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Abstract Bioactive molecules in tick saliva are considered to be key to successful feeding and further the transmission of tick-borne pathogens. Problems such as pathogen transmission and animal weight loss result in tick infestation can cause tremendous economic losses to the livestock industry. Therefore, the development of a universal tick vaccine is urgently needed. In this paper, three serine protease inhibitor (serpin) proteins RMS-3, L7LRK7 and L7LTU1 were analyzