Point mutations in the voltage-gated sodium channel gene conferring pyrethroid resistance in China populations of the Dermanyssus gallinae.

BACKGROUND: Dermanyssus gallinae, the poultry red mite (PRM), is a worldwide ectoparasite posing significant economic challenges in poultry farming. The extensive use of pyrethroids for PRM control has led to the emergence of pyrethroid resistance. The objective of this study is to detect the pyrethroid resistance and explore its associated point mutations in the voltage-gated sodium channel (VGSC) gene among PRM populations in China. RESULTS: Several populations of D. gallinae, namely CJF-1, CJP-2, CJP-3, CSD-4 and CLD-5, displayed varying degrees of resistance to beta-cypermethrin compared to a susceptible field population (CBP-5). Mutations of VGSC gene in populations of PRMs associated with pyrethroid resistance were identified through sequencing its fragments IIS4-IIS5 and IIIS6. The mutations I917V, M918T/L, A924G and L925V were present in multiple populations, while no mutations were found at positions T929, I936, F1534 and F1538. CONCLUSION: The present study confirmed the presence of extremely high levels of pyrethroid resistance in PRM populations in China, and for the first time detected four pyrethroid resistance mutations in the VGSC gene. Identifying pyrethroid resistance in the field population of PRM in China can be achieved through screening for VGSC gene mutations as an early detection method. Our findings underscore the importance of implementing chemical PRM control strategies based on resistance evidence, while also considering the management of acaricide resistance in the control of PRMs. © 2024 Society of Chemical Industry.

Self-nanomicellizing solid dispersion: A promising platform for oral drug delivery.

Amorphous solid dispersion (ASD) has been widely used to enhance the oral bioavailability of water-insoluble drugs for oral delivery because of its advantages of enhancing solubility and dissolution rate. However, the problems related to drug recrystallization after drug dissolution in media or body fluid have constrained its application. Recently, a self-nanomicellizing solid dispersion (SNMSD) has been developed by incorporating self-micellizing polymers as carriers to settle the problems, markedly improving the ability of supersaturation maintenance and enhancing the oral bioavailability of drug. Spontaneous formation and stability of the self-nanomicelle (SNM) have been proved to be the key to supersaturation maintenance of SNMSD system. This offers a novel research direction for maintaining supersaturation and enhancing the bioavailability of ASDs. To delve into the advantages of SNMSDs, we provide a concise review introducing the formation mechanism, characterization methods and stability of SNMs, emphasizing the advantages of SNMSDs for oral drug delivery facilitated by SNM formation, and discussing relevant research prospects.

Identification and transcriptional response of ATP-binding cassette transporters to beta-cypermethrin in the poultry red mite, Dermanyssus gallinae.

Dermanyssus gallinae, a worldwide pest in birds, has developed varying degrees of resistance to insecticides. The ATP-binding cassette (ABC) transporters are essential for the removal of xenobiotics from arthropods. However, our knowledge about ABC transporter proteins in D. gallinae is limited. Forty ABC transporters were identified in the transcriptome and genome of D. gallinae. The resistant population displayed an augmented metabolic rate for beta-cypermethrin compared to the susceptible group, with a remarkable increase in the content of ABC transporters. Verapamil was found able to increase the toxicity of beta-cypermethrin in the resistant population. Results from qRT-PCR analysis showed that eleven ABC transcripts were more highly expressed in the resistant population than the susceptible group at all stages of development, and beta-cypermethrin was observed to be able to induce the expression of DgABCA5, DgABCB4, DgABCD3, DgABCE1 and DgABCG5 in D. gallinae. RNAi-mediated knockdown of the five genes was observed to increase the susceptibility of resistant mites to beta-cypermethrin. These results suggest that ABC transporters, DgABCA5, DgABCB4, DgABCD3, DgABCE1 and DgABCG5 genes, may be related to beta-cypermethrin resistance in D. gallinae. This research will serve as a foundation for further studies on mechanism of insecticide resistance, which could be beneficial for controlling D. gallinae.

Vitellogenin and its upstream gene TOR play essential roles in the reproduction of Dermanyssus gallinae.

