Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens.

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

TITLE: Caractérisation d'une cystéine protéase des poux rouges de la volaille et son utilisation potentielle comme vaccin pour les poulets. ABSTRACT: Les acariens communément appelés poux rouges de la volaille (PRV, Dermanyssus gallinae) sont des ectoparasites qui affectent négativement les poulets d'élevage, entraînant de graves pertes économiques au niveau mondial. Des acaricides ont été utilisés pour contrôler les PRV dans les poulaillers. Cependant, certains PRV ont développé une résistance aux acaricides, et par conséquent, différentes approches sont nécessaires pour gérer les problèmes qu'ils causent. La vaccination des poulets est l'une des méthodes envisagées pour réduire le nombre de PRV dans les poulaillers. Dans une étude précédente, une cystéine protéase, Deg-CPR-1, a été identifiée comme un vaccin candidat contre les PRV distribués en Europe. Dans cette étude, nous avons étudié les caractéristiques de Deg-CPR-1. L'analyse phylogénétique a révélé que Deg-CPR-1 est étroitement liée aux cystéine protéases digestives d'autres espèces d'acariens, et elle a été classée dans un groupe différent de celui des cathepsines de poulet. La Deg-CPR-1 des PRV au Japon a une substitution d'acide aminé par rapport à celle des PRV en Europe, mais elle a montré une efficacité en tant que vaccin, conformément aux résultats précédents. Deg-CPR-1 a présenté une activité enzymatique de type cathepsine L. De plus, l'ARNm de Deg-CPR-1 était exprimé dans l'intestin moyen et à tous les stades où les PRV se nourrissent de sang. Ces résultats impliquent que Deg-CPR-1 dans l'intestin moyen peut avoir des fonctions importantes dans les processus physiologiques, et que l'inhibition de son expression peut contribuer à l'efficacité d'un vaccin basé sur Deg-CPR-1. Des recherches supplémentaires sont nécessaires pour comprendre pleinement les mécanismes de l'efficacité du vaccin.

Short term transcriptional responses of P450s to phytochemicals in insects and mites.

Cytochrome P450 monooxygenases (P450s) play a key role in the detoxification of phytochemicals in arthropod herbivores. We present here an overview of recent progress in understanding the breadth and specificity of gene expression plasticity of P450s in response to phytochemicals. We discuss experimental setups and new findings in mechanisms of P450 regulation. Whole genome transcriptomic analysis of arthropod herbivores, either after direct administration of phytochemicals or after host plant shifts, allowed to integrate various levels of chemical complexity and lead to the unbiased identification of responsive P450 genes. However, despite progress in identification of inducible P450s, the link between induction and metabolism is still largely unexplored, and to what extent the overall response is biologically functional should be further investigated. In the near future, such studies will be more straightforward as forward and reverse genetic tools become more readily available.

Molecular and biochemical characterization of enolase from Dermanyssus gallinae.

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305 bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae.

Integrative taxonomy of Abacarus mites (Eriophyidae) associated with hybrid sugarcane plants, including description of a new species.

Phytophagous mites belonging to the Eriophyoidea are extremely diverse and highly host-specific. Their accurate morphological identification is hampered by their reduced size and simplified bodies and by the existence of cryptic species complexes. Previous studies have demonstrated the urgency of applying multisource methods to accurate taxonomic identification of eriophyoid mites, especially species belonging to the genus Abacarus. This genus comprises 65 species, of which 37 are associated with grasses and four with sugarcane Saccharum (Poaceae). Recently, Abacarus specimens very similar to Abacarus sacchari were collected from the sugarcane crop in Brazil; however, their taxonomic placement was uncertain. In this study, we used an integrative approach to determine whether A. aff. sacchari specimens belong to A. sacchari or constitute a cryptic species. Morphological data were combined with molecular phylogeny based on the nucleotide sequences of three markers, one mitochondrial (COI) and two nuclear (D2 region of 28S and ITS). Morphological differences were observed between A. aff. sacchari, A. sacchari and A. doctus. The phylogenetic relationships among these three taxa and the genetic distances separating them revealed an interspecific divergence. The results of the morphological and molecular methods were congruent and supported the existence of a new species: Abacarus neosacchari n. sp. Duarte and Navia, herein described. This species belongs to the Abacarus cryptic species complex associated with sugarcane in the Americas. The results of this study, presenting the occurrence of multiple Abacarus species associated with sugarcane, contribute to the knowledge on plants and mites diversity by adding up one more clue highlighting that plant hybridization can be an important mechanism contributing to the speciation of plant-feeding arthropods.

