The poultry red mite, Dermanyssus gallinae, is a haematophagous ectoparasite causing significant economic losses in the commercial egg-laying sector. Blood meal digestion by D. gallinae is required for nutrient acquisition, with acidic lysosomal proteinases such as cathepsin L and cathepsin D playing a critical role in haemoglobin digestion. This study investigated the role of a cathepsin D-like aspartyl proteinase, Dg-CatD-1, in the haemoglobin digestion cascade. Haemoglobin processing was investigated by RNA interference (RNAi)-mediated silencing of Dg-CatD-1 and assessing the impact on haemoglobin digestion. RNAi-mediated knockdown of Dg-CatD-1 was achieved by feeding a target-specific double-stranded RNA (dsRNA) to D. gallinae in a blood meal. The minimum length and concentration of Dg-CatD-1 dsRNA for effective knockdown was determined. In addition, the effect of Dg-CatD-1 knockdown on mite digestive physiology, haemoglobin digestion, and egg-laying by adult female mites was assessed. Feeding Dg-CatD-1 dsRNAs via a blood meal to adult female D. gallinae mites resulted in a substantial knockdown of target gene expression. The minimum length and concentration of dsRNA required for effective Dg-CatD-1 knockdown were 25 base pairs (bp, at 200 ng/μl) (61

The performance of 21 synthetic peptides for serological identification of Borrelia burgdoferi sensu stricto, B. garinii and B. afzelii was assessed in this study using two ELISA protocols based on (1) conventional passive binding onto 96-well Greiner Microloon®600 High-binding microplates and (2) covalent binding onto 96-well surfaced-activated Nunc™ Immobilizer Amino Plates. Sensitivity, specificity and accuracy was initially assessed testing 13 follow-up positive sera from seroconverted patients bitten by ticks that had tested positive for Borrelia burgdorferi sensu stricto (N = 2), B. garinii (N = 2), B. afzelii (N = 9). The optimized protocol was then applied to two cohorts of samples including plasma from Danish patients with Lyme borreliosis (LB) (N = 32) and positive samples from Danish healthy blood donors (HBD) (N = 44). Use of covalent binding resulted in higher OD values and significantly increased the accuracy of the test. Results of the seroprevalence study applied to LB and HBD samples showed different distribution of the three Bbsl. Borrelia burgdorferi sensu stricto (Bbss), B. garinii and B. afzelii were identified in 3 out 24 (12.5%), 13 out of 24 (54.2%) and 7 out of 24 (29.2%) LB samples, respectively. B. garinii was confirmed to be to the most prevalent species also in the second cohort of HBD samples as it was identified in 20 out 32 samples (62.5%) followed by Borrelia burgdorferi sensu stricto and B. afzelii and which were identified in 5 out of 32 (15.6%) and 4 out of 32 (12.5%) samples, respectively.

Quantifying the strengths of interactions in complex vector-borne disease ecological systems is challenging. Yet, overcoming this challenge is fundamental for understanding the ecological mechanisms shaping disease hazard. Here we quantified the strengths of the hypothesised direct and indirect mechanistic pathways through which deer affect ticks and one of the Lyme borreliosis pathogens, Borrelia afzelii, by conducting a combined analysis of three previously published datasets from 39 sites across Scotland. Structural equation modelling revealed that, as predicted, deer had a strong positive overall effect (direct and indirect pathways combined) on questing Ixodes ricinus nymph density and a weak, non-significant, negative overall effect on B. afzelii prevalence. This resulted in an overall weak, non-significant, positive effect of deer on B. afzelii hazard (the density of infected nymphs), indicating that their negative effect on B. afzelii prevalence was weaker than their positive effect on questing nymph density. A key novelty of this study was being able to tease apart the direct and indirect pathways for each of these overall effects and demonstrating that they were primarily driven by direct mechanisms, such as deer driving nymph density. Although deer negatively affected rodent abundance, the hypothesised indirect pathways from deer to ticks and pathogen, acting through vegetation and/or rodents, were weak. This could result from low densities of rodents relative to deer in Scotland, consistent with Scotland having among the lowest nymphal Lyme borreliosis pathogen prevalences in Europe. Applying the methodological framework used in this study would be useful for teasing apart complex interactions in other vector-borne disease systems.

