Identification and CRISPR-Cas9 validation of a novel ß-adrenergic-like octopamine receptor mutation associated with amitraz resistance in Varroa destructor.

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the ß-adrenergic-like octopamine receptor (Octß2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octß2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octß2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.

Comparison of Brain Gene Expression Profiles Associated with Auto-Grooming Behavior between Apis cerana and Apis mellifera Infested by Varroa destructor.

The grooming behavior of honeybees serves as a crucial auto-protective mechanism against Varroa mite infestations. Compared to Apis mellifera, Apis cerana demonstrates more effective grooming behavior in removing Varroa mites from the bodies of infested bees. However, the underlying mechanisms regulating grooming behavior remain elusive. In this study, we evaluated the efficacy of the auto-grooming behavior between A. cerana and A. mellifera and employed RNA-sequencing technology to identify differentially expressed genes (DEGs) in bee brains with varying degrees of grooming behavior intensity. We observed that A. cerana exhibited a higher frequency of mite removal between day 5 and day 15 compared to A. mellifera, with day-9 bees showing the highest frequency of mite removal in A. cerana. RNA-sequencing results revealed the differential expression of the HTR2A and SLC17A8 genes in A. cerana and the CCKAR and TpnC47D genes in A. mellifera. Subsequent homology analysis identified the HTR2A gene and SLC17A8 gene of A. cerana as homologous to the HTR2A gene and SLC17A7 gene of A. mellifera. These DEGs are annotated in the neuroactive ligand-receptor interaction pathway, the glutamatergic synaptic pathway, and the calcium signaling pathway. Moreover, CCKAR, TpnC47D, HTR2A, and SLC17A7 may be closely related to the auto-grooming behavior of A. mellifera, conferring resistance against Varroa infestation. Our results further explain the relationship between honeybee grooming behavior and brain function at the molecular level and provide a reference basis for further studies of the mechanism of honeybee grooming behavior.

Molecular phylogeny reveals Varroa mites are not a separate family but a subfamily of Laelapidae.

Varroa mites, notorious for parasitizing honeybees, are generally classified as Varroidae. Their extremely modified morphologies and behaviors have led to debates regarding their phylogenetic position and classification as an independent family. In this study, two different datasets were employed to reconstruct the phylogenies of Varroa mites and related Laelapidae species: (1) 9257 bp from the whole 13 mitochondrial protein-coding genes of 24 taxa, (2) 3158 bp from 113 taxa using Sanger sequencing of four nuclear loci. Both mitochondrial and nuclear analyses consistently place Varroa mites within the Laelapidae. Here we propose to place Varroa mites in the subfamily Varroinae stat. nov., which represents a highly morphologically adapted group within the Laelapidae. Ancestral state reconstructions reveal that bee-associated lifestyles evolved independently at least three times within Laelapidae, with most phoretic traits originating from free-living ancestors. Our revised classification and evolutionary analyses will provide new insight into understanding the Varroa mites.

RNA interference as a next-generation control method for suppressing Varroa destructor reproduction in honey bee (Apis mellifera) hives.

BACKGROUND: The Varroa mite (Varroa destructor) is considered to be the greatest threat to apiculture worldwide. RNA interference (RNAi) using double-stranded RNA (dsRNA) as a gene silencing mechanism has emerged as a next-generation strategy for mite control. RESULTS: We explored the impact of a dsRNA biopesticide, named vadescana, designed to silence the calmodulin gene in Varroa, on mite fitness in mini-hives housed in a laboratory. Two dosages were tested: 2 g/L dsRNA and 8 g/L dsRNA. Vadescana appeared to have no effect on mite survival, however, mite fertility was substantially reduced. The majority of foundress mites exposed to vadescana failed to produce any offspring. No dose-dependent effect of vadescana was observed, as both the low and high doses inhibited mite reproduction equally well in the mini-hives and neither dose impacted pupal survival of the honey bee. Approximately 95% of bee pupae were alive at uncapping across all treatment groups. CONCLUSION: These findings suggest that vadescana has significant potential as an effective alternative to conventional methods for Varroa control, with broader implications for the utilization of RNAi as a next-generation tool in the management of pest species. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Population-wide modelling reveals prospects of marker-assisted selection for parasitic mite resistance in honey bees.