Effects of UV-B radiation on the survival, egg hatchability and transcript expression of antioxidant enzymes in a high-temperature adapted strain of Neoseiulus barkeri.

Biological control of spider mites in hot and dry weather is a serious technical issue. A high-temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes was selected from its conventional strain (CS), via long-term heat acclimation and frequent heat hardenings in our previous studies. However, the environment of high temperature is usually associated with enhanced ultraviolet (UV) radiation. In the present study, the physiological effects of UV-B radiation on survival rate and egg damage of N. barkeri were investigated, as well as the activities and expression profiles of antioxidant enzymes to UV-B radiation stress. UV-B radiation had deleterious effects on egg hatchability and survival of N. barkeri. Adults of the HTAS strain were less UV-B resistant than those of the CS strain; they also had lower levels of enzymatic activity of superoxide dismutase (SOD) and catalase against oxidative damage and weaker upregulation of SOD genes. The mRNA expression of three SOD genes of CS adult females immediately increased whereas that of HTAS showed almost no difference under UV-B stress for 1 h. The results showed the HTAS of N. barkeri had lower fitness under UV-B stress compared with the CS of N. barkeri. These results suggested that long-term heat acclimation may exert a profound impact on the developmental physiology of N. barkeri.

Proximate mechanisms of the differences in reproductive success of males bearing different alleles of Pgdh - a gene involved in a sexual conflict in bulb mite.

Enzyme polymorphism in phosphogluconate dehydrogenase (Pgdh) is a striking example of single gene polymorphism involved in sexual conflict in bulb mite Rhizoglyphus robini. Males homozygous for the S Pgdh allele were shown to achieve higher reproductive success than FF homozygous males, while negatively influencing fecundity of their female partners. Here, we investigate proximate mechanisms responsible for the increased reproductive success of SS males and find that the S allele is associated with shorter time until copulation, higher copulation frequency and increased sperm production. We also show that Pgdh alleles are probably codominant, with SS males gaining the highest reproductive success, FF males - the lowest - and FS-heterozygous males taking an intermediate position in all fitness parameters differentiating males of different genotypes. Additionally, we confirm the negative effect that S-bearing males impose on the fecundity of females they mate with, showing a clear pattern of interlocus sexual conflict. We discuss that this effect is probably associated with increased copulation frequency. Whereas, contrary to what we have predicted, the S allele does not cause increased general male mobility, we speculate that the S allele-bearing males are more efficient in forcing copulation and/or detecting females.

The genetic structure of hypoderatid mites (Actinotrichida: Astigmata) parasitizing great cormorant (Phalacrocorax carbo) during host post-breeding dispersal in Milicz, SW Poland.

The astigmatid family Hypoderatidae includes over 80 mite species exhibiting peculiar life cycle. Deutonymphs are parasitic instars inhabiting subcutaneous or visceral tissues of birds and rodents, while all other instars are nidicolous forms. In this study we investigated genetic diversity of deutonymphs of two hypoderatid species, Neottialges evansi and Phalacrodectes gaudi, collected from 16 individuals of the great cormorant Phalacrocorax carbo in SW Poland during host post-breeding dispersal. The initial alternative hypotheses were: (1) populations of deutonymphs in both mite species found in tissues of particular bird individuals form genetically distinct populations, (2) mites are panmictic among hosts. The topologies of NJ phylogenetic trees and median-joining haplotype networks reconstructed for COI haplotypes revealed lack of hostdependent genetic structure in populations of N. evansi and P. gaudi. Furthermore, high haplotype diversity (Hd) and low nucleotide diversity (Pi) prove high genetic differentiation of both mite species. We concluded, that unlimited dispersal of mites among cormorant specimens could be explained by host specific breeding behavior: free mating between cormorants originating from different European populations and low contribution of reunited pairs in breeding colonies in subsequent breeding seasons, reuse of the same nest material by different members of the colony, and host behavior called prospecting.

Analysis of Genetic Variation in Brevipalpus yothersi (Acari: Tenuipalpidae) Populations from Four Species of Citrus Host Plants.

We studied species diversity and genetic variation among populations of Brevipalpus mites from four species of citrus host plants. We sampled mites on orange, lime, grapefruit and mandarin trees from orchards at six localities distributed in the five most important citrus producing states in Mexico. Genetic variation among citrus host plants and localities were assessed by analysis of nucleotide sequence data from fragments of the mitochondrial cytochrome oxidase subunit I (COI). Both Brevipalpus yothersi and B. californicus were found at these sites, and B. yothersi was the most abundant species found on all citrus species and in all localities sampled. B. californicus was found mainly on orange and mandarin and only in two of the states sampled. AMOVA and haplotype network analyses revealed no correlation between B. yothersi genetic population structure and geographical origin or citrus host plant species. Considering that a previous study reported greater genetic diversity in B. yothersi populations from Brazil than we observed in Mexico, we discuss the possibility that the Mexican populations may have originated in the southern region of America.