A novel panel of peptide for serological identification of Borrelia burgdoferi sensu stricto, Borrelia garinii and Borrelia afzelii was developed and assessed in this study. The diagnostic algorithm of the novel test was initially trained testing 10 US human sera including 3 early-stage and 3 late-stage Lyme disease positive sera, 2 sera positive for Babesia and 2 sera positive for Syphilis, all purchased from a private biorepository. Findings were then corroborated testing (a) 33 additional EU follow-up positive sera from seroconverted patients bitten by ticks that tested positive for B. burgdorferi sensu stricto (No 2), Borrelia garinii (No 14), Borrelia afzelii (No 15) Borrelia valaisiana (No 2), and (b) 40 negative sera from US healthy donors. Results of preliminary US sera testing showed successful detection of IgM and IgG antibodies and correct identification of Borrelia burgdorferi sensu stricto in all the samples tested. Analysis of EU follow-up sera showed much higher sensitivity and accuracy when IgM and IgG were tested combined together rather than separately. Sensitivity and accuracy in species identification of the anti-IgM + IgG multiplex peptide ELISA was 93.5 % and 96.5 % respectively; lower test performance was observed when IgM (i.e. sensitivity = 58.1 %; correct identification = 88.8 %) and IgG testing (i.e. sensitivity = 74.1 %; correct identification = 96.5 %) were carried out separately. Overall specificity of the anti-IgM, anti-IgG and anti-IgM + IgG multiplex peptide ELISA calculated on a total number of 46 negative sera included in this study was 91.3 %, 95.6 and 93.4 %, respectively.

Background The poultry red mite, Dermanyssus gallinae , is a haematophagous ectoparasite causing significant economic losses in the commercial egg laying sector. Blood meal digestion by D. gallinae is required for nutrient acquisition, with acidic lysosomal proteinases such as cathepsin L and cathepsin D playing a critical role in haemoglobin digestion. This study investigated the role of a cathepsin D-like aspartyl proteinase, Dg-CatD-1, in initiating the haemoglobin digestion cascade. Methods Haemoglobin processing was investigated by RNA interference (RNAi) mediated silencing of Dg-CatD-1 and assessing the impact on haemoglobin digestion. RNAi-mediated knockdown of Dg-CatD-1 was achieved by feeding a target-specific dsRNA to D. gallinae in a blood-meal. The minimum length and concentration of Dg-CatD-1 dsRNA for effective knockdown was determined. In addition, the effect of Dg-CatD-1 knockdown on mite digestive physiology, haemoglobin digestion, and egg laying by adult female mites was assessed. Results Feeding Dg-CatD-1 dsRNAs via a blood meal to adult female D. gallinae mites resulted in a substantial knockdown of target gene expression. The minimum length and concentration of dsRNA required for effective Dg-CatD-1 knockdown were 25 bp (at a 200 ng/µl) (61% knockdown) and 25 ng/µl (at 500 bp) (42% knockdown), respectively. When Dg-CatD-1 dsRNA was delivered as a single feed it resulted in up to 91% reduction in Dg-CatD-1 expression, although no observable effect on blood digestion was observed. The phenotypic impact of Dg-CatD-1 knockdown was demonstrated following two consecutive rounds of Dg-CatD-1 dsRNA feeding (“double-dsRNA-fed”) where knockdown reduced the ability of mites to process and clear their blood meal relative to control non-specific dsRNA fed mites. Conclusions This work highlights the importance of Dg-CatD-1 as an essential enzyme in the initiation of the haemoglobin digestion pathway of D. gallinae . These findings open avenues for the development of targeted control strategies aimed at disrupting the digestive processes of D. gallinae . Furthermore, this research suggests that reductions in gene expression via RNAi do not always lead to corresponding decreases in protein levels or observable phenotypes. Repeated exposure to dsRNA may be necessary to reveal phenotypic effects of gene knockdown.

Honey bees (Apis mellifera) provide important ecosystem services to both natural and human-managed environments, but are increasingly threatened by a variety of pathogens, the most common of which is deformed wing virus (DWV). DWV is known to replicate in the honey bee brain and has been documented as both improving and impairing olfactory learning and memory. We examined the transcriptomic response of the honey bee mushroom bodies - an area of the insect brain associated with higher cognitive functions - in bees with naturally occurring DWV infections, which varied in their ability to perform an associative learning task. RNA-sequencing analysis detected increased expression of genes involved in the immune response, including important antimicrobial peptides such as hymenoptaecin, apidaecin, and abaecin, and the downregulation of lysozyme, prophenoloxidase, and other genes associated with responses to a range of stressors. Additionally, gene ontology enrichment analysis revealed overrepresentation of key biological processes that form part of the immune response. We also noted significant differential expression of long non-coding RNAs (lncRNAs) presumed to be acting in a regulatory manner, and used these lncRNAs to construct gene regulatory networks. Strikingly, in contrast to previous studies on bees with artificially induced infections that have examined viral loads in the abdomen and non-specific areas of the brain, no correlation between DWV load in the mushroom bodies and cognitive function was noted. This highlights the complexity of host-pathogen interactions in honey bee neural tissues and the benefits of a spatially refined approach to brain transcriptomics in naturally occurring infections.

