Molecular detection of avian pathogens in poultry red mite (Dermanyssus gallinae) in Algerian layer farms as a potential predictive tool.

The poultry red mite Dermanyssus gallinae is a hematophagous ectoparasite of layer hens. Infestations with poultry red mites pose an increasing threat to the egg production industry, causing serious problems to animal health and welfare, directly or indirectly as a vector of several infectious agents. In this study, we aimed to investigate common avian pathogens in mites. The mite samples were collected from 58 poultry farms in 7 regions accounting for more than 70 % of the national egg production in Algeria. The presence of 13 avian pathogens was detected using DNA and RNA samples from mites collected. Results revealed significant associations between PRM and potential pathogens such as Escherichia coli, Salmonella enterica, fowlpox virus, and gallid herpesvirus 1. Pathogen detection in Dermanyssus gallinae could serve as an early diagnostic or a risk analysis tool for infectious diseases in poultry farms, facilitating effective disease management strategies. Despite further research being necessary to address uncertainties, such a strategy could be used to enhance the integrated management of poultry health.

Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus-Vector System Threatening Solanaceous Crops Worldwide.

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus-vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

ICTV Virus Taxonomy Profile: Fimoviridae 2024.

Members of the family Fimoviridae are plant viruses with a multipartite negative-sense enveloped RNA genome (-ssRNA), composed of 4-10 segments comprising 12.3-18.5 kb in total, within quasi-spherical virions. Fimoviruses are transmitted to plants by eriophyid mites and induce characteristic cytopathologies in their host plants, including double membrane-bound bodies in the cytoplasm of virus-infected cells. Most fimoviruses infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the ICTV Report on the family Fimoviridae, which is available at ictv.global/report/fimoviridae.

Helper Component-Proteinase of Triticum Mosaic Virus Is a Viral Determinant of Wheat Curl Mite Transmission.

Triticum mosaic virus (TriMV; genus Poacevirus; family Potyviridae) is an economically important virus in the Great Plains region of the United States. TriMV is transmitted by the wheat curl mite (Aceria tosichella) Type 2 genotype but not by Type 1. Helper component-proteinase (HC-Pro) is a vector transmission determinant for several potyvirids, but the role of HC-Pro in TriMV transmission is unknown. In this study, we examined the requirement of the HC-Pro cistron of TriMV for wheat curl mite (Type 2) transmission through deletion and point mutations and constructing TriMV chimeras with heterologous HC-Pros from other potyvirids. TriMV with complete deletion of HC-Pro failed to be transmitted by wheat curl mites at detectable levels. Furthermore, TriMV chimeras with heterologous HC-Pros from aphid-transmitted turnip mosaic virus and tobacco etch virus, or wheat curl mite-transmitted wheat streak mosaic virus, failed to be transmitted by wheat curl mites. These data suggest that heterologous HC-Pros did not complement TriMV for wheat curl mite transmission. A decreasing series of progressive nested in-frame deletions at the N-terminal region of HC-Pro comprising amino acids 3 to 125, 3 to 50, 3 to 25, 3 to 15, 3 to 8, and 3 and 4 abolished TriMV transmission by wheat curl mites. Additionally, mutation of conserved His20, Cys49, or Cys52 to Ala in HC-Pro abolished TriMV transmissibility by wheat curl mites. These data suggest that the N-terminal region of HC-Pro is crucial for TriMV transmission by wheat curl mites. Collectively, these data demonstrate that the HC-Pro cistron of TriMV is a viral determinant for wheat curl mite transmission.

Dermanyssus gallinae: the long journey of the poultry red mite to become a vector.

The possibility that Dermanyssus gallinae, the poultry red mite, could act as a vector of infectious disease-causing pathogens has always intrigued researchers and worried commercial chicken farmers, as has its ubiquitous distribution. For decades, studies have been carried out which suggest that there is an association between a wide range of pathogens and D. gallinae, with the transmission of some of these pathogens mediated by D. gallinae as vector. The latter include the avian pathogenic Escherichia coli (APEC), Salmonella enterica serovars Enteritidis and Gallinarum and influenza virus. Several approaches have been adopted to investigate the relationship between D. gallinae and pathogens. In this comprehensive review, we critically describe available strategies and methods currently available for conducting trials, as well as outcomes, analyzing their possible strengths and weaknesses, with the aim to provide researchers with useful tools for correctly approach the study of the vectorial role of D. gallinae.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Unravelling the involvement of cilevirus p32 protein in the viral transport.