In Dermanyssus gallinae, a hematophagous mite, the initiation of vitellogenesis induced by blood feeding is essential for its reproduction. However, the precise gene structures and physiological functions of Vg in D. gallinae and its upstream gene, Target of Rapamycin (TOR), have not been fully understood. This study revealed the presence of four homologous genes within D. gallinae, named Dg-Vg1, Dg-Vg1-like, Dg-Vg2, and Dg-Vg2-like, especially, Dg-Vg2-like was firstly identified in the mites. The expression levels of all these Vg genes were significantly higher in adult females than other stages. Following blood feeding, the expression levels of these genes increased significantly, followed by a subsequent decrease, aligning with egg production. Silencing Dg-Vgs by RNA interference (RNAi) led to decreased fecundity and egg hatching rates, as well as abnormal embryonic development, suggesting a vital role for Dg-Vgs in both egg formation and embryonic development. Furthermore, the knockdown of Dg-TOR significantly reduced the expression of Dg-Vgs and negatively impacted the reproductive capabilities of PRMs, indicating that TOR influences PRM reproduction by regulating the expression of Dg-Vgs. In summary, these findings demonstrated the crucial roles of Dg-Vgs and Dg-TOR in PRM reproduction, highlighting their potential as targets for pest control.

Activation of CncC pathway by ROS burst regulates ABC transporter responsible for beta-cypermethrin resistance in Dermanyssus gallinae (Acari:Dermanyssidae).

The drug resistance of poultry red mites to chemical acaricides is a global issue in the control of the mites, which presents an ongoing threat to the poultry industry. Though the increased production of detoxification enzymes has been frequently implicated in resistance development, the overexpression mechanism of acaricide-resistant related genes in mites remains unclear. In the present study, it was observed that the transcription factor Cap 'n' Collar isoform-C (CncC) and its partner small muscle aponeurosis fibromatosis (Maf) were highly expressed in resistant strains compared to sensitive strains under the stress of beta-cypermethrin. When the CncC/Maf pathway genes were down-regulated by RNA interference (RNAi), the expression of the ABC transporter genes was down-regulated, leading to a significant increase in the sensitivity of resistant strains to beta-cypermethrin, suggesting that CncC/Maf played a crucial role in mediating the resistance of D.gallinae to beta-cypermethrin by regulating ABC transporters. Furthermore, it was observed that the content of H2O2 and the activities of peroxidase (POD) and catalase (CAT) enzymes were significantly higher in resistant strains after beta-cypermethrin stress, indicating that beta-cypermethrin activates reactive oxygen species (ROS). In ROS scavenger assays, it was found that the expression of CncC/Maf significantly decreased, along with a decrease in the ABC transporter genes. The present study showed that beta-cypermethrin seemed to trigger the outbreak of ROS, subsequently activated the CncC/Maf pathway, as a result induced the ABC transporter-mediated resistance to the drug, shedding more light on the resistance mechanisms of D.gallinae to pyrethroids.

Toltrazuril alkalizer-modifying solid dispersions against Toxoplasma gondii: A pharmacotechnical strategy to improve the efficacy of the drug.

Toxoplasma gondii is a zoonotic protozoan that can parasitize nucleated cells of all warm-blooded animals, and seriously harm human and livestock. Toltrazuril (TOL) has insecticidal activity against parasites of the phylum Apicomplexan at multiple development stages, but the clinical application is limited by its poor water solubility. To improve the dissolution of TOL, nine ternary solid dispersions (SD) were prepared with PEG6000 as the carrier and various alkalizers as the pH modifier. Compared with the binary SD, all ternary SDs had improved TOL dissolution although dissolution rates differed. The complete dissolution was achieved for the Ca(OH)2-SD, associated with a gradual release of the alkalizer and adequate pH regulation of the microenvironment. DSC, PXRD and FTIR analyses indicated that TOL in the Ca(OH)2-SD was present in an amorphous form and had a strong hydrogen bond with Ca(OH)2. Within the drug concentration of 100 µg/mL, Ca(OH)2-SD was proved to have no damage to host cells by in vitro cytotoxicity analysis, and its anti-T. gondii efficacy was significantly higher than that of TOL and binary SD. The in vivo efficacy of Ca(OH)2-SD against T. gondii in mice further confirmed that Ca(OH)2-SD could be used as a new strategy to prevent T. gondii from killing mice and treat toxoplasmosis. In conclusion, Ca(OH)2-SD is expected to eventually turn into a clinical candidate for toxoplasmosis treatment in the future.

