New records of soft ticks (Acari: Argasidae) from caves in Brazil, with a morphological study of Ornithodoros fonsecai and an analysis of the taxonomic status of Antricola inexpectata.

In this study, we report soft ticks from bat-inhabiting caves in different areas of Brazil. From 2010 to 2019, we collected 807 tick specimens from nine caves located in four Brazilian states among two biomes. Ticks were morphologically identified as Antricola guglielmonei (282 specimens), Ornithodoros cavernicolous (260 specimens), and Ornithodoros fonsecai (265 specimens). Whereas A. guglielmonei was collected on bat guano in hot caves, O. cavernicolous and O. fonsecai were collected in cracks and crevices on the walls of cold caves, sometimes in the same chamber. Morphological identifications were corroborated by molecular and phylogenetic analyses inferred from tick mitochondrial 16S rRNA gene partial sequences. The sequences of A. guglielmonei, O. cavernicolous and O. fonsecai collected in this study clustered with conspecific GenBank sequences from different localities of Brazil. Remarkably, a clade containing 12 sequences of O. fonsecai was clearly bifurcated, denoting a degree of genetic divergence (up to 5 %) of specimens from Cerrado/Atlantic Forest biomes with the specimens from the Caatinga biome. To further evaluate this divergence, we performed morphometric analysis of the larval stage of different O. fonsencai populations by principal component analysis, which indicated that the larvae from Caatinga populations were generally smaller than the larvae from other biomes. Some of the present A. guglielmonei specimens were collected from the type locality of Antricola inexpectata. Comparisons of these specimens with the type specimens of A. inexpectata and A. guglielmonei indicated that they could not be separated by their external morphology. Hence, we are relegating A. inexpectata to a synonym of A. guglielmonei. This proposal is corroborated by our phylogenetic analysis.

Molecular detection of Borrelia sp. in Ornithodoros cavernicolous (Acari: Argasidae) in midwestern Brazil.

Ticks are obligate hematophagous parasites that can transmit to vertebrate hosts several pathogens, including viruses, bacteria, protozoa and helminths. Among these agents, some Borrelia species some Borrelia species cause disease in humans and other vertebrate hosts; therefore, they have medical and veterinary health importance. To gather additional information on Borrelia species in Brazil, the current study aimed to detect the presence of these species in Ornithodoros cavernicolous ticks collected in September 2019 from cement pipes that are used by bats as shelter in a farm located in the midwestern region of Brazil. DNA samples obtained from 18 specimens of O. cavernicolous were subjected of two polymerase chain reactions, targeting a segment of the Borrelia fla B gene. Of the samples tested, only one (6 %, 1/18) showed amplification. The nucleotide sequence of the amplified DNA showed more than 97 % (293/300) identity with a sequence of a Borrelia sp. detected in blood collected from a bat from Macaregua Cave, Colombia, and more than 97 % (292/300) detected in lungs from vampire bats from northeastern Brazil. The deduced amino acid sequences were identical to each other. Phylogenetic analysis indicated that these sequences formed a group of Borrelia species (putatively associated with bats) that is closely related to sequences of Borrelia species of the Lyme borreliosis group. Further investigations should be carried out in order to determine whether the sequence of the Borrelia sp. we found belongs to a new taxon. It will also be of great importance to determine which vertebrate hosts, besides bats, O. cavernicolous ticks can parasitize in order to investigate whether the Borrelia sp. we found may be transmitted and cause disease to the other vertebrate hosts.

[Genetic variations in four geographical isolates of Gohieria fusca based on cytochrome b and internal transcribed spacer genes].