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

Molecular detection of vector-borne agents in ectoparasites and reptiles from Brazil.

Trombidiformes and Mesostigmata mites, as well as Ixodida ticks, infest ectothermic tetrapods worldwide, potentially acting as vectors of bacteria, viruses and protozoa. The relationship among ectoparasites, transmitted pathogenic agents (e.g., Borrelia spp., Coxiella spp., Hepatozoon spp., and Rickettsia spp.) and ectothermic hosts has been scarcely investigated. This research focuses on a large collection of Brazilian herpetofauna screened for the presence of arthropod ectoparasites and vector-borne microbial agents. Reptiles (n = 121) and amphibians (n = 49) from various locations were infested by ectoparasites. Following genomic extraction, microbial agents were detected in 81 % of the Acari (i.e. n = 113 mites and n = 26 ticks). None of the mites, ticks and tissues from amphibians yielded positive results for any of the screened agents. Blood was collected from reptiles and processed through blood cytology and molecular analyses (n = 48). Of those, six snakes (12.5 %) showed intraerythrocytic alterations compatible with Hepatozoon spp. gamonts and Iridovirus inclusions. Hepatozoon spp. similar to Hepatozoon ayorgbor and Hepatozoon musa were molecularly identified from seven hosts, two mite and two tick species. Rickettsia spp. (e.g., Rickettsia amblyommatis, Rickettsia bellii-like, Rickettsia sp.) were detected molecularly from four mite species and Amblyomma rotundatum ticks. Phylogenetic analyses confirmed the molecular identification of the above-mentioned microbial agents of mites and ticks related to snakes and lizards. Overall, our findings highlighted that the Brazilian herpetofauna and its ectoparasites harbour potentially pathogenic agents, particularly from the northern and south-eastern regions. The detection of several species of spotted fever group Rickettsia pointed out the potential role of ectothermic hosts and related arthropod ectoparasites in the epidemiological cycle of these bacteria in Brazil.

Survey of the citrus leprosis vector (Brevipalpus yothersi) and phytoseiids in spontaneous plants of an organic citrus orchard.

Citrus leprosis (CL) is one of the most important viral diseases in sweet orange orchards in Latin America. It is caused by members of at least five species of the so-called Brevipalpus-transmitted viruses (BTV), and the prevalent is Citrus leprosis virus C (CiLV-C). This virus has the broadest host range amongst all CL-associated viruses and is transmitted by Brevipalpus yothersi, a polyphagous mite that can colonize a large variety of host plants, including some spontaneous ground cover plants. But if, on one hand, spontaneous plants can host CL virus and vector, on the other hand, they can offer alternative food for predators, equally common in organic citrus orchards. Brevipalpus yothersi and predator mites were surveyed in 33 spontaneous plants of a Westin sweet orange orchard conducted under organic production system in Brazil, from June 2010 to April 2011. Predatory mites were identified as phytoseiids, and Iphiseiodes zuluagai was the prevalent species, representing 58% of all predators. Other phytoseiids were considered accidental species in the area. Ageratum conyzoides and Alternanthera tenella were the most represented plant host species to predators, comprising 28 and 10% of the total surveyed plants, respectively. Brevipalpus yothersi specimens were detected on various spontaneous species: A. conyzoides, A. tenella, Amaranthus deflexus, Bidens pilosa, Ipomoea quamoclit, I. cairica, Merremia cissoides, Solanum americanum, Panicum maximum, and, predominantly, Commelina benghalensis. The latter has been previously reported as host of CiLV-C as well and, therefore, it is recommended to eliminate this species from citrus orchards.

Modeling Aceria tosichella biotype distribution over geographic space and time.