Acaricidal activity of bioactive compounds isolated from Aspergillus oryzae against poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae).

Dermanyssus gallinae, the poultry red mite (PRM), is an obligate ectoparasite feeding on poultry blood, seriously affecting the health of layers and egg production. The control of PRMs mainly relies on chemical drugs, which is facing several challenges such as the environment pollution and drug resistance. Using fungal metabolites is an environmentally friendly alternative for the control of pests. However, few studies have been conducted on the efficacy of fungal metabolites against D. gallinae. In this study, five strains of fungi were isolated from D. gallinae under laboratory conditions, and their extracts with ethyl acetate were tested for acaricidal activity on D. gallinae. The crude extract of Aspergillus oryzae caused 75.55 ± 6.94% mortality of mites at a concentration of 12.5 mg/mL, showing the highest acaricidal effect in all extracts. Subsequently, the extract of A. oryzae was isolated by bio-guided fractionation, and ten major compounds were identified by LC-MS/MS analysis. The results of bioassays indicated that five compounds exhibited acaricidal activity against D. gallinae. N, N-dimethyldecylamine N-oxide was the optimal acaricidal compound with LC50 of 0.568 mg/mL. Additionally, palmitic acid, triethanolamine, cuminaldehyde, and 2,4-dimethylbenzaldehyde also showed acaricidal activity. These compounds have great application potential in the mite control, and the analysis of these fungal acaricidal substances provides a new idea and basis for the subsequent development of PRM control technology.

Function of glycogen synthase kinase3 in embryogenesis of Dermanyssus gallinae.

In the life cycle of Dermanyssus gallinae, the embryo is a developmental stage that does not require blood meals, but needs glucose to produce adenosine triphosphate (ATP) through glycolysis or oxidative phosphorylation, providing energy for embryonic development. Glycogen synthase kinase 3 (GSK3), belonging to the serine/threonine kinase family, is a key enzyme involved in glycogen metabolism in many eukaryotes, but not be described in D. gallinae. The present study was conducted to explore the role of Dg-GSK3 in the embryogenesis of D. gallinae. The results of qPCR showed that Dg-GSK3 mRNA was expressed in different development stages of D. gallinae embryos. RNA interference (RNAi) was performed on the female mites and eggs by immersion, and it was found that lowering GSK3 expression level could significantly decrease the female egg laying rate and egg hatching rate (P < 0.05). Some eggs became shrunken and shriveled in appearance. The fecundity of female D. gallinae obtained from the rDg-GSK3-immunized group of chickens (2.56 ± 0.35 eggs per mite, P < 0.0001) decreased significantly from that of the control group (3.49 ± 0.35). The oviposition rate of rDg-GSK3-immunized group (75.94 ± 7.28 %, P = 0.0003）was significantly lower that of the control group (89.69 ± 2.63 %). In conclusion, Dg-GSK3 is a crucial gene during the embryogenesis of D. gallinae, which can affect both the female fecundity and the egg hatching, which help us understand the function of GSK3 gene in the embryogenesis of mites.

Sex dimorphism in the deutonymphs of Dermanyssus gallinae (De Geer, 1778) based on geometric morphometrics.

The lifecycle of poultry red mite (PRM), Dermanyssus gallinae,includes several stages and only the adult has been reported to have sex discrimination based on body structures and color patterns. Currently, it's still unknown how to distinguish two sexes of deutonymphs. We measured body length of 254 engorged deutonymphs and examined body size and shape variation of 104 engorged deutonymphs using geometric morphometric techniques. Our findings showed that deutonymph females (with average values of 813.08 µm) had a longer body length than deutonymph males (713.39 µm). Additionally, deutonymph females were found to had a narrow and elongated posterior body shape while deutonymph males had a suboval shape, and the former was bigger than the latter. These results suggest that there is sexual dimorphism in PRM deutonymphs, and the differentiation of deutonymph females and males based on their body length, shape, and size will facilitate a better understanding of reproductive behavior and the accurate population dynamics of PRMs.

Microenvironment pH modified solid dispersion of Toltrazuril as a new strategy to improve the treatment of experimental Apicomplexan infection.