Honey bees ( Apis mellifera ) provide important ecosystem services to both natural and human-managed environments, but are increasingly threatened by a variety of pathogens, the most common of which is deformed wing virus (DWV). DWV is known to replicate in the honey bee brain and has been documented as both improving and impairing olfactory learning and memory. We examined the transcriptomic response of the honey bee mushroom bodies—an area of the insect brain associated with higher cognitive functions—in bees with naturally occurring DWV infections who varied in their ability to perform an associative learning task. RNA-seq analysis detected increased expression of genes involved in the immune response, including important antimicrobial peptides (AMPs) such as hymenoptaecin, apidaecin , and abaecin , and the downreguation of lysozyme , PPO , and other genes associated with responses to a range of stressors. Additionally, gene ontology (GO) enrichment analysis revealed overrepresentation of key biological processes which form part of the immune response. We also noted significant differential expression of long non-coding RNAs (lncRNAs) presumed to be acting in a regulatory manner, and used these lncRNAs to construct gene regulatory networks (GRNs). Strikingly, in contrast to previous studies on bees with artificially-induced infections that have examined viral loads in the abdomen and non-specific areas of the brain, no correlation between DWV load in the mushroom bodies and cognitive function was noted. This highlights the complexity of host-pathogen interactions in honey bee neural tissues and the benefits of a spatially-refined approach to brain transcriptomics in naturally-occurring infections. ### Competing Interest Statement The authors have declared no competing interest. Internal Funding to Pump-Prime Research and Research Networks CB Dennis British Beekeepers’ Research Trust

ABSTRACT Viral infections can be detrimental to the foraging ability of the western honey bee, Apis mellifera. The deformed wing virus (DWV) is the most common honey bee virus and has been proposed as a possible cause of learning and memory impairment. However, evidence for this phenomenon so far has come from artificially infected bees, while less is known about the implications of natural infections with the virus. Using the proboscis extension reflex (PER), we uncovered no significant association between a simple associative learning task and natural DWV load. However, when assessed through a reversal associative learning assay, bees with higher DWV load performed better in the reversal learning phase. DWV is able to replicate in the honey bee mushroom bodies, where the GABAergic signalling pathway has an antagonistic effect on associative learning but is crucial for reversal learning. Hence, we assessed the pattern of expression of several GABA-related genes in bees with different learning responses. Intriguingly, mushroom body expression of selected genes was positively correlated with DWV load, but only for bees with good reversal learning performance. We hypothesise that DWV might improve olfactory learning performance by enhancing the GABAergic inhibition of responses to unrewarded stimuli, which is consistent with the behavioural patterns that we observed. However, at higher disease burdens, which might be induced by an artificial infection or by a severe, natural Varroa infestation, this DWV-associated increase in GABA signalling could impair associative learning as previously reported by other studies.

Varroa destructor is an ectoparasitic mite associated with significant losses of honeybee colonies globally. The mite vectors a range of pathogenic viruses, the most important of which is the Deformed wing virus (DWV). In the absence of Varroa, DWV exists as a low-level, highly diverse virus population. However, when transmitted by Varroa, certain variants become highly elevated, and may become near-clonal and cause symptomatic infections. Mite transmission between colonies can occur when parasitised workers drift from or rob adjacent hives. These activities can result in elevated mite levels, but the resulting change in the DWV population, the primary determinant of winter colony losses, has not been determined. In reciprocal studies, we investigated the influence of the removal of mites, or their acquisition, on the DWV population. When mites were removed from heavily infested colonies, there was a striking and rapid reduction in virus load. Conversely, siting Varroa-naïve colonies in a mite-infested apiary resulted in the acquisition of mites and concomitant changes in the virus population. We observed both near-clonal and highly divergent virus populations regardless of titre, suggesting changes were stochastic and colony-specific. Our findings have implications for the outcome of strategies in areas with total or patchy implementation of Varroa control plans.