OBJECTIVE: To investigate the genetic diversity and genetic differentiation of different geographical isolates of Gohieria fusca. METHODS: G. fusca isolates were sampled from Wuhu (WH), Bengbu (BB) and Bozhou cities (BZ) of Anhui Province and Jiaxing City of Zhejiang Province (JX). Mitochondrial cytochrome b (Cytb) and ribosomal internal transcribed spacer (ITS) genes were amplified in WH, BB, BZ and JX isolates of G. fusca using PCR assay. The gene sequences were edited and aligned using the software Chromas 2 and DNASTAR 1.00, and the haplotype, haplotype diversity (Hd) and nucleotide polymorphism (Pi) of each isolate were calculated using the software DnaSP 5.10.00. The genetic differentiation among isolates (Fst) and gene flow value (Nm) were estimated using the software MEGA 10.2, and a phylogenetic tree was built. Tests of neutrality and analysis of molecular variance (AMOVA) were performed using the software Arlequin 3.1 and a haplotype network was built based on the Median-Joining network using the software Network 10.2. RESULTS: PCR assay showed that the sizes of the Cytb and ITS genes were 372 bp and 1 301 to 1 320 bp, respectively. All four isolates of G. fusca presented high genetic diversity based on mitochondrial Cytb and ITS genes (Hd = 0.804, Pi = 0.006 91). AMOVA showed genetic differentiation among geographical isolates of G. fusca (Fst = 0.202 40, P < 0.05), and the genetic variation was mainly caused by intra-population variations (79.76%). Gene flow analysis showed a high level of gene flow among G. fusca isolates (Nm > 1). Tests of neutrality based on Cytb gene measured a Tajima's D value of -1.796 31 (P < 0.05) and a Fu's FS value of -3.293 98 (P < 0.05) in WH isolate of G. fusca, indicating population expansion in WH isolate of G. fusca. Haplotype network analysis and phylogenetic analysis revealed no remarkable geographical distribution pattern among different geographical isolates of G. fusca. All four isolates of G. fusca presented high genetic diversity (Hd = 0.985, Pi = 0.011 97). AMOVA showed moderate level of genetic differentiation between four isolates (Fst = 0.104 62, P < 0.05). The tests of neutrality based on ITS genes measured a Tajima's D value of -6.088 20 and a Fu's FS value of -1.935 99 (both P > 0.05) in the whole isolate of G. fusca, indicating no obviously population expansion. CONCLUSIONS: The four geographical isolates of G. fusca have high genetic diversity and remarkable genetic differentiation. Since a high level of gene flow is detected among different geographical isolates of G. fusca, no obvious geographical distribution pattern of G. fusca is found.

Morphological and molecular confirmation of Ornithodoros hasei (Schulze, 1935) (Acari: Argasidae) in Colombia.

A large number of tick species are proven vectors for the transmission of bacteria, protozoa, and viruses. Soft ticks (Acari: Argasidae) in South America have been found to be the most frequent carriers of borreliae of the relapsing fever group (RFG); however, there are several information gaps specially on the taxonomy and distribution of some tick species. Here, we used light microscopy, scanning electron microscopy, and PCR amplification of a fragment of the mitochondrial 16S rRNA gene to evaluate 174 larvae of Ornithodoros (Argasidae) collected from three bat species (Eptesicus orinocensis, Molossus rufus and Noctilio albiventris) in the Orinoquia Region of Colombia. The morphological and molecular results confirmed that all the analyzed larvae corresponded to Ornithodoros hasei. Comparisons of mitochondrial 16S rDNA sequences showed low genetic divergence (0% - 0.3%) between larvae of the Department of Arauca in the Orinoquia Region and higher genetic divergence (3.4 - 4.7%) in sequences from other American countries. Our work represents the most recent collection of this species in Colombia and provides a molecular evaluation for the first time. Moreover, a new association of O. hasei with bats such as E. orinocensis is documented. Considering the wide distribution of O. hasei in the American Continent, and its putative role as vector for Borrelia, integrative studies that involve morphological, morphometric, molecular data and experimental crosses are needed to determine if the higher genetic distances are associated with cryptic speciation, as detected in other tick complexes, or represent genetic divergences among geographically different populations of O. hasei.

Molecular characterization and phylogenetic analysis of Argas persicus (Oken, 1818) (Acari: Argasidae) from domestic birds in eastern Algeria.

Argas persicus (the fowl tick) is a species of soft tick commonly associated with poultry farms. It has a wide geographic distribution and colonizes different climate regions. Morphological identification of A. persicus has been reported worldwide, but genetic data regarding its molecular characterization is limited. The present study provides data for morphological identification and genetic characterization of A. persicus collected from domestic birds in traditional farms from east Algeria (Setif region). Additionally, A. persicus samples originating from Gansu province in China were included for comparative molecular study. In total, 1518 ticks collected from 30 infested farms were examined and morphologically identified as A. persicus. Furthermore, the 14 tick samples obtained from China were morphologically identified as A. persicus. Molecular analysis of 30 ticks from Algeria (one tick from each infested farm) and the 14 Chinese samples based on PCR, sequencing, and phylogenetic analysis of three mitochondrial genetic markers (16S rRNA, 12S rRNA, and cox1) confirmed morphological results where all samples belonged to the A. persicus group. However, phylogenetic analysis showed that all Algerian samples and two Chinese samples belong to A. persicus sensu stricto (s.s.), while the remaining Chinese samples represented A. persicus sensu lato (s.l.) (divergent lineage). The present study confirms the occurrence of A. persicus s.s. both in Algeria and China, as well as provides novel molecular data for a distinct Chinese lineage of A. persicus.

