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.

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.

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.

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.

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.

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.

Low-temperature storage of the poultry red mite, Dermanyssus gallinae, facilitates laboratory colony maintenance and population growth.

The poultry red mite, Dermanyssus gallinae, is currently the most common ectoparasite affecting egg-laying hens. Since continuous culture of D. gallinae on birds is a biologically and economically costly endeavour, storage techniques for mites are urgently needed. Effects of temperature on adult and nymph survival were first studied to optimize storage conditions. Then, fecundity of D. gallinae was studied after mites were stored at optimal storage conditions. Results showed the survival rates of protonymphs (42.11%), deutonymphs (8.19%) and females (19.78%) at 5°C after 84 days were higher than those at 0, 25 and 30°C. Thereafter the fecundity and the capability of re-establishing colonies of D. gallinae were evaluated after they were stored for 40 and 80 days at 5°C. After storage, the mean number of eggs showed no statistical difference between treated (5°C for 40 or 80 days) and control groups (25°C for 7 days), while the hatching rates of eggs were in all cases above 97%. The dynamic changes of mite populations and egg numbers showed similar trends to the control group after the stored adult or nymph mites were fed on chicks. Dermanyssus gallinae can be successfully stored at 5°C for 80 days with no interference with the fecundity of mites, and the stored mites could re-establish colonies successfully. Adults and nymphs were two main stages with capability for low temperature storage. These results suggest that low temperature storage is a viable option for colony maintenance of D. gallinae under laboratory conditions.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

Formulation and Evaluation of a Novel Oral Oil-Based Suspension Using Micro-environmental pH-Modifying Solid Dispersion.

Drugs with pH-dependent solubility that have poor water solubility can be identified in the drug discovery pipeline. Some of them have poor oral absorption, which can result in insufficient efficacy. Micro-environmental pH-modifying solid dispersion (micro pHm SD) is a promising approach to overcome the poor oral absorption of these drugs. In the present study, toltrazuril (TOL), a weakly acidic drug with poor aqueous and pH-dependent solubility, was used as a model drug. Using micro pHm SD, a novel oral oil-based suspension of TOL SD (TSDS) was developed, and the stability of this formulation was evaluated based on particle size, settling volume ratio, redispersibility, thermal stability, and drug content. The optimized soybean oil-based TSDS (S-TSDS) had high physicochemical stability and good histocompatibility with common inflammatory reactions. The results of the in vitro dissolution analysis showed that S-TSDS rapidly and markedly released the drug and provided higher efficacy and longer persistence against coccidiosis (above 90.9%) in rabbits. This technique could increase the oral absorption and bioavailability of new drug candidates.

A single subcutaneous administration of a sustained-release ivermectin suspension eliminates Psoroptes cuniculi infection in a rabbit farm.

BACKGROUND: Psoroptes cuniculi mites are the most common ear parasites infesting breeding female rabbits. The suffering rabbits show cutaneous signs of the infestation in the ears and are prone to secondary infections. OBJECTIVES: This trial was conducted to eliminate P. cuniculi in farm rabbits with a sustained-release ivermectin-loaded solid dispersion suspension (IVM-SD) suspension, and studied the stability of the formulation. ANIMALS: There were 986 breeding female Hyplus rabbits naturally infected with P. cuniculi. METHODS: All rabbits infected with P. cuniculi were subcutaneously administered with a single dose of IVM-SD suspension at 2 mg/kg body weight. Twenty-seven rabbits with severe infections were observed daily and examined on days 0 and 14 to score the lesions and count mites in crusts. RESULTS: Fourteen days after the treatment no live mites were detected, demonstrating 100% therapeutic efficacy. The mean lesion scores decreased from 4.33 to 0.11 in the left ears and from 4.22 to 0.22 in the right ears. No reinfection occurred within 60 days of treatment. CONCLUSIONS: A single subcutaneous administration of the IVM-SD suspension at 2 mg/kg was effective in eliminating P. cuniculi infection in the rabbit farm.

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.

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.

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.

Eimeria tenella: interleukin 17 contributes to host immunopathology in the gut during experimental infection.

Although IL-17 is a key factor in Th17 lineage host responses and plays critical roles in immunological control of a variety of infectious diseases, the contribution of IL-17 to immune function during Eimeria tenella infection is unknown. In the present study, we used an experimental E. tenella infection model to clarify the role of Th17-associated response in the resulting immune response by quantitative real-time PCR assays. We observed robust production of STAT-3 (the transcription factors), IL-1ß, IL-6 and IL-17 in cecal intraepithelial lymphocytes during the early infection, peaking at 6h p.i. and declining thereafter. The expression of TGF-ß was moderately upregulated and had 2 peaks at 6 and 72h p.i. during the early infection. To further investigate the role of chIL-17 during the infection, we treated the infected chickens with IL-17 and its neutralized antibody. As a result, the reduced fecal oocyst shedding and cecal lesion scores, but enhanced body weight gains were observed in IL-17 neutralized chickens. The results of histopathology showed that the neutrophils recruitment diminished and the parasite burden in IL-17 neutralized chickens decreased. These results may be due to the significant decrease in the production of IL-17, IL-6 and TGF-ß, but enhanced IL-12 and IFN-γ expression in IL-17 neutralized chickens. The converse results were shown in IL-17 treated infected-chickens in which chickens showed increased fecal oocyst shedding, exacerbated lesion scores, and reduced body weight gains. These results suggested that chicken IL-17 might mediate E. tenella - induced immunopathology during the infection.

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.

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.

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.

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.

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.

Eimeria tenella: expression profiling of toll-like receptors and associated cytokines in the cecum of infected day-old and three-week old SPF chickens.

Coccidiosis is an economically important protozoan disease worldwide caused by Eimeria parasites. Toll-like receptors (TLRs), a family of highly conserved proteins, are involved in pathogen detection by initiating host responses, and play important roles in the reduction and clearance of pathogens. Little is known about the roles of chicken TLRs during Eimeria tenella infection. We detected the dynamic changes in the expression of TLRs and associated cytokines in the cecum of E. tenella-infected chickens during the early stage of infection. Day-old (Experiment 1) and three-week-old (Experiment 2) chickens were orally gavaged with 10,000 oocysts (30 chickens each experiment), and their cecum intraepithelial lymphocytes were collected at 3, 6, 12, 24, 48, and 72h post-infection (hpi). Expression profiling of TLR1LA, TLR4, TLR5, TLR7, TLR21, and IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-12 genes were analyzed using quantitative real-time polymerase chain reaction. Almost all TLR transcripts were transiently increased at 3hpi in Experiment 1. In three-week-old chickens, TLR1LA, TLR4, TLR5, TLR7, and TLR21 expression was upregulated at 12hpi, and TLR1LA, TLR5, and TLR21 were highly expressed at 72hpi. In day-old chickens, IFN-α, IFN-ß, IFN-γ, IL-1ß, and IL-12 expression was significantly upregulated at 3hpi (156.1-1117.1-fold change), in comparison to the different peak level times and relatively small changes for these cytokines in the three-week-old chickens. Our results provide a valuable overview for the expression pattern of TLRs and associated cytokines during the early stage of E. tenella infection in chickens.