The wheat curl mite, Aceria tosichella Keifer, one of the most destructive arthropod pests of bread wheat worldwide, inflicts significant annual reductions in grain yields. Moreover, A. tosichella is the only vector for several economically important wheat viruses in the Americas, Australia and Europe. To date, mite-resistant wheat genotypes have proven to be one of the most effective methods of controlling the A. tosichella-virus complex. Thus, it is important to elucidate A. tosichella population genetic structure, in order to better predict improved mite and virus management. Two genetically distinct A. tosichella lineages occur as pests of wheat in Australia, Europe, North America, South America and the Middle East. These lineages are known as type 1 and type 2 in Australia and North America and in Europe and South America as MT-8 and MT-1, respectively. Type 1 and type 2 mites in Australia and North America are delineated by internal transcribed spacer 1 region (ITS1) and cytochrome oxidase I region (COI) sequence differences. In North America, two A. tosichella genotypes known as biotypes are recognized by their response to the Cmc3 mite resistance gene in wheat. Aceria tosichella biotype 1 is susceptible to Cmc3 and biotype 2 is virulent to Cmc3. In this study, ITS1 and COI sequence differences in 25 different populations of A. tosichella of known biotype 1 or biotype 2 composition were characterized for ITS1 and COI sequence differences and used to model spatio-temporal dynamics based on biotype prevalence. Results showed that the proportion of biotype 1 and 2 varies both spatially and temporally. Greater ranges of cropland and grassland within 5000m of the sample site, as well as higher mean monthly precipitation during the month prior to sampling appear to reduce the probability of occurrence of biotype 1 and increase the probability of occurrence of biotype 2. The results suggest that spatio-temporal modeling can effectively improve A. tosichella management. Continual integration of additional current and future precipitation and ground cover data into the existing model will further improve the accuracy of predicting the occurrence of A. tosichella in annual wheat crops, allowing producers to make informed decisions about the selection of varieties with different A. tosichella resistance genes.

Lack of transmission of Sugarcane yellow leaf virus in Florida from Columbus grass and sugarcane to sugarcane with aphids or mites.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane (Saccharum spp.), the weed Columbus grass (Sorghum almum) and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18-28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.

Tropilaelaps species identification and viral load evaluation of Tropilaelaps and Varroa mites and their Apis mellifera hosts in Palawan, Philippines.

Apis mellifera pupae and their parasites Tropilaelaps and Varroa destructor were collected from honey bee hives in Palawan, Philippines for species identification of the Tropilaelaps and viral analyses. Genetic analysis identified Tropilaelaps mercedesae infesting A. mellifera on the island. Viral analyses showed that all pupae and their infesting Tropilaelaps or Varroa shared the same Deformed Wing Virus (DWV) variant infections with DWV-B being more prevalent than DWV-A. Pupae infested with either Varroa or Tropilaelaps had higher levels of both DWV variants than uninfested pupae. Vigilance is needed to prevent the spread of Tropilaelaps clareae into Palawan and T. mercedesae and DWV variants from Palawan to other provinces.

RNA virome screening in diverse but ecologically related citrus pests reveals potential virus-host interactions.

As an evergreen ecosystem, citrus orchards have specialized pest species and stable ecological homeostasis; thus, they provide an ideal model for investigating RNA viromes in diverse but ecologically related species. For this purpose, we collected specialized citrus pests from three classes of invertebrates, Insecta, Arachnida, and Gastropoda and we constructed two kinds of libraries (RNA and small RNA) for the pests by deep sequencing. In total, six virus-derived sequences were identified, including four Picornavirales, one Jingchuvirales and one Nidovirales. The picornavirus-derived small RNAs showed significant small RNA peaks and symmetric distribution patterns along the genome, which suggests these viruses infected the hosts and triggered host antiviral immunity RNA interference. Screening of virus-derived sequences in multiple species of citrus pests (n = 10 per species) showed that Eotetranychus kankitus picorna-like virus and Tetranychus urticae mivirus may be present in multiple pests. Our investigation in citrus pests confirmed that RNA viruses revealed by metagenomics could impact host immunity (e.g. RNAi). An approach with parallel deep sequencing of RNAs and small RNAs is useful not only for viral discoveries but also for understanding virus-host interactions of ecologically related but divergent pest species.

Morphological and molecular characterization of the Colomerus vitis erineum strain (Trombidiformes: Eriophyidae) from grapevine erinea and buds.