Complete mitochondrial genome of Hygrobates turcicus Pesic, Esen & Dabert, 2017 (Acari, Hydrachnidia, Hygrobatoidea).

The aim of the study was sequencing of the mitogenome of Hygrobates turcicus Pesic, Esen & Dabert, 2017 to expand knowledge of the polymorphism and cryptic or pseudocryptic diversity within Hydrachnidia. The samples originated from Bulgaria, Vidima River near Debnewo, 42°56'41.4''N, 24°48'44.6''E, depth 0.4 m, stones on the bottom, water flow 0.71 m/s, temperature 10 °C, pH 8.53, oxygen 110%, conductivity 279 µS/cm, hardness 121 CaO mg/l; 11 males, 27 females, 2 deutonymphs 12.x.2019 leg. Zawal, Michonski & Bankowska; one male and one female dissected and slides mounted. The study was carried out using the following methods: DNA extraction, sequencing, assembly and annotation, comparison with other populations of H. turcicus, and multigene phylogeny. As a result of the study, it was determined that the mitogenome is 15,006 bp long and encodes for 13 proteins, 2 rRNAs, and 22 tRNAs. The genome is colinear with those of H. longiporus and H. taniguchii, the difference in size originating from a non-coding region located between protein-coding genes ND4L and ND3. Five genes have alternative start-codon, and four display premature termination. The multigene phylogeny obtained using all mitochondrial protein-coding genes unambiguously associates H. turcicus with the cluster formed by H. longiporus and H. taniguchii.

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.

Haplotype divergence supports long-term asexuality in the oribatid mite Oppiella nova.

Sex strongly impacts genome evolution via recombination and segregation. In the absence of these processes, haplotypes within lineages of diploid organisms are predicted to accumulate mutations independently of each other and diverge over time. This so-called "Meselson effect" is regarded as a strong indicator of the long-term evolution under obligate asexuality. Here, we present genomic and transcriptomic data of three populations of the asexual oribatid mite species Oppiella nova and its sexual relative Oppiella subpectinata We document strikingly different patterns of haplotype divergence between the two species, strongly supporting Meselson effect-like evolution and long-term asexuality in O. nova: I) variation within individuals exceeds variation between populations in O. nova but vice versa in O. subpectinata; II) two O. nova sublineages feature a high proportion of lineage-specific heterozygous single-nucleotide polymorphisms (SNPs), indicating that haplotypes continued to diverge after lineage separation; III) the deepest split in gene trees generally separates the two haplotypes in O. nova, but populations in O. subpectinata; and IV) the topologies of the two haplotype trees match each other. Our findings provide positive evidence for the absence of canonical sex over evolutionary time in O. nova and suggest that asexual oribatid mites can escape the dead-end fate usually associated with asexual lineages.

DNA barcodes enable higher taxonomic assignments in the Acari.

Although mites (Acari) are abundant in many terrestrial and freshwater ecosystems, their diversity is poorly understood. Since most mite species can be distinguished by variation in the DNA barcode region of cytochrome c oxidase I, the Barcode Index Number (BIN) system provides a reliable species proxy that facilitates large-scale surveys. Such analysis reveals many new BINs that can only be identified as Acari until they are examined by a taxonomic specialist. This study demonstrates that the Barcode of Life Datasystem's identification engine (BOLD ID) generally delivers correct ordinal and family assignments from both full-length DNA barcodes and their truncated versions gathered in metabarcoding studies. This result was demonstrated by examining BOLD ID's capacity to assign 7021 mite BINs to their correct order (4) and family (189). Identification success improved with sequence length and taxon coverage but varied among orders indicating the need for lineage-specific thresholds. A strict sequence similarity threshold (86.6%) prevented all ordinal misassignments and allowed the identification of 78.6% of the 7021 BINs. However, higher thresholds were required to eliminate family misassignments for Sarcoptiformes (89.9%), and Trombidiformes (91.4%), consequently reducing the proportion of BINs identified to 68.6%. Lineages with low barcode coverage in the reference library should be prioritized for barcode library expansion to improve assignment success.