In 2019, a joint eight-variant model was published in which eight single nucleotide polymorphisms (SNPs) in seven Apis mellifera genes were associated with Varroa destructor drone brood resistance (DBR, i.e. mite non-reproduction in drone brood). As this model was derived from only one Darwinian Black Bee Box colony, it could not directly be applied on a population-overarching scale in the northern part of Belgium (Flanders), where beekeepers prefer the carnica subspecies. To determine whether these eight SNPs remained associated with the DBR trait on a Flemish colony-broad scope, we performed population-wide modelling through sampling of various A. mellifera carnica colonies, DBR scoring of Varroa-infested drone brood and variant genotyping. Novel eight-variant modelling was performed and the classification performance of the eight SNPs was evaluated. Besides, we built a reduced three-variant model retaining only three genetic variants and found that this model classified 76% of the phenotyped drones correctly. To examine the spread of beneficial alleles and predict the DBR probability distribution in Flanders, we determined the allelic frequencies of the three variants in 292 A. mellifera carnica queens. As such, this research reveals prospects of marker-assisted selection for Varroa drone brood resistance in honeybees.

Geographical distribution of pyrethroid resistance mutations in Varroa destructor across Türkiye and a European overview.

Varroa destructor Anderson & Trueman (Acari: Varroidae) is of paramount significance in modern beekeeping, with infestations presenting a primary challenge that directly influences colony health, productivity, and overall apicultural sustainability. In order to control this mite, many beekeepers rely on a limited number of approved synthetic acaricides, including the pyrethroids tau-fluvalinate, flumethrin and organophosphate coumaphos. However, the excessive use of these substances has led to the widespread development of resistance in various beekeeping areas globally. In the present study, the occurrence of resistance mutations in the voltage-gated sodium channel (VGSC) and acetylcholinesterase (AChE), the target-site of pyrethroids and coumaphos, respectively, was examined in Varroa populations collected throughout the southeastern and eastern Anatolia regions of Türkiye. All Varroa samples belonged to the Korean haplotype, and a very low genetic distance was observed based on cytochrome c oxidase subunit I (COI) gene sequences. No amino acid substitutions were determined at the key residues of AChE. On the other hand, three amino acid substitutions, (L925V/I/M), previously associated with pyrethroid resistance, were identified in nearly 80% of the Turkish populations. Importantly, L925M, the dominant mutation in the USA, was detected in Turkish Varroa populations for the first time. To gain a more comprehensive perspective, we conducted a systematic analysis of the distribution of pyrethroid resistance mutations across Europe, based on the previously reported data. Varroa populations from Mediterranean countries such as Türkiye, Spain, and Greece exhibited the highest frequency of resistance mutation. Revealing the occurrence and geographical distribution of pyrethroid resistance mutations in V. destructor populations across the country will enhance the development of more efficient strategies for mite management.

Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites.

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Genetic variability of the honey bee mite, Varroa destructor, from a humid continental climatic region of Canada, and temperate and tropical climatic regions of Mexico.

Varroa destructor is a damaging mite of Western honey bees (Apis mellifera). Genetic variability of the mite in different regions of the world could be related to the movement of infested bees or other factors, such as climate. In this study, V. destructor samples were collected from tropical and temperate climate regions of Mexico, and a humid continental climate region of Canada. COX-1 AFLPs showed that all the mites were the Korean haplotype. Four microsatellites revealed nine haplogroups from the continental climate region of Canada, compared to three haplogroups from the tropical and temperate climate regions of Mexico. CytII-ATP sequences showed seven haplogroups from the humid continental climate region vs. two haplogroups from the temperate region and one haplogroup from the tropical region. CytB sequences revealed seven haplogroups from Canada vs. three from Mexico. A comparison of the cytB sequences of the samples from Canada and Mexico to those from a worldwide collection showed that one sequence, designated the cytB1 type, predominated, comprising 57% of the 86 sequences; it clustered with similar sequences that comprised 80% of the sequences, designated family A. CytB1 was predominant in Mexico, but not in Canada. The other 20% of sequences were in families B and C, and all those samples originated from East and Southeast Asia. The microsatellite, cytII-ATP, and cytB markers, all showed higher variability in mites collected in Canada than in Mexico, which could be related to the cooler climate or an earlier invasion and/or multiple mite invasions in Canada.