The phylum Apicomplexa contains some of the most serious human and veterinary parasites, including Eimeria magna, Toxoplasma gondii, and many others. Toltrazuril (TOL) has activity against multiple stages of Apicomplexan parasites, but its clinical use is limited by low bioavailability. In present study, we prepared one new formulation named the microenvironment pH modified solid dispersion (pHM-SD), which was composed of three components including Ca(OH)2, TOL, and PVPk30 with the weight ratio of 1:8:8. In vivo evaluation for bioavailability and efficacy of the pHM-SD was conducted following oral administration and hypodermic injection. The performance of the pHM-SD was also contrast to corresponding results of raw material drug and commercial Baycox® to evaluate the advantages for clinical application. The results showed that the bioavailability of prototype TOL and its active metabolites toltrazuril sulfoxide (TOLSO), toltrazuril sulfone (TOLSO2) in rabbits were improved remarkably after oral administration of the pHM-SD. The safety of the pHM-SD via oral administration was adequately verified via the histopathological examination. We subsequently evaluated effects of the pHM-SD on Eimeria magna oocysts and Toxoplasma gondii tachyzoites. In vivo anti-coccidia efficacy further confirmed that the pHM-SD could be used as a strategy to minimize the oocyst exposure. In vitro cytotoxicity and anti-Toxoplasma tests showed that the pHM-SD had little damage to host cells within the concentration of 100 µg/ mL, and the anti-Toxoplasma efficacy was significantly improved compared with TOL. Combined with the above-mentioned experimental results, we conclude that the pHM-SD maybe a promising candidate for providing better clinical outcomes.

Identification of guanine and hematin as arrestment pheromones of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae) and their application in mite control.

Dermanyssus gallinae, also known as the poultry red mite (PRM), is one of widespread ectoparasite in the poultry industry worldwide, causing direct and indirect detriments to poultry as well as substantial financial losses. Novel control methods are urgently needed to improve the current acaricide-based control of D. gallinae. The control approach based on arrestment pheromone is environment-friendly but the related research is limited in PRMs. In the present study we found two compounds from the mite feces acting as arrestment pheromones of D. gallinae, which could lead to mite arrestment upon contact. One is guanine, which was also found in unfed female mites' acid-saline extract. The other is hematin. Moreover, it was found that the ferric ion of hematin played a pivotal role in stimulating the arrestment of mites. Finally, it was found the combination of guanine or/and hematin plus cypermethrin led to significantly improved mite-killing performance compared with cypermethrin, showing a promising potential of novel control method based on the arrestment pheromone.

A new method using quail (Coturnix coturnix) as a suitable host for laboratory rearing of Dermanyssus gallinae.

The poultry red mite (PRM) is a detrimental blood-feeding ectoparasite infesting poultry and sylvatic birds. A traditional rearing system of PRMs was usually established by using chickens as the host. However, the system with chickens had some defects, such as high feed consumption, large amount of feces, high cost, and intensive labor. In present study, we used quails as the host to rear mites, and compared the reproductive efficiency of this new system with that of the chicken system. The results showed that the number of mites increased 96-fold and the number of eggs increased 50-fold after four weeks in the quail system, which were significantly higher than those in the system with chickens. The survival rates of mites in both systems were higher than 95%, and the value in the quail system at the fourth week was significantly better than that of the chicken system. The statistical results of feed consumption, live weight, and daily excrement of chickens and quails showed that the quail system was more stable with less feed consumption and less waste excretion than the chicken system. Comparing the number of eggs laid by each female mite, hatching rates and molting rates in both systems, we can conclude that the breeding system, in which quails replaced chickens as hosts, had no effect on the fecundity of D. gallinae. In conclusion, the new system established using quails as the host, is a highly efficient alternative for largely rearing of mites under laboratory conditions.

A blood digestion scoring method for poultry red mites, Dermanyssus gallinae.

The poultry red mite (PRM), Dermanyssus gallinae, is one of the most detrimental ectoparasite on poultry farms worldwide. The blood fed on birds provides the mites with nutrition and energy for their activities, development and reproduction. In the evaluation of the efficacy of novel drugs or vaccines against PRMs, their effects on blood digestion are generally used as a key parameter. The blood digestion of haematophagous arthropods (including D. gallinae) is usually assessed by weighing; however, this method shows some limitations. The main objective of the present study was to develop a scoring method that can quickly and visually evaluate the blood digestion status of PRMs. A 0­4 point scoring criterion was established to describe the blood digestion status of D. gallinae based on the changes in appearance in the intestinal tract of PRMs during the blood digestion process. There was a good consistency between the results obtained by the blood digestion scoring and the weighing, indicating the reliability of this new method. The results obtained from volunteers were consistent with the results from researchers with low coefficient of variation, indicating that the scoring method has good practicability. The applicability of the scoring method was confirmed in an efficacy study, where it was found that doramectin could significantly inhibit the blood digestion of PRMs, lowering the blood digestion score.