Genetic Diversity of Hard Ticks (Acari: Ixodidae) in the South and East Regions of Kazakhstan and Northwestern China.

To date, there is no report on the genetic diversity of ticks in these regions. A total of 370 representative ticks from the south and east regions of Kazakhstan (SERK) and Xinjiang Uygur Autonomous Region (XUAR) were selected for molecular comparison. A fragment of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, ranging from 631 bp to 889 bp, was used to analyze genetic diversity among these ticks. Phylogenetic analyses indicated 7 tick species including Hyalomma asiaticum, Hyalomma detritum, Hyalomma anatolicum, Dermacentor marginatus, Rhipicephalus sanguineus, Rhipicephalus turanicus and Haemaphysalis erinacei from the SERK clustered together with conspecific ticks from the XUAR. The network diagram of haplotypes showed that i) Hy. asiaticum from Almaty and Kyzylorda Oblasts together with that from Yuli County of XUAR constituted haplogroup H-2, and the lineage from Chimkent City of South Kazakhstan was newly evolved; and ii) the R. turanicus ticks sampled in Israel, Almaty, South Kazakhstan, Usu City, Ulugqat and Baicheng Counties of XUAR were derivated from an old lineage in Alataw City of XUAR. These findings indicate that: i) Hy. asiaticum, R. turanicus and Ha. erinacei shared genetic similarities between the SERK and XUAR; and ii) Hy. marginatum and D. reticulatus show differences in their evolution.

Testing for phylogenetic signal in claws suggests great influence of ecology on Caribbean intertidal arthropods (Acari, Oribatida).

Claws are common biological attachment devices that can be found in a wide variety of animal groups. Their curvature and size are supposed to be parameters related to ecological aspects. Mites, known as very small arthropods, occupy a wide range of ecological niches and are a perfect model system to investigate correlations of claw morphology with ecology. There is only one study regarding this question in littoral mites but the phylogenetic impact, which plays an important role in the evolution of morphological traits, was not tested. We investigated claw shapes of different Caribbean populations of five species showing different substrate/habitat preferences. We used geometric morphometrics to quantify claw shape and tested for phylogenetic signal within this morphological trait. Even in closely related populations, we found clear claw shapes for hard versus soft substrate, confirming previous findings. Surprisingly, we found no phylogenetic signal within the trait, which demonstrates that ecology (different surfaces and substrates) has acted as one of the primary selective forces in the diversification of claw shapes. Considering that the basic claw design may be the same in the majority of arthropods, our results have important implications for further investigations of claw morphology and its ecological relevance within this phylum.

Identification and Molecular Analysis of Ixodid Ticks (Acari: Ixodidae) Infesting Domestic Animals and Tick-Borne Pathogens at the Tarim Basin of Southern Xinjiang, China.

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

Genomic insights into mite phylogeny, fitness, development, and reproduction.

BACKGROUND: Predatory mites (Acari: Phytoseiidae) are the most important beneficial arthropods used in augmentative biological pest control of protected crops around the world. However, the genomes of mites are far less well understood than those of insects and the evolutionary relationships among mite and other chelicerate orders are contested, with the enigmatic origin of mites at one of the centres in discussion of the evolution of Arachnida. RESULTS: We here report the 173 Mb nuclear genome (from 51.75 Gb pairs of Illumina reads) of the predatory mite, Neoseiulus cucumeris, a biocontrol agent against pests such as mites and thrips worldwide. We identified nearly 20.6 Mb (~ 11.93% of this genome) of repetitive sequences and annotated 18,735 protein-coding genes (a typical gene 2888 bp in size); the total length of protein-coding genes was about 50.55 Mb (29.2% of this assembly). About 37% (6981) of the genes are unique to N. cucumeris based on comparison with other arachnid genomes. Our phylogenomic analysis supported the monophyly of Acari, therefore rejecting the biphyletic origin of mites advocated by other studies based on limited gene fragments or few taxa in recent years. Our transcriptomic analyses of different life stages of N. cucumeris provide new insights into genes involved in its development. Putative genes involved in vitellogenesis, regulation of oviposition, sex determination, development of legs, signal perception, detoxification and stress-resistance, and innate immune systems are identified. CONCLUSIONS: Our genomics and developmental transcriptomics analyses of N. cucumeris provide invaluable resources for further research on the development, reproduction, and fitness of this economically important mite in particular and Arachnida in general.