Mite non-reproduction, recapping behavior, and hygienic behavior (freeze-kill method) linked to Varroa destructor infestation levels in selected Apis mellifera colonies.

The genetic selection of honey bees (Apis mellifera) possessing specific social hygienic behaviors offers the beekeeping industry the possibility of controlling the Varroa destructor parasite and thus reducing its dependence on acaricides. However, the links between these behavioral traits are not yet well defined, which limits genetic progress in breeding programs. We measured the following behavioral varroa resistance traits: freeze-kill brood (FKB) and pin-kill brood (PKB) assays, varroa-sensitive hygiene (VSH), pupae removal, mite non-reproduction (MNR), and recapping activity. We found 2 negative and significant relationships: 1) between the recapping of cells infested with varroa and the total number of recapped cells, and 2) between the recapping of cells infested with varroa and VSH. We also selected the best predictive model of varroa infestation levels using the "step-wise" approach based on the Akaike information criterion. Our model revealed that MNR and FKB were significantly related to the varroa population levels, with a negative relationship; recapping was significantly related to mite infestation levels, with a positive relationship. Thus, a higher MNR or FKB score was linked to lower levels of mite infestation in colonies on August 14 (prior to fall infestation treatments); a higher recapping activity was linked to a higher level of mite infestation. Recapping behavior could be a useful trait to aid the selection of varroa-resistant bee lineages.

First large-scale genomic prediction in the honey bee.

Genomic selection has increased genetic gain in several livestock species, but due to the complicated genetics and reproduction biology not yet in honey bees. Recently, 2970 queens were genotyped to gather a reference population. For the application of genomic selection in honey bees, this study analyzes the accuracy and bias of pedigree-based and genomic breeding values for honey yield, three workability traits, and two traits for resistance against the parasite Varroa destructor. For breeding value estimation, we use a honey bee-specific model with maternal and direct effects, to account for the contributions of the workers and the queen of a colony to the phenotypes. We conducted a validation for the last generation and a five-fold cross-validation. In the validation for the last generation, the accuracy of pedigree-based estimated breeding values was 0.12 for honey yield, and ranged from 0.42 to 0.61 for the workability traits. The inclusion of genomic marker data improved these accuracies to 0.23 for honey yield, and a range from 0.44 to 0.65 for the workability traits. The inclusion of genomic data did not improve the accuracy of the disease-related traits. Traits with high heritability for maternal effects compared to the heritability for direct effects showed the most promising results. For all traits except the Varroa resistance traits, the bias with genomic methods was on a similar level compared to the bias with pedigree-based BLUP. The results show that genomic selection can successfully be applied to honey bees.

Confirmation of the Y215H mutation in the ß2 -octopamine receptor in Varroa destructor is associated with contemporary cases of amitraz resistance in the United States.

BACKGROUND: The parasitic mite, Varroa destructor (Anderson and Trueman), is a leading cause of honey bee colony losses around the world. Application of miticides such as amitraz are often the primary method of Varroa control in commercial beekeeping operations in the United States. It is likely that excessive and exclusive amitraz application has led to the development of amitraz resistance in Varroa. A mutation of tyrosine at amino acid position 215 to histidine (Y215H) in the ß2 -octopamine receptor was identified in putatively amitraz-resistant Varroa in the United States. This research investigated the presence of the Y215H mutation in quantitatively confirmed amitraz-resistant Varroa from the United States. RESULTS: There was a strong association of susceptible and resistant phenotypes with the corresponding susceptible and resistant genotypes respectively, and vice versa. The resistance bioassay may understate resistance levels because of the influence of environmental conditions on the outcome of the test, whereby Varroa with an amitraz-resistant genotype may appear with a susceptible phenotype. CONCLUSION: Confirmation of the Y215H mutation in the ß2 -octopamine receptor of amitraz-resistant Varroa encourages the development and validation of low-cost, high-throughput genotyping protocols to assess amitraz resistance. Resistance monitoring via genotyping will allow for large-scale passive monitoring to accurately determine the prevalence of amitraz resistance rather than directed sampling of apiaries with known resistance issues. Genotyping of Varroa for amitraz resistance early in the beekeeping season may predict late-season resistance at the colony level and provide beekeepers with enough time to develop an effective Varroa management strategy. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Genomic signatures underlying the oogenesis of the ectoparasitic mite Varroa destructor on its new host Apis mellifera.

INTRODUCTION: Host shift of parasites may have devastating effects on the novel hosts. One remarkable example is that of the ectoparasitic mite Varroa destructor, which has shifted its host from Eastern honey bees (Apis cerana) to Western honey bees (Apis mellifera) and posed a global threat to apiculture. OBJECTIVES: To identify the genetic factors underlying the reproduction of host-shifted V. destructor on the new host. METHODS: Genome sequencing was conducted to construct the phylogeny of the host-shifted and non-shifted mites and to screen for genomic signatures that differentiated them. Artificial infestation experiment was conducted to compare the reproductive difference between the mites, and transcriptome sequencing was conducted to find differentially expressed genes (DEGs) during the reproduction process. RESULTS: The host-shifted and non-shifted V. destructor mites constituted two genetically distinct lineages, with 15,362 high-FST SNPs identified between them. Oogenesis was upregulated in host-shifted mites on the new host A. mellifera relative to non-shifted mites. The transcriptomes of the host-shifted and non-shifted mites differed significantly as early as 1h post-infestation. The DEGs were associated with nine genes carrying nonsynonymous high-FST SNPs, including mGluR2-like, Lamb2-like and Vitellogenin 6-like, which were also differentially expressed, and eIF4G, CG5800, Dap160 and Sas10, which were located in the center of the networks regulating the DEGs based on protein-protein interaction analysis. CONCLUSIONS: The annotated functions of these genes were all associated with oogenesis. These genes appear to be the key genetic determinants of the oogenesis of host-shifted mites on the new host. Further study of these candidate genes will help elucidate the key mechanism underlying the success of host shifts of V. destructor.

Vector-virus interaction affects viral loads and co-occurrence.

BACKGROUND: Vector-borne viral diseases threaten human and wildlife worldwide. Vectors are often viewed as a passive syringe injecting the virus. However, to survive, replicate and spread, viruses must manipulate vector biology. While most vector-borne viral research focuses on vectors transmitting a single virus, in reality, vectors often carry diverse viruses. Yet how viruses affect the vectors remains poorly understood. Here, we focused on the varroa mite (Varroa destructor), an emergent parasite that can carry over 20 honey bee viruses, and has been responsible for colony collapses worldwide, as well as changes in global viral populations. Co-evolution of the varroa and the viral community makes it possible to investigate whether viruses affect vector gene expression and whether these interactions affect viral epidemiology. RESULTS: Using a large set of available varroa transcriptomes, we identified how abundances of individual viruses affect the vector's transcriptional network. We found no evidence of competition between viruses, but rather that some virus abundances are positively correlated. Furthermore, viruses that are found together interact with the vector's gene co-expression modules in similar ways, suggesting that interactions with the vector affect viral epidemiology. We experimentally validated this observation by silencing candidate genes using RNAi and found that the reduction in varroa gene expression was accompanied by a change in viral load. CONCLUSIONS: Combined, the meta-transcriptomic analysis and experimental results shed light on the mechanism by which viruses interact with each other and with their vector to shape the disease course.

Varroa destructor resistance to tau-fluvalinate: relationship between in vitro phenotypic test and VGSC L925V mutation.