[Isolation, identification and biodegradation characterization of a novel monensin-degrading bacterial strain].

Biodegradation of antibiotic pollutants by microorganisms has received widespread attention, to which the identification of microorganisms capable of efficiently degrading antibiotics is a key. In this study, a strain DM-1 with high degradation capability was successfully isolated from monensin-contaminated chicken manure by using monensin as the sole carbon source. The strain was further identified basing on morphological, physiological and biochemical characteristics and 16S rRNA gene sequence-based phylogenetic analysis. The degradation efficiency of DM-1 for monensin was determined by HPLC post-column derivatization, and then the degradation conditions of DM-1 were optimized. DM-1 was identified as a strain of Acinetobacter and named as Acinetobacter baumannii DM-1. The optimal conditions for monensin degradation by strain DM-1 were pH 7.0, 30 ℃, and initial monensin concentration of 50 mg/L. The strain DM-1 degraded more than 87.51% of monensin at an initial concentration of 10 mg/L in 28 days, while only a slight decrease in monensin concentration was observed in the control without monensin-degrading strain. This study indicates that the strain DM-1 has a promising application prospect in the bioremediation of monensin-contaminated environment.

Two ferritins from Dermanyssus gallinae: characterization and in vivo assessment as protective antigens.

BACKGROUND: The poultry red mite, Dermanyssus gallinae is recognized worldwide as the most important bloodsucking ectoparasite in layer and breeder flocks. In bloodsucking ectoparasites, ferritins (FERs), the iron-storage proteins, play a pivotal role in dealing with the challenge of large amounts of released iron during the digestion of blood meal. However, no information is available concerning FERs of D. gallinae. The aim of the present study was to investigate the characteristics, functions and the vaccine efficacy of FERs in D. gallinae. RESULTS: Two heavy-chain FERs of D. gallinae were identified and characterized. Phylogenetic analysis indicated that Dg-FER1 may be a secretory FER and Dg-FER2 an intracellular one. RNAi results demonstrated that Dg-fers play key roles in mite survival, successful reproduction and blood digestion. Immunization with rDg-FER1 or rDg-FER2 successfully induced chickens to produce high levels of antigen-specific IgY, resulting in a significant increase in mite mortality (by 58.67% on Day 5) and decreases in oviposition (by 42.15%) and fecundity (by 68.97%) in the rDg-FER1 group, and a 13.73% increase in mite mortality and a 20.89% decrease in fecundity in the rDg-FER1 group. The overall immunization efficacy of rDg-FER1 was 93.51%. CONCLUSION: Two Dg-FERs are crucial to the survival, reproduction and blood digestion of D. gallinae. This study has provided preliminary evidence demonstrating the potential of rDg-FER1 as a vaccine antigen for D. gallinae. © 2021 Society of Chemical Industry.

Enteric and hydrophilic polymers enhance dissolution and absorption of poorly soluble acidic drugs based on micro-environmental pH-modifying solid dispersion.