Molecular characterization and genetic diversity of Ornithonyssus sylviarum in chickens (Gallus gallus) from Hainan Island, China.

BACKGROUND: The northern fowl mite (NFM), Ornithonyssus sylviarum, is an obligatory hematophagous ectoparasite of birds and one of the most important pests in the poultry industry on several continents. Although NFM poses a serious problem, it remains a neglected pest of poultry in China and other Asian countries. Therefore, a molecular analysis was conducted to provide baseline information on the occurrence, genetic diversity and emergence of NFM in poultry farms from China. METHODS: This study focused on morphological description and identification of adults based on electron microscopy, molecular sequencing of the mitochondrial cox1 gene and phylogenetic analysis. We have also used the DNA sequences of the cox1 gene to study the genetic diversity, population structure and demographic history. The neutrality tests were used to analyze signatures of historical demographic events. RESULTS: The mites collected were identified as the northern fowl mite Ornithonyssus sylviarum based on external morphological characterization using electron microscopy. Molecular analysis using a 756-bp long partial fragment of the cox1 gene revealed 99-100% sequence identity with NFM and phylogenetic inferences showed a bootstrap value of 99% indicating a well-supported monophyletic relationship. Molecular diversity indices showed high levels of haplotype diversity dominated by private haplotypes, but low nucleotide divergence between haplotypes. The Tajima's D test and Fu's Fs test showed negative value, indicating deviations from neutrality and both suggested recent population expansion of mite populations supported by a star-like topology of the isolates in the network analysis. Our genetic data are consistent with a single introduction of NFM infestations and the spread of NFM infestation in Hainan poultry farms and a private haplotype dominance, which suggest that infestations are recycled within the farms and transmission routes are limited between farms. CONCLUSIONS: To our knowledge, this is the first time a molecular report of NFM in chicken from China including other Asian countries using DNA barcoding. The findings have potential implications with respect to understanding the transmission patterns, emergence and populations trends of parasitic infestations of poultry farms that will help for setting the parameters for integrated pest management (IPM) tactics against mite infestations.

Increasing species sampling in chelicerate genomic-scale datasets provides support for monophyly of Acari and Arachnida.

Chelicerates are a diverse group of arthropods, represented by such forms as predatory spiders and scorpions, parasitic ticks, humic detritivores, and marine sea spiders (pycnogonids) and horseshoe crabs. Conflicting phylogenetic relationships have been proposed for chelicerates based on both morphological and molecular data, the latter usually not recovering arachnids as a clade and instead finding horseshoe crabs nested inside terrestrial Arachnida. Here, using genomic-scale datasets and analyses optimised for countering systematic error, we find strong support for monophyletic Acari (ticks and mites), which when considered as a single group represent the most biodiverse chelicerate lineage. In addition, our analysis recovers marine forms (sea spiders and horseshoe crabs) as the successive sister groups of a monophyletic lineage of terrestrial arachnids, suggesting a single colonisation of land within Chelicerata and the absence of wholly secondarily marine arachnid orders.

Advancing mite phylogenomics: Designing ultraconserved elements for Acari phylogeny.

Mites (Acari) are one of the most diverse groups of life on Earth; yet, their evolutionary relationships are poorly understood. Also, the resolution of broader arachnid phylogeny has been hindered by an underrepresentation of mite diversity in phylogenomic analyses. To further our understanding of Acari evolution, we design targeted ultraconserved genomic elements (UCEs) probes, intended for resolving the complex relationships between mite lineages and closely related arachnids. We then test our Acari UCE baits in-silico by constructing a phylogeny using 13 existing Acari genomes, as well as 6 additional taxa from a variety of genomic sources. Our Acari-specific probe kit improves the recovery of loci within mites over an existing general arachnid UCE probe set. Our initial phylogeny recovers the major mite lineages, yet finds mites to be non-monophyletic overall, with Opiliones (harvestmen) and Ricinuleidae (hooded tickspiders) rendering Parasitiformes paraphyletic.