BACKGROUND: Varroa destructor is a parasitic mite of the honey bee, Apis mellifera. Its presence in colonies can lead to a collapse within a few years. The use of acaricides has become essential to manage the hive infestation. However, the repeated and possibly incorrect use of acaricide treatments, as tau-fluvalinate, has led to the development of resistance. The in vitro phenotypic test allows the proportion of susceptible or resistant individuals to be known following an exposure to an active substance. In Varroa mites, resistance to tau-fluvalinate is associated with the presence of mutations at the position 925 of the voltage-gated sodium channel (VGSC). RESULTS: Here, we compared the results obtained with an in vitro phenotypic test against tau-fluvalinate and those obtained with an allelic discrimination assay on 13 treated and untreated Varroa populations in France. The correlation between the phenotype and the genetic profile rate is found to be 0.89 Varroa mites having resistant phenotypic profile have a probability of 63% to present the L925V mutation (resistance detection reliability). However, 97% of the Varroa mites having the susceptible phenotype do not present the L925V mutation (susceptible detection reliability). CONCLUSION: The L925V mutation explains most of the resistance to tau-fluvalinate in V. destructor in the populations tested. However, other mutations or types of resistance may also be involved to explain the survival of Varroa mites in the phenotypic test. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Marker assisted selection for Varroa destructor resistance in New Zealand honey bees.

Varroa destructor is a honey bee (Apis mellifera) parasite identified as one of the leading causes of overwintering colony loss in New Zealand. It has been shown that a naturally occurring heritable trait, "Varroa Sensitive Hygiene" (VSH), confers an advantage to colonies by increasing behaviours that limit the survival and reproduction of Varroa mites. The SNP 9-9224292 is an adenine/guanine (A/G) polymorphism on chromosome 9 of Apis mellifera where the G allele was observed to be associated with VSH behaviour in North American honey bees. In this study, we sought to determine if selection for the G allele of SNP 9-9224292 could decrease Varroa mite infestation of New Zealand honey bee (Apis mellifera ligustica) colonies. We genotyped queens and tracked their colonies over summer before measuring Varroa levels at the point of autumn Varroa treatment. The mean Varroa population level in colonies headed by queens that carry two copies of VSH associated G allele of SNP 9-9224292 was 28.5% (P<0.05) lower compared with colonies headed by queens with two copies of non-VSH associated A alleles. Although a significant reduction in mite infestation was achieved in treatment colonies, conventional Varroa treatment was still required for adequate Varroa control. Considering the open mating of queens used and a lack of drift control in this study, this VSH SNP shows promise for marker assisted selection of New Zealand honey bees when aiming for innate Varroa control traits.

Effects of Niemann-Pick type C2 (NPC2) gene transcripts silencing on behavior of Varroa destructor and molecular changes in the putative olfactory gene networks.

To understand the role of two Niemann-Pick type C2 (NPC2) transcripts, Vd40090 (NP1) and Vd74517 (NP5), in the chemosensing pathway of Varroa destructor, we evaluated the impact of NP5 silencing on mites behavior and compared the effect of silencing of either transcripts on the interaction between chemosensory transcripts. In contrast to silencing NP1, which reduced feeding and reproduction by the mite (Nganso et al., 2021), silencing of NP5 reduced significantly the host reaching ability, but it did not affect the feeding on nurse bee. However, silencing of either transcript changed dramatically the co-expression patterns among the putative chemosensory genes, binding proteins and receptors. The results suggest the role of gustatory receptors in the detection of long-range chemical cues in the chemosensory cascade of the Varroa mite.

Study of pyrethroid resistance mutations in populations of Varroa destructor across Spain.