Background: The avian haematophagous ectoparasite, Dermanyssus gallinae or the poultry red mite, causes significant economic losses to the egg laying industry worldwide and also represents a significant welfare threat. Current acaricide-based controls are unsustainable due to the mite's ability to rapidly develop resistance, thus developing a novel sustainable means of control for D. gallinae is a priority. RNA interference (RNAi) mediated gene silencing is a valuable tool for studying gene function in non-model organisms, but is also emerging as a novel tool for parasite control. Methods: Here we use an in silico approach to identify core RNAi pathway genes in the recently sequenced D. gallinae genome. In addition we utilise an in vitro feeding device to deliver dsRNA to D. gallinae targeting the D. gallinae vATPase subunit A ( Dg vATPase A ) gene and monitor gene knockdown using quantitive PCR (qPCR). Results: We identified core components of the small interfering RNA (siRNA) and micro RNA (miRNA) pathways in D. gallinae , which indicate these gene silencing pathways are likely functional. Strikingly, the Piwi-interacting RNA (piRNA) pathway was absent in D. gallinae . In addition, we demonstrate that feeding Dg vATPase A dsRNA to adult female D. gallinae results in silencing of the targeted gene compared to control mites fed non-specific lacZ dsRNA. In D. gallinae , dsRNA mediated gene knockdown is rapid, detectable 24 hours after oral delivery of dsRNA and persisted for at least 120 hours. Conclusions: This study has shown the presence of core RNAi machinery components in the D. gallinae genome. In addition, we have developed a robust RNAi methodology for targeting genes in D. gallinae , which will be of value for studying genes of unknown function and validating potential control targets in D. gallinae .

Varroa destructor is an ectoparasite of honey bees and an active disease vector, which represents one of the most severe threats for the beekeeping industry. This parasitic mite feeds on the host’s body fluids through a wound in the cuticle, which allows food uptake by the mother mite and its progeny, offering a potential route of entrance for infecting microorganisms. Mite feeding is associated with saliva injection, whose role is still largely unknown. Here we try to fill this gap by identifying putative host regulation factors present in the saliva of V. destructor and performing a functional analysis for one of them, a chitinase (Vd-CHIsal) phylogenetically related to chitinases present in parasitic and predatory arthropods, which shows a specific and very high level of expression in the mite’s salivary glands. Vd-CHIsal is essential for effective mite feeding and survival, since it is apparently involved both in maintaining the feeding wound open and in preventing host infection by opportunistic pathogens. Our results show the important role in the modulation of mite-honey bee interactions exerted by a host regulation factor shared by different evolutionary lineages of parasitic arthropods. We predict that the functional characterization of Varroa sialome will provide new background knowledge on parasitism evolution in arthropods and the opportunity to develop new bioinspired strategies for mite control based on the disruption of their complex interactions with a living food source.

This dataset represents raw data and metadata underlying the findings (and the figures) reported in the manuscript "A salivary chitinase of Varroa destructor regulates mite-honey bee interactions". Fig 2A. Relative expression data of 3 selected candidates are presented as mean fold changes of 3-4 independent biological replicates. Each replicate consisted of a pool of 5-10 mites, from which the RNA was extracted from, salivary glands (SG), and from the rest of the whole body, deprived of salivary glands (Whole Body – SG). Values on Y-axis are reported in Log10 scale. Error bars represent standard error of the mean. Statistically significant differences are denoted with an asterisk (P < 0.005). Fig 4. Survival of V. destructor as affected by RNAi-mediated silencing of the gene encoding VdCHIsal. (A) Relative expression of Vd-CHIsal after soaking in a dsRNA solution. qRT-PCR data are presented as mean fold changes of 2-6 independent biological replicates. Each replicate consisted in a pool of 2-3 mites. Each time point was separately analyzed and the control sample was used as calibrator. Mean values within each time point were compared by Student’s t-test. Error bars represent the standard error of the mean. Mean values denoted with asterisks are significantly different (*p<0.05; ** p<0.01). (B) Kaplan-Meier survival curves of mites soaked in a solution of dsRNA targeting Vd-CHIsal. Host pupae were maintained throughout the feeding or replaced every 24 h. Error bars represent the standard error of the mean. Fig 5. Differentially expressed genes in honey bee pupae artificially infested with mites delivering saliva with the full repertoire of proteins or lacking Vd-CHIsal. (A) Differential expression of 13 honey bee genes, as affected by presence of Vd-CHIsal in the saliva (KD/WS). DESeq2 adjusted P was < 0.05 and FDR was set at 5%. Log transformed mean FPKM values are reported on Y axis. Error bars represent SEM. Summary tables of differential expression analysis are presented in S2 and S3 Tables. (B) Relative expression of immune-related genes in honey bee pupae as affected by Vd-CHIsal expression in Varroa destructor infesting mites. Results of qRT-PCR are presented as mean fold changes relative to non-infested pupae used as calibrator. Values on Y axis are reported in Log10 scale. Error bars represent the standard error of the mean. Mean values were compared by one-way ANOVA, followed by Tukey post-hoc test, and values statistically different denoted with different letters (P<0.05). Detailed results of statistical analyses are presented in S4 Table. NP: non-parasitized controls; WS: pupae infested with mites soaked in saline solution; KD: pupae infested with mites soaked in Vd-CHIsal dsRNA solution.