The oral bioavailability of poorly water-soluble active pharmaceutical ingredient (API) is often inadequate for the desired therapeutic effect. Micro-environmental pH-modifying solid dispersion (micro pHm SD) is an effective method for enhancing the dissolution of pH-dependent soluble APIs. However, erratic bioavailability of these drugs was often found when the micro pHm SD of the drugs was orally administrated and passed through the gastrointestinal tract. Because the added alkalizer in micro pHm SD could be neutralized by the acid in the stomach, as a result not enough alkalizer is left to form alkaline micro-environment around the drug in the intestine, leading to poor dissolution and bioavailability of API. Enteric polymers are applicable materials for site-specific drug delivery that are insoluble in gastric tract but soluble in the intestine targeted for drug release. In this study, a poorly water-soluble model drug, toltrazuril (TOL), was prepared as enteric micro pHm SD with enteric, hydrophilic polymers and alkalizer. The surface of enteric micro pHm SD tablets staining and alkalizer protection test in the acid dissolution medium qualitatively and quantitatively confirmed the protective effects of the enteric polymer on the alkalizer. Dissolution studies revealed that the drug release from the enteric micro pHm SDs was improved significantly compared with micro pHm SD with no enteric polymer. The pH-dependent solubility of enteric polymer had effects on the dissolution of APIs from the SDs in neutral medium. Enteric micro pHm SDs with higher proportion of enteric polymer showed higher Cmax and dissolution rate of TOL. The physicochemical characterization and the molecular interaction between drug and matrix were analyzed by electron microscopy (SEM), differential scanning calorimetry (DSC), and fourier transform infrared spectroscopy (FTIR), finding that the formation of hydrogen bonds between TOL and matrix was helpful to promote dissolution of TOL. Ca(OH)2-TOL-PVPk30-HPMCAS 8: 8: 18: 6 was determined as the most optimal enteric micro pHm SD, which significantly improved the bioavailability of TOL and its active metabolism (TOLSO, TOLSO2) in pharmacokinetic study and could effectively reduce the irritation of the gastrointestinal mucosa caused by the alkalizer Ca(OH)2 when the SD was orally administrated to rabbits. The present study demonstrates that formulating APIs with poor water solubility as enteric micro pHm SD is an effective method for protecting the alkalizer in SD and improving the dissolution of APIs and the bioavailability following oral administration.

Pharmacokinetics of toltrazuril and its metabolites after oral and parenteral administration of novel oil-based suspension based on micro-environmental pH-modifying solid dispersion in rabbits.

Toltrazuril (TOL) is a broad-spectrum anticoccidial drug which is widely used in poultry and livestock. A novel oral suspension based on soybean oil-based TOL micro-environmental pH-modifying solid dispersion (micro pHm SD) and a novel injectable suspension based on white oil-based TOL micro pHm SD were developed, showing high physicochemical stability and high drug release in vitro with good histocompatibility. The present study is to evaluate the pharmacokinetic profiles of TOL and its major metabolites, e.g. toltrazuril sulfoxide (TOLSO) and toltrazuril sulfone (TOLSO2) in rabbits following oral or subcutaneous administration with these two TOL SD suspensions. The plasma concentrations of TOL, TOLSO and TOLSO2 were determined by high performance liquid chromatography (HPLC). Plasma concentration-time data were analyzed by a non-compartmental model analysis. The soybean oil-based TOL suspension after single oral administration at 20 mg/kg body weight (bw) significantly increased the plasma concentrations of TOL, TOLSO and TOLSO2 compared with Baycox® 5 % suspension. Following subcutaneous administration of the white oil-based TOL suspension (20 mg/kg bw), TOL was well absorbed and metabolized more slowly to TOLSO and TOLSO2, compared with oral administration, resulting in the significantly prolonged residence time in rabbits. The two suspensions significantly improved the relative bioavailability of TOL and its two metabolites, showing their potential usage in the control of coccidian in poultry and livestock.

Susceptibility of Dermanyssus gallinae from China to acaricides and functional analysis of glutathione S-transferases associated with beta-cypermethrin resistance.

Dermanyssus gallinae poses a significant threat to poultry production, and the resistance to pyrethroids has been identified worldwide. Periodic monitoring of acaricide resistance in D. gallinae is very important for its control, and molecular mechanism associated with beta-cypermethrin resistance in D. gallinae is not fully clear. Results showed, four field isolates of CBP-1, CBP-2, CBP-5 and CBY-1 from China remained either susceptible or with decreased susceptibility (resistance ratio < 5.0) to phoxim, amitraz, propoxur and carbaryl. Four field isolates of CBP-1, CBP-3, CBY-2 and CBH-1 had developed high or extremely high level of resistance (resistance ratio ≥ 40.0) to beta-cypermethrin or permethrin. Detoxification enzyme activity of GSTs was significantly higher in beta-cypermethrin resistant (RS) than susceptible strain (SS), indicating that GSTs are probably involved in beta-cypermethrin resistance in D. gallinae. The recombinant GSTs (rGST-1, 2, 3) showed a pronounced activity toward the conjugates of 1-chloro-2, 4 dinitrobenzene (CDNB) and glutathione (GSH), with rGST-1 presenting the highest enzymatic activity. Constitutive over-expression of Deg-GST-2 was detected in RS strain, and GSTs genes were all inducible with the treatment of beta-cypermethrin in SS and RS strains. More importantly, knocking down Deg-GST-2 gene expression by RNAi increased the susceptibility of RS strain to beta-cypermethrin. HPLC analysis indicated that rGST-1 protein could metabolize phoxim directly, but rGSTs could not directly metabolize beta-cypermethrin. Our results indicated that some field isolates of D. gallinae from China had developed high level of resistance to pyrethroids, and elevated GSTs activity as well as increased GSTs expression levels were involved in beta-cypermethrin resistance, but the three evaluated GSTs did not play a direct role in the metabolism of beta-cypermethrin.