The Varroa destructor mite is a serious worldwide pest of honeybees that is usually controlled with pyrethroid-based acaricides. However, the intensive use of these substances over the past decades has led to the development of resistance in these mites. Here, Varroa samples collected between 2006 and 2021 from apiaries across Spain were studied to evaluate the presence of mutations producing pyrethroid resistance, particularly those in the gene encoding the voltage-gated sodium channel (VGSC). Genotyping of the IIS4-IIS5 region of this gene detected the L925V (Leucine 'CTG' to valine 'GTG') mutation at position 925 and confirmed the presence of the M918L (Methionine 'ATG' to Leucine 'TTG') mutation at position 918 in these Spanish Varroa mites. Interestingly, the M918L mutation was always found in combination with L925V, both of which were always homozygous. Over and above the high frequency of pyrethroid-resistant mutations in Spanish Varroa populations, this apparently recent association of the M918L and L925V point mutations is a combination that appears to trigger greater resistance than that produced by L925V alone.

Genetic diversification of an invasive honey bee ectoparasite across sympatric and allopatric host populations.

Invasive parasites are major threats to biodiversity. The honey bee ectoparasite, Varroa destructor, has shifted host and spread almost globally several decades ago. This pest is generally considered to be the main global threat to Western honey bees, Apis mellifera, although the damages it causes are not equivalent in all its new host's populations. Due to the high virulence of this parasite and the viruses it vectors, beekeepers generally rely on acaricide treatments to keep their colonies alive. However, some populations of A. mellifera can survive without anthropogenic mite control, through the expression of diverse resistance and tolerance traits. Such surviving colonies are currently found throughout the globe, with the biggest populations being found in Sub-Saharan Africa and Latin America. Recently, genetic differences between mite populations infesting surviving and treated A. mellifera colonies in Europe were found, suggesting that adaptations of honey bees drive mite evolution. Yet, the prevalence of such co-evolutionary adaptations in other invasive populations of V. destructor remain unknown. Using the previous data from Europe and novel genetic data from V. destructor populations in South America and Africa, we here investigated whether mites display signs of adaptations to different host populations of diverse origins and undergoing differing management. Our results show that, contrary to the differences previously documented in Europe, mites infesting treated and untreated honey bee populations in Africa and South America are genetically similar. However, strong levels of genetic differentiation were found when comparing mites across continents, suggesting ongoing allopatric speciation despite a recent spread from genetically homogenous lineages. This study provides novel insights into the co-evolution of V. destructor and A. mellifera, and confirms that these species are ideal to investigate coevolution in newly established host-parasite systems.

Selection of stable reference genes for quantitative real-time PCR in the Varroa mite, Varroa destructor.

To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real-time polymerase chain reaction (qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase [NADHD], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], eukaryotic translation elongation factor 1 α 1 [eEF1A1], eukaryotic translation elongation factor 2 [eEF2], ribosomal protein L5 [RpL5], Actin, tubulin α-1D chain [α-tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α-tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α-tubulin and RpL5 are recommended for investigating the tissue-specific expression profiles of target genes.

An Insight Into the microRNA Profile of the Ectoparasitic Mite Varroa destructor (Acari: Varroidae), the Primary Vector of Honey Bee Deformed Wing Virus.

The remarkably adaptive mite Varroa destructor is the most important honey bee ectoparasite. Varroa mites are competent vectors of deformed wing virus (DWV), and the Varroa-virus complex is a major determinant of annual honey bee colony mortality and collapse. MicroRNAs (miRNAs) are 22-24 nucleotide non-coding RNAs produced by all plants and animals and some viruses that influence biological processes through post-transcriptional regulation of gene expression. Knowledge of miRNAs and their function in mite biology remains limited. Here we constructed small RNA libraries from male and female V. destructor using Illumina's small RNA-Seq platform. A total of 101,913,208 and 91,904,732 small RNA reads (>18 nucleotides) from male and female mites were analyzed using the miRDeep2 algorithm. A conservative approach predicted 306 miRNAs, 18 of which were upregulated and 13 downregulated in female V. destructor compared with males. Quantitative real-time PCR validated the expression of selected differentially-expressed female Varroa miRNAs. This dataset provides a list of potential miRNA targets involved in regulating vital Varroa biological processes and paves the way for developing strategies to target Varroa and their viruses.