Involvement of Three Esterase Genes from Panonychus citri (McGregor) in Fenpropathrin Resistance.

The citrus red mite, Panonychus citri (McGregor), is a major citrus pest with a worldwide distribution and an extensive record of pesticide resistance. However, the underlying molecular mechanism associated with fenpropathrin resistance in this species have not yet been reported. In this study, synergist triphenyl phosphate (TPP) dramatically increased the toxicity of fenpropathrin, suggesting involvement of carboxylesterases (CarEs) in the metabolic detoxification of this insecticide. The subsequent spatiotemporal expression pattern analysis of PcE1, PcE7 and PcE9 showed that three CarEs genes were all over-expressed after insecticide exposure and higher transcripts levels were observed in different field resistant strains of P. citri. Heterologous expression combined with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay in Spodoptera frugiperda (Sf9) cells revealed that PcE1-, PcE7- or PcE9-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against fenpropathrin, demonstrating that PcEs probably detoxify fenpropathrin. Moreover, gene silencing through the method of leaf-mediated dsRNA feeding followed by insecticide bioassay increased the mortalities of fenpropathrin-treated mites by 31% (PcE1), 27% (PcE7) and 22% (PcE9), respectively, after individual PcE gene dsRNA treatment. In conclusion, this study provides evidence that PcE1, PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P. citri and enrich molecular understanding of CarEs during the resistance development of the mite.

The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis.

Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus) occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J' and J" clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J' and J" clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis.

Characteristics of carboxylesterase genes and their expression-level between acaricide-susceptible and resistant Tetranychus cinnabarinus (Boisduval).

Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation.

Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins.

BACKGROUND: Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry. METHODS: A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, -2 and -3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, -2 and -3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs. RESULTS: 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and -3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17% respectively. Phoxim also inhibited rDeg-2-GST (14.8%) and rDeg-GST-3 (20.6%) activities. CONCLUSIONS: Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite, Neoseiulus cucumeris (Acari: Phytoseiidae).

Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Thermal stress responses are usually associated with the increased generation of reactive oxygen species (ROS), resulting in oxidative damage. In this study, we examined the time-related effect (durations for 1, 2, 3, and 5 h) of thermal stress conditions-i.e., relatively low (0, 5, 10, and 15 °C) or high (35, 38, 41, and 44 °C) temperatures-on the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), and total antioxidant capacity (T-AOC) of the predatory mite Neoseiulus cucumeris. Also the lipid peroxidation (LPO) levels of the predatory mite were measured under thermal stress conditions. The results confirmed that thermal stress results in a condition of so-called oxidative stress and the four antioxidant enzymes play an important role in combating the accumulation of ROS in N. cucumeris. CAT and POX activity changed significantly when the mites were exposed to cold and heat shock, respectively. The elevated levels of SOD and GSTs activity, expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. However, the levels of LPO in N. cucumeris were high, serving as an important signal that these antioxidant enzyme-based defense mechanisms were not always adequate to counteract the surplus ROS. Thus, we hypothesize that thermal stress, especially extreme temperatures, may contribute much to the generation of ROS in N. cucumeris, and eventually to its death.

Effects of heat stress on development, reproduction and activities of protective enzymes in Mononychellus mcgregori.

Mononychellus mcgregori is a pest mite of cassava. Since its invasion into China in 2008 it has spread rapidly. In order to determine the potential distribution and to analyze its invasion, diffusion and ecological adaptation mechanisms, we investigated the effect of high-temperature stress (30, 33, 36, 39 and 42 °C) on its development and reproduction, and the activity of protective enzymes in the mite. The results indicated significant influences: (1) adults could not lay eggs after they had been exposed to 42 °C for 4 h or longer; (2) egg development was slower and egg hatchability decreased after exposure of adults to 33-42 °C for 1 h; (3) offspring development (all stages) was slower after exposure of adults to 33-42 °C for 2 h or more; and (4) polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) activities in the adults increased to high levels after exposure to 33-42 °C for 1 h, and superoxide dismutase activity increased only after exposure to 42 °C for 1 h. In conclusion, exposure to high temperatures for only 1 h probably has an important impact on the mite's population growth. The significant increase of PPO, POD, APX, and CAT activities in adults may partially explain how M. mcgregori survive exposure to a relatively high temperature.