Varroa destructor is an ectoparasitic mite associated with significant losses of honey bee colonies globally. The mite vectors a range of pathogenic viruses, most important of which is Deformed Wing Virus (DWV, (+)ssRNA). Overwintering colony losses, accounting for the death of ∼25% of all colonies each year, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continued unchecked. We aim to determine whether rational, coordinated treatment is beneficial, using features of the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland). It is reported that a high level of a near-clonal virus population is associated with Varroa infestation and colony losses, whereas Varroa-free healthy colonies carry only low levels of a diverse population of DWV. The study area contains 50-85 colonies and 25 beekeepers. Sampling and virus analysis – strain diversity and viral loads – have been conducted before and after treatment. Changes in virus diversity are quantified by next generation sequencing analysis to determine population diversity. In the first two years we have observed a geographic decrease in Varroa and changes in the composition of the virus population. This study will inform our development of rational Varroa control strategies for beekeepers in temperate regions and could be used to inform policy changes regarding treatment regimes in Scotland and elsewhere for this global pathogen.

This dataset represents raw data underlying the findings (and the figures) reported in the manuscript "A salivary chitinase of Varroa destructor influences host immunity and mite’s survival". Fig 2A. Salivary gland expression of putative host regulation factors present found in the predicted secretome of Varroa destructor. (A) Relative expression data of 3 selected candidates are presented as mean fold changes of 3-4 independent biological replicates. Each replicate consisted of a pool of 5-10 mites and comprised , from which the RNA was extracted from,two samples: salivary glands (SG), and from the rest of the whole body, deprived of salivary glands (Whole Body – SG). Values on Y-axis are reported in Log10 scale. Error bars represent standard error of the meandeviation (SD). Statistically significant differences are denoted with an asterisk (P < 0.005). Fig 4. Survival of Varroa. destructor as affected by RNAi-mediated silencing of the gene encoding VdCHIsal. (A) Relative expression of Vd-CHIsal after mite soaking in a dsRNA solution. qRT-PCR data are presented as mean fold changes of 2-67 independent biological replicates. Each replicate consisted in a pool of 2-3 mites. Each time point was separately analyzed and the 0.9% NaCl 0.9% control sample was used as calibrator. Mean dCt values within each time point were compared by Student’s t-testone-way ANOVA followed by Tukey’s post-hoc test. Mean values denoted with different letters are significantly different. Error bars represent standard deviation (the standard error of the meanSD). Mean values denoted with asterisks are significantly different (*p<0.05; ** p<0.01). (B) Kaplan-Meier survival curves of mites soaked in a solution of dsRNA targeting Vd-CHIsal. Saline controls (0.9% NaCl), GFP dsRNA and Vd-CHIsal dsRNA soaked mites (blue rhombus, green circles and orange squares, respectively) were individually maintained on the same host pupa throughout the whole duration of the assayP. Subjects at risk were 18, 30 and 45 for 0.9% NaCl, GFP dsRNA and Vd-CHIsal dsRNA treatments, respectively. Survival curve of dsVd-CHIsal was significantly different from dsGFP (log rank test: X2= 6.086; P=0.0136) and from NaCl 0.9 (log rank test: X2=6.611; P=0.0101), while no difference was observed between dsGFP and NaCl 0.9% (log rank test: X2= 0.46; P=0.49). Statistical significance was set at 0.016 (Bonferroni correction). aIn a separateconcurrent set of trials, host pupae were replaced every 24 h for both saline controls (0.9% NaCl) and Vd-CHIsal dsRNA soaked mites (violet down-pointing and red up-pointing triangles, respectively). Subjects at risk were 29 and 22 for 0.9% NaCl and Vd-CHIsal dsRNA, respectively. Host pupae were maintained throughout the feeding or replaced every 24 h. Survival curve of dsVd-CHIsal was significantly different from (log rank test: X2= 18.21; P<0.0001), Statistical significance was set at 0.05.Statistical details are in the text. Error bars represent the standard error of the mean. Fig 5. Differentially expressed