development. CONCLUSIONS: In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic brain development.

A virulent strain of deformed wing virus (DWV) of honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission.

The globally distributed ectoparasite Varroa destructor is a vector for viral pathogens of the Western honeybee (Apis mellifera), in particular the Iflavirus Deformed Wing Virus (DWV). In the absence of Varroa low levels DWV occur, generally causing asymptomatic infections. Conversely, Varroa-infested colonies show markedly elevated virus levels, increased overwintering colony losses, with impairment of pupal development and symptomatic workers. To determine whether changes in the virus population were due Varroa amplifying and introducing virulent virus strains and/or suppressing the host immune responses, we exposed Varroa-naïve larvae to oral and Varroa-transmitted DWV. We monitored virus levels and diversity in developing pupae and associated Varroa, the resulting RNAi response and transcriptome changes in the host. Exposed pupae were stratified by Varroa association (presence/absence) and virus levels (low/high) into three groups. Varroa-free pupae all exhibited low levels of a highly diverse DWV population, with those exposed per os (group NV) exhibiting changes in the population composition. Varroa-associated pupae exhibited either low levels of a diverse DWV population (group VL) or high levels of a near-clonal virulent variant of DWV (group VH). These groups and unexposed controls (C) could be also discriminated by principal component analysis of the transcriptome changes observed, which included several genes involved in development and the immune response. All Varroa tested contained a diverse replicating DWV population implying the virulent variant present in group VH, and predominating in RNA-seq analysis of temporally and geographically separate Varroa-infested colonies, was selected upon transmission from Varroa, a conclusion supported by direct injection of pupae in vitro with mixed virus populations. Identification of a virulent variant of DWV, the role of Varroa in its transmission and the resulting host transcriptome changes furthers our understanding of this important viral pathogen of honeybees.

Disease profiles of juvenile edible crabs (Cancer pagurus L.) differ at two geographically-close intertidal sites.

The prevalence of disease in edible crabs (Cancer pagurus) was assessed at two sites in South West Wales; one estuarine (Pembroke Ferry) and another facing open water (Freshwater East). Diseases included pink crab disease caused by Hematodinium sp., an infection of the antennal gland caused by Paramikrocytos canceri and an idiopathic inflammatory condition of the connective tissue surrounding the anterior ganglionic masses. This latter condition was only found in crabs from Pembroke Ferry. There was a significantly higher prevalence of pink crab disease at Freshwater East than Pembroke Ferry, although both sites had similar levels of infection by P. canceri.

A strong immune response in young adult honeybees masks their increased susceptibility to infection compared to older bees.

Honeybees, Apis mellifera, show age-related division of labor in which young adults perform maintenance ("housekeeping") tasks inside the colony before switching to outside foraging at approximately 23 days old. Disease resistance is an important feature of honeybee biology, but little is known about the interaction of pathogens and age-related division of labor. We tested a hypothesis that older forager bees and younger "house" bees differ in susceptibility to infection. We coupled an infection bioassay with a functional analysis of gene expression in individual bees using a whole genome microarray. Forager bees treated with the entomopathogenic fungus Metarhizium anisopliae s.l. survived for significantly longer than house bees. This was concomitant with substantial differences in gene expression including genes associated with immune function. In house bees, infection was associated with differential expression of 35 candidate immune genes contrasted with differential expression of only two candidate immune genes in forager bees. For control bees (i.e. not treated with M. anisopliae) the development from the house to the forager stage was associated with differential expression of 49 candidate immune genes, including up-regulation of the antimicrobial peptide gene abaecin, plus major components of the Toll pathway, serine proteases, and serpins. We infer that reduced pathogen susceptibility in forager bees was associated with age-related activation of specific immune system pathways. Our findings contrast with the view that the immunocompetence in social insects declines with the onset of foraging as a result of a trade-off in the allocation of resources for foraging. The up-regulation of immune-related genes in young adult bees in response to M. anisopliae infection was an indicator of disease susceptibility; this also challenges previous research in social insects, in which an elevated immune status has been used as a marker of increased disease resistance and fitness without considering the effects of age-related development.

Development and laboratory validation of a plant-derived repellent blend, effective against Aedes aegypti [Diptera: Culicidae], Anopheles gambiae [Diptera: Culicidae] and Culex quinquefasciatus [Diptera: Culicidae].

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market.

Evaluation of Metarhizium brunneum- and Metarhizium-Derived VOCs as Dual-Active Biostimulants and Pest Repellents in a Wireworm-Infested Potato Field.

Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities.

Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives.

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.

Grand challenges in entomology: Priorities for action in the coming decades.