Suppression of Plant Defenses by Herbivorous Mites Is Not Associated with Adaptation to Host Plants.

Some herbivores suppress plant defenses, which may be viewed as a result of the coevolutionary arms race between plants and herbivores. However, this ability is usually studied in a one-herbivore-one-plant system, which hampers comparative studies that could corroborate this hypothesis. Here, we extend this paradigm and ask whether the herbivorous spider-mite Tetranychus evansi, which suppresses the jasmonic-acid pathway in tomato plants, is also able to suppress defenses in other host plants at different phylogenetic distances from tomatoes. We test this using different plants from the Solanales order, namely tomato, jimsonweed, tobacco, and morning glory (three Solanaceae and one Convolvulaceae), and bean plants (Fabales). First, we compare the performance of T. evansi to that of the other two most-commonly found species of the same genus, T. urticae and T. ludeni, on several plants. We found that the performance of T. evansi is higher than that of the other species only on tomato plants. We then showed, by measuring trypsin inhibitor activity and life history traits of conspecific mites on either clean or pre-infested plants, that T. evansi can suppress plant defenses on all plants except tobacco. This study suggests that the suppression of plant defenses may occur on host plants other than those to which herbivores are adapted.

A genomic approach to identify and monitor a novel pyrethroid resistance mutation in the redlegged earth mite, Halotydeus destructor.

Resistance mechanisms are typically uncovered by identifying sequence variation in known candidate genes, however this strategy can be problematic for species with no reference data in known relatives. Here we take a genomic approach to identify resistance to pyrethroids in the redlegged earth mite, Halotydeus destructor, a member of the Penthalidae family of mites that are virtually uncharacterized genetically. Based on shallow genome sequencing followed by a genome assembly, we first identified contigs of the H. destructor parasodium channel gene. By linking variation in this gene to known resistant phenotypes, we located a single nucleotide polymorphism in resistant mites. This polymorphism results in a leucine (L) to phenylalanine (F) amino acid substitution in the II6 region (predicted) of the gene (L1024F). This novel mutation has not previously been linked to pyrethroid resistance, although other polymorphisms have been identified in the two-spotted spider mite, Tetranychus urticae at the same locus (L1024V). The sequencing approach was successful in generating a candidate polymorphism that was then validated using laboratory bioassays and field surveys. A high throughput Illumina-based sequencing diagnostic was developed to rapidly assess resistance allele frequencies in pools of mites sourced from hundreds of populations across Australia. Resistance was confirmed to be widespread in the southern wheatbelt region of Western Australia. Two different resistance mutations were identified in field populations, both resulting in the same amino acid substitution. The frequency and distribution of resistance amplicon haplotypes suggests at least two, and probably more independent origins of resistance.

Gene cloning and difference analysis of vitellogenin in Neoseiulus barkeri (Hughes).

Neoseiulus barkeri (HUGHES) is the natural enemy of spider mites, whiteflies and thrips. Screening for chemically-resistant predatory mites is a practical way to balance the contradiction between the pesticide using and biological control. In this study, the number of eggs laid by fenpropathrin-susceptible and resistant strains of N. barkeri was compared. Additionally, we cloned three N. barkeri vitellogenin (Vg) genes and used quantitative real-time polymerase chain reaction to quantify Vg expression in susceptible and resistant strains. The total number of eggs significantly increased in the fenpropathrin-resistant strain. The full-length cDNA cloning of three N. barkeri Vg genes (NbVg1, NbVg2 and NbVg3) revealed that the open reading frames of NbVg1, NbVg2 and NbVg3 were 5571, 5532 and 4728 bp, encoding 1856, 1843 and 1575 amino acids, respectively. The three N. barkeri Vg possessed the Vitellogenin-N domain (or lipoprotein N-terminal domain (LPD_N)), von Willebrand factor type D domain (VWD) and the domain with unknown function 1943 (DUF1943). The NbVg1 and NbVg2 expression levels were significantly higher in the resistant strain than in the susceptible strain, while the NbVg3 expression level was lower in the resistant strain. Thus, we speculate that the increased number of eggs laid by the fenpropathrin-resistant strain of N. barkeri may be a consequence of changes in Vg gene expression.

RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: A comparative analysis of five methods for gene silencing.

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health.