The grapevine erineum mite strain (GEM) of Colomerus vitis (Pagenstecher) has spread throughout the main viticultural areas worldwide and was recently demonstrated to be a vector of Grapevine pinot gris virus (GPGV) and Grapevine inner necrosis virus (GINV). Its females mainly overwinter under the outer bud scales as winter morphs (deutogynes). Goals of this study were to characterize the morphology of protogynes (spring-summer morphs) and deutogynes (winter morphs), to confirm their genetic similarity, and to establish the seasonal period of the deutogyne occurrence. Buds or leaves from a single vineyard (cv. Luisa), Bari area, Apulia, Italy, infested with GEM were sampled 6 × from December 2015 to January 2017. Sixty-six traits commonly used for taxonomic identification were analysed on females. The length of the tibial setae l' on leg I and the tarsal setae ft' on leg II, as well as the number of smooth dorsal semiannuli differed significantly between protogynes and deutogynes, and were easier to detect than other significantly distinctive traits. ITS1 was investigated in individuals collected from buds and erinea, and the sequences confirmed that these two morphs have identical ITS1 fragments. The 1-year study demonstrated the simultaneous presence of protogynes and deutogynes in July and September 2016, whereas only protogynes were found in April and May 2016, and only deutogynes in December 2015 and January 2017.

Incidence of Mite-Vectored Viruses of Wheat in the Texas High Plains and Interactions With Their Host and Vector.

Mite-vectored virus diseases of wheat are common throughout the Great Plains and cause significant economic losses to growers each year. These diseases are caused by Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and Wheat mosaic virus (WMoV), all of which are transmitted by the wheat curl mite (WCM), Aceria tosichella Keifer. New wheat cultivars with tolerance or resistance to WSMV have been released recently, but their widespread cultivation and potential impact on mite-transmitted virus incidence in the Texas Panhandle was unknown. A total of 648 symptomatic wheat samples were collected from 26 counties, predominately in the Texas Panhandle, and tested by enzyme-linked immunosorbent assay (ELISA) for WSMV, TriMV, and WMoV. Samples that tested negative by ELISA were subsequently tested by real-time quantitative PCR (qPCR) for each virus. Approximately 93% of the samples tested by ELISA were positive for WSMV, 43% were positive for TriMV, and 7% were positive for WMoV. Eleven samples tested positive only for TriMV, but none were positive only for WMoV. When samples that tested negative for the different viruses by ELISA were retested by real-time qPCR, detection of each virus was significantly increased. When results of the ELISA test and qPCR were combined, 100% of the 648 samples tested positive for WSMV, approximately 94% were positive for TriMV, and 23% were positive for WMoV. This demonstrated that the incidence of TriMV in the Texas High Plains is much greater than previously reported. The fact that real-time qPCR revealed over a 2-fold increase in the incidence of TriMV and a 3-fold increase in WMoV demonstrated that the ELISA test, which is commonly used by diagnostic laboratories in the Great Plains, should not be used for studies requiring a high degree of sensitivity and accuracy in virus detection. After initial virus infection status was determined, samples that tested positive for WSMV and TriMV were further observed for WCM infestation. A total of 292 samples were inspected and a total of 101 mites were collected from 40 tillers. Individual mites and the tillers from which they were recovered were tested by real-time qPCR to determine how copy numbers of WSMV and TriMV in mites and host tissue compared, and whether the WSMV/TriMV copy number ratio in individual mites was similar to that of the host tissue from which they were collected. In all mites and tillers tested, the WSMV copy number was always higher than that of TriMV and copy numbers of both viruses were always higher in plant tissue than in mites. Although there was a significant correlation between the WSMV/TriMV copy number ratio in plant tissue and in associated mites, the correlation coefficient was very low (r = 0.31, P = 0.0248). In the majority of comparisons, the WSMV/TriMV ratio was higher in individual mites than in the tiller from which they were recovered. The reason for this increase is unknown but indicates that mites may preferentially acquire WSMV from tillers coinfected with WSMV and TriMV, a finding that could have significant implications for virus transmission and disease epidemiology.

Questions surrounding the taxonomic validity of the species Garlic mite-borne filamentous virus (genus Allexivirus).

Garlic mite-borne filamentous virus is one of the oldest recognized allexivirus species but, paradoxically, one with the least well studied member viruses. In this paper, we review the history of this taxon and highlight problems in designating a holotype (exemplar isolate). Analyses are presented that suggest that GarMbFV is conspecific with Garlic virus A, and therefore the former taxon should be abolished.

Raman spectroscopy as an early detection tool for rose rosette infection.