Entomology is key to understanding terrestrial and freshwater ecosystems at a time of unprecedented anthropogenic environmental change and offers substantial untapped potential to benefit humanity in a variety of ways, from improving agricultural practices to managing vector-borne diseases and inspiring technological advances.We identified high priority challenges for entomology using an inclusive, open, and democratic four-stage prioritisation approach, conducted among the membership and affiliates (hereafter 'members') of the UK-based Royal Entomological Society (RES).A list of 710 challenges was gathered from 189 RES members. Thematic analysis was used to group suggestions, followed by an online vote to determine initial priorities, which were subsequently ranked during an online workshop involving 37 participants.The outcome was a set of 61 priority challenges within four groupings of related themes: (i) 'Fundamental Research' (themes: Taxonomy, 'Blue Skies' [defined as research ideas without immediate practical application], Methods and Techniques); (ii) 'Anthropogenic Impacts and Conservation' (themes: Anthropogenic Impacts, Conservation Options); (iii) 'Uses, Ecosystem Services and Disservices' (themes: Ecosystem Benefits, Technology and Resources [use of insects as a resource, or as inspiration], Pests); (iv) 'Collaboration, Engagement and Training' (themes: Knowledge Access, Training and Collaboration, Societal Engagement).Priority challenges encompass research questions, funding objectives, new technologies, and priorities for outreach and engagement. Examples include training taxonomists, establishing a global network of insect monitoring sites, understanding the extent of insect declines, exploring roles of cultivated insects in food supply chains, and connecting professional with amateur entomologists. Responses to different challenges could be led by amateur and professional entomologists, at all career stages.Overall, the challenges provide a diverse array of options to inspire and initiate entomological activities and reveal the potential of entomology to contribute to addressing global challenges related to human health and well-being, and environmental change.

Wasting disease regulates long-term population dynamics in a threatened seagrass.

The role of disease in the long-term dynamics of threatened species is poorly quantified, as well as being under-represented in ecology and conservation management. To understand persistent host-pathogen interaction operating in a vulnerable habitat, we quantified dynamics driving patterns of seagrass density using a longitudinal study in a relatively pristine site (Isles of Scilly, UK). Replicated samples of eelgrass (Zostera marina) density and wasting disease prevalence, presumably caused by Labyrinthula zosterae, were taken from five meadows at the height of the growing season, over the years 1997-2010. Data were used to parameterise a population dynamic model, incorporating density-dependent factors and sea temperature records. We found that direct density and disease-mediated feedback operate within a network of local populations. Furthermore, our results indicate that the strength of limitation to seagrass growth by disease was increased at higher temperatures. This modification of the coupled host-pathogen dynamics forms a novel hypothesis to account for dramatic die-backs of Z. marina widely reported elsewhere. Our findings highlight the importance of disease in structuring distributions of vulnerable species, as well as the application of population modelling in order to reveal ecological processes and prioritize future mechanistic investigation.

Environment predicts seagrass genotype, phenotype, and associated biodiversity in a temperate ecosystem.

Coastal vegetative ecosystems are among the most threatened in the world, facing multiple anthropogenic stressors. A good example of this is seagrass, which supports carbon capture, coastal stabilization, and biodiversity, but is declining globally at an alarming rate. To understand the causes and consequences of changes to these ecosystems, we need to determine the linkages between different biotic and abiotic components. We used data on the seagrass, Zostera marina, collected by citizen scientists across 300 km of the south coast of the United Kingdom as a case study. We assembled data on seagrass genotype, phenotype, infauna, and associated bathymetry, light, sea surface temperature, and wave and current energy to test hypotheses on the distribution and diversity of this temperate sub-tidal ecosystem. We found spatial structure in population genetics, evident through local assortment of genotypes and isolation by distance across a broader geographic scale. By integrating our molecular data with information on seagrass phenotype and infauna, we demonstrate that these ecosystem components are primarily linked indirectly through the effects of shared environmental factors. It is unusual to examine genotypic, phenotypic, and environmental data in a single study, but this approach can inform both conservation and restoration of seagrass, as well as giving new insights into a widespread and important ecosystem.

Volatiles of the entomopathogenic fungus, Metarhizium brunneum, attract and kill plant parasitic nematodes.

Root knot nematodes (RKNs) cause significant crop losses. Although RKNs and entomopathogenic fungi, such as Metarhizium brunneum, are associated with plant roots, very little is known about the interactions between these two organisms. This study showed that conidia and VOCs of Me. brunneum influenced the behaviour of M. hapla. The response was dependent on the fungal strain, VOC, concentration of both VOC and conidia, and time. Tomatoes planted in soil treated with the highest doses of conidia usually had a higher number of nematodes than untreated control plants. This was particularly obvious for Me. brunneum strain ARSEF 4556, 7 and 14-days post-treatment. The VOCs, 1-octen-3-ol and 3-octanone, lured M. hapla to plants when used at low doses and repelled them at high doses. In Petri dish assays. the VOCs 1-octen-3-ol and 3-octanone, caused 100% mortality of M. hapla at the highest dose tested (20 µl). Very few live M. hapla were recovered from soil treated with the VOC 1-octen-3-ol, especially at the highest doses tested.

Unravelling the Spatial and Temporal Plasticity of Eelgrass Meadows.

The phenotypic plasticity of seagrasses enables them to adapt to changes in environmental conditions and withstand or recover from disturbance. This plasticity was demonstrated in the large variation recorded throughout a suite of bioindicators measured within Zostera marina meadows around Wales and SW England, United Kingdom. Short-term spatial data were analysed alongside long-term monitoring data to determine which bioindicators best described the status of eelgrass meadows subjected to a range of environmental and anthropogenic drivers. Shoot density, leaf length, leaf nutrients (C:N ratio, %N, %P) including stable isotope of δ13C and δ15N provided insight into the longer-term status of the meadows studied and a good indication of the causes of long-term decline. Meadows ranged from those in the Isles of Scilly with little evidence of impact to those in Littlewick in Milford Haven, Wales that showed the highest levels of impacts of all sites. Bioindicators at Littlewick showed clear warning signs of nutrient loading reflected in the long-term decline in shoot density, and prevalence of wasting disease. This study highlights the need for continuous consistent monitoring and the benefits of using extra tools in the form of shoot nutrient analysis to determine causes of decline.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.