Northern fowl mite (Ornithonyssus sylviarum) in Sweden.

Haematophagous mites were collected from the vent region and plumage of chickens in six hobby flocks of ornamental breeds in Sweden, one of which included turkeys. Soiled vent skin and feathers, dermatitis, hyperkeratosis, skin necroses and ulcers were observed in 12 necropsied birds from two of the flocks. The mites were identified as the northern fowl mite Ornithonyssus sylviarum (Mesostigmata: Macronyssidae). This was supported by sequence analysis of a 642-bp region in the mitochondrial cytochrome oxidase subunit 1 (COI) gene (COI) in mites collected from five flocks, which showed 97-99% sequence similarity to O. sylviarum by blast analysis. Pairwise sequence comparisons revealed nucleotide variations in the range of 0-2.8%, whereas amino acid sequences were highly conserved. This paper represents one of very few records of O. sylviarum in European poultry, and is the first to report COI sequence data for O. sylviarum from poultry in Europe.

Determination of the inheritance, cross-resistance and detoxifying enzyme levels of a laboratory-selected, spiromesifen-resistant population of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae).

BACKGROUND: Neoseiulus californicus of the family Phytoseiidae is an effective predatory mite species that is used to control pest mites. RESULTS: The LC50 and LC60 values of spiromesifen were determined on N. californicus using a leaf-disc method and spraying tower. A laboratory selection population designated SPR13 was found to have a 52.08-fold resistance to spiromesifen following 13 selection cycles. This population developed low resistance to hexythiazox and moderate cross-resistance to propargite, clofentezine, spirodiclofen, etoxazole and milbemectin. PBO, IBP and DEM synergised resistance 3.75-, 2.54- and 1.93-fold respectively. Crossing experiments revealed that spiromesifen resistance in the SPR13 population was intermediately dominant and monogenic. In addition, detoxifying enzyme activities were increased 2.74-fold for esterase, 3.09-fold for glutathione S-transferase and 2.17-fold for cytochrome P450 monooxygenase in the SPR13 population. CONCLUSIONS: Selection for spiromesifen under laboratory conditions resulted in the development of spiromesifen resistance in the predatory mite N. californicus. Predatory mites that are resistant to pesticides are considered valuable for use in resistance management programmes within integrated pest control strategies.

Identification and characterization of seven glutathione S-transferase genes from citrus red mite, Panonychus citri (McGregor).

The citrus red mite, Panonychus citri (McGregor), is a global citrus pest, and has developed severe resistance to several types of acaricides. However, the molecular mechanisms of resistance in this mite remain unknown. In this study, seven full-length cDNAs encoding glutathione S-transferases (GSTs) genes were identified and characterized in P. citri. The effects of pyridaben and fenpropathrin exposure on the expression of these genes were also investigated. Phylogenetic analysis revealed that the seven GSTs genes in P. citri cloned in this study belong to three different cytosolic classes, including four in mu, two in delta and one in zeta. Among these seven GSTs genes, the relative expression level of PcGSTm1 was significantly higher in adult than in the other life stages (egg, larvae and nymph). Compared with the control, the mRNA levels of the seven GST genes did not change significantly following exposure to pyridaben at LC10. However, RT-qPCR results showed that, when exposed to LC10 of fenpropathrin, six GSTs gene (PcGSTm1, PcGSTm3, PcGSTm4, PcGSTd1, PcGSTd2 and PcGSTz1) transcripts increased in a time-dependent manner. This is the first insight into the molecular characteristics of GSTs gene cDNAs in P. citri. The elevated GSTs gene transcripts following exposure to fenpropathrin might be one of the mechanisms involved in detoxification of this acaricide.

Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences.

For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum. Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids.

Intestinal proteases of free-living and parasitic astigmatid mites.

Among arthropod pests, mites are responsible for considerable damage to crops, humans and other animals. However, detailed physiological data on these organisms remain sparse, mainly because of their small size but possibly also because of their extreme diversity. Focusing on intestinal proteases, we draw together information from three distinct mite species that all feed on skin but have separately adapted to a free-living, a strictly ecto-parasitic and a parasitic lifestyle. A wide range of studies involving immunohistology, molecular biology, X-ray crystallography and enzyme biochemistry of mite gut proteases suggests that these creatures have diverged considerably as house dust mites, sheep scab mites and scabies mites. Each species has evolved a particular variation of a presumably ancestral repertoire of digestive enzymes that have become specifically adapted to their individual environmental requirements.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites.

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi'an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi'an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%-99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.