genes in honey bee pupae artificially infested with mites delivering saliva with the full repertoire of proteins or lacking Vd-CHIsal. (A) Differential expression of 13 honey bee genes, as affected by presence of Vd-CHIsal in the saliva (KD/WS). DESeq2 adjusted P was < 0.05 and FDR was set at 5%. Log transformed mean FPKM values are reported on Y axis. For each Each group representsexperimental condition 3 individually analyzedseparate pupae were analyzed. Error bars represent SEMSD. Summary tables data sheets of differential expression analysis are presented in S2 and S3 Tables. (B) Relative expression of immune-related genes in honey bee pupae as affected by Vd-CHIsal expression in Varroa destructor infesting mites. Each groupmean value representsis obtained on 7-10 individually analyzed pupae, individually analyzed. Results of qRT-PCR are presented as mean fold changes relative to non-infested pupae used as calibrator. Values on Y axis are reported in Log10 scale. Error bars represent standard deviation (the standard error of the meanSD). Mean values were compared by one-way ANOVA, followed by Tukey post-hoc test, and values statistically different are denoted with different letters (P<0.05). Detailed resultsDetails of statistical analyses are presented in S4 Table. NP: non-parasitized controls; WS: pupae infested with mites soaked in saline solution; KD: pupae infested with mites soaked in a solution of dsRNA Vd-CHIsal dsRNA dsRNA solution. S1 File. Table resulting from Trinotate in-house annotation of the putatively secreted components of Varroa destructor predicted proteome. The annotation was performed following the protocol described in Materials and Methods section. Content of columns is hereafter described. A: GenBank accession number of the original transcript of V. destructor; B: GenBank accession number of the matching protein in V. destructor; C: ID name assigned by Transdecoder software; D: coordinates of the translation; E: top BLASTp hits in Swiss-Prot db; F: top BLASTp hits in the database of Hymenoptera venom; G: top BLASTp hits in the database of Acarina saliva; H: Pfam hits; I,: SignalP results indicating the presence of the signal peptide; J: TMHMM results indicating the presence of transmembrane domains; K: eggnog db hits; L: Kegg db hits; M: gene ontology retrieved from BLASTp; N: gene ontology retrieved from Pfam.

Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.

OBJECTIVES:Current serological methods cannot distinguish active from past infection with Borrelia burgdorferi sensu lato. The aim of this study was to develop an IgG avidity Western blot and assess its potential to differentiate patients with early and late Lyme borreliosis (LB) i.e. active disease, from those infected in the past.METHODS:An IgG avidity Western blot was developed. Penalized linear discriminant analysis (PLDA) was employed to compare the Western blot/avidity Western blot profiles of an evaluation panel consisting of 75 sera from patients with early (n = 26) and late (n = 24) LB and past infection (n = 25). The PLDA models produced were used to predict infection stage for 20 well characterised sera from the Centers for Disease Control and Prevention (CDC) Lyme disease serum repository and 112 routine seropositive sera (disease stage unknown), to validate and assess the usefulness of the avidity Western blot/avidity Western blot and PLDA approach.RESULTS:PLDA correctly classified 40/51 (78%) of patients when early LB and past infection groups in the evaluation panel were compared. Likewise, when late LB and past infection groups were compared, 34/49 (69%) were correct. The resultant PLDA models correctly predicted infection stage for 18/20 (90%) of the CDC sera, validating the use of the avidity Western blot/avidity Western blot and PLDA approach. When tested with the routine sera, 21/29 (72%) tested with the early LB vs. past infection model were correct but only 32/83 (39%) with the late LB vs. past infection model. Past infection was predicted for 40/112 (35%) of the routine sera, 80% of which correlated with the clinical picture.CONCLUSION:The Western blot/avidity Western blot with PLDA approach shows exciting potential for being able to predict disease stage in some patients with LB, which could improve patient management.

Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.

European honey bees (Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.