MAIN CONCLUSION: Hand-held Raman spectroscopy is a potential tool for a confirmatory, non-invasive, and non-destructive detection and identification of rose rosette disease. Using this spectroscopic approach, structural changes in roses that are associated with this viral infection can be revealed. The commercial rose shrub industry in the United States is one of the largest of its kind. All commercial rose varieties are susceptible to rose rosette disease (RRD), a deadly viral disease vectored by eriophyid mites. This disease is typically diagnosed visually and/or by PCR-based detection assays. The present work demonstrates that Raman spectroscopy can detect RRD in intact leaf tissue. It is shown that chemometric analysis can distinguish between spectra collected from symptomatic and asymptomatic tissue, as well as between healthy and asymptomatic tissue. This method will be useful as an initial screen for RRD prior to PCR analysis to help conserve reagents and save time.

A new, sensitive and efficient method for taxonomic placement in the Eriophyoidea and virus detection in individual eriophyoids.

Eriophyoids affect crops around the globe directly or indirectly as virus vectors. Eriophyoid systematics initiated over a century ago, yet more than 90% of their fauna remain undescribed. Morphological identification is challenging because of a limited number of traits, cryptic speciation and complex life cycle reported for many species in the group. Nucleic acids extraction for mite identification is challenging due to their microscopic size with researchers using pooled samples leading to polymorphisms and inconclusive results. Identification of mite virus vectors is a tiresome task that could be simplified with a protocol that allows for the detection of viruses in the individual specimen. This communication describes an innovative, highly efficient extraction and detection pipeline. Direct Reverse Transcriptase - Polymerase Chain Reaction (Drt-PCR) assays were implemented in the molecular identification of eriophyoids and detection of viruses present in their bodies. The reverse transcription step allows for amplification from a single mite or egg, as in addition to the genomic DNA, it incorporates the abundant transcripts of targeted genes, whereas it also allows for the amplification of viruses. This communication provides an efficient, sensitive and cost-effective alternative that can be implemented in pest identification and detection as well as biological and ecological studies.

Bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker and its effects on predatory mites in citrus groves.

The acaricidal bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker (Acari: Tenuipalpidae), a vector mite of the Citrus leprosis virus (CiLV), and its impact on predatory mites were assessed. For this purpose, laboratory and field assays using bioacaricide concentrations ranging from 0.5 to 2.0 mg L-1 of oxymatrine were performed during the years from 2015 to 2016. Laboratory results showed that the oxymatrine-based commercial formulation does not cause deleterious effects on B. yothersi eggs; however, it causes high larval mortality. For adult females, the bioacaricide caused high acute toxicity and residual effect for at least 5 days after application. In the field, the bioacaricide exhibited high acaricidal activity against B. yothersi, with efficacy levels similar to that of synthetic acaricide spirodiclofen (48 mg L-1) until 49 days after the application. The application of the bioacaricide did not negatively affect the population levels of phytoseiid predatory mites. Therefore, our results suggest that the oxymatrine-based commercial formulation is an important tool for management of the citrus leprosis mite in citrus groves.

Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus.

Quantitative reverse transcription PCR (RT-qPCR) is a high-throughput method to analyze the transcriptional expression of genes. Currently, no reference genes have been described for evaluating gene expression in Brevipalpus yothersi, the false spider mite, a polyphagous that act as vector of the citrus leprosis virus C (CiLV-C), an important citrus disease. This study aimed to identify the most stable reference genes in B. yothersi. The RT-qPCR expression data for selected genes were evaluated from three conditions: different developmental stages, plant hosts and acquisition of CiLV-C. To analyze the stability of the candidate reference genes we used ΔCq method, GeNorm, NormFinder, BestKeeper and RefFinder. Ubiq and GAPDH are best suited for normalizing gene expression data in viruliferous and non-viruliferous mites. Ubiq, EF1α and GAPDH are the most stable for different developmental stages. RPL13 and RPL32 are the best reference genes for approaches to B. yothersi in different host plants. Considering all the experimental conditions, Ubiq, EF1α, and GAPDH were the most stable genes. Here we developed an accurate and comprehensive RT-qPCR strategy for use in B. yothersi gene expression analysis. These results will improve the understanding of the biology of the false spider mites and their role as virus vectors.