Evaluation of the vaccine efficacy of three digestive protease antigens from Dermanyssus gallinae using an in vivo rearing system.

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite considered as the major pest in the egg-laying industry. Vaccination is feasible strategy for controlling the haematophagous PRMs. Cathepsin D (CatD), cathepsin L (CatL) and legumain (Lgm) are three endopeptidases participating in digestion of hemoglobin in ticks. The in vitro test and the on-hen feeding device have been used to evaluate the efficacy of vaccines against D. gallinae, however they lacked some of the natural feeding cues for mites, resulting in unreliable results. In the present study, a reliable in vivo rearing system which was nearly close to the natural infestation status of mites was applied to evaluate the efficacy of vaccines against D. gallinae. After vaccinations with rDg-CatD-1, rDg-CatL-1 or rDg-Lgm, chicks developed the antigen-specific IgY immune response to each antigen. The survival rates of D. gallinae in three groups decreased significantly after they fed on the immunized birds. And the oviposition rate and fecundity were significantly reduced by 13.18% and 49.90% in the rDg-CatD-1 immunized group, 5.49% and 38.55% in the rDg-CatL-1 immunized group, respectively. Moreover, immunization with rDg-CatD-1 or rDg-CatL-1 significantly decreased the blood digestion rate of D. gallinae. However, no statistically significant effects on reproduction performance and blood digestion rate of mite were observed in group immunized with rDg-Lgm. Our results demonstrated that immunization with rDg-CatD-1 or rDg-CatL-1 could prevent and control D. gallinae by reducing the survival, reproductive capacity and blood digestion of mite. Importantly, the evaluation system based on the in vivo rearing system was reliable and practical, and it can accurately evaluate the effects of immunization on D. gallinae for pre-screening of potential novel antigens.

De novo assembly and discovery of genes related to blood digestion in the transcriptome of Dermanyssus gallinae (Acari: Dermanyssidae).

Dermanyssus gallinae is an economically important blood-feeding ectoparasite affecting layer chicken farms in many countries. Similar to other blood-feeding arthropods, the blood-meal digestion plays a key role in the survival and reproduction of D. gallinae. The knowledge of the genes involved in blood digestion processes may provide new targets for drug and vaccine against the red mites. In the present study, we sequenced and de novo assembled the transcriptomes of unfed and fed adult red mites using Illumina RNA sequencing (RNA-seq) technology. Up to 40,506 unigenes were assembled, and 13,018 unigenes were identified and annotated. GO analysis of the annotated unigenes clustered into three main GO terms. The dominant GO terms of biological processes were cellular process and metabolic process, the prevailing GO terms of the cellular component were cell part and membrane part, and the dominant GO terms of molecular functions were catalytic and binding activities. Up to 6,443 annotated sequences were assigned to 246 active pathways by KEGG analysis. Differentially expressed genes (DEGs) analysis identified 2,877 unigenes with upregulated 2,094 and downregulated 783 in fed female mites compared with unfed female mites. The biological function of these DEGs was further investigated using the KEGG and GO databases. The upregulated DEGs were potentially involved in nutrient metabolism, highlighting their importance in red mite biology. Quantitative reverse transcription real-time PCR (qRT-PCR) validated that the expression levels of the selected six upregulated DEGs were consistent with those in RNA-seq, indicating that the transcriptomic data are reliable. The present study provides valuable and fundamental knowledge that improves our understanding of the physiology of D. gallinae digestion at a molecular level. Moreover, these transcriptomic data will facilitate the identification of novel function genes and candidate antigens for the development of effective vaccines or drug targets to control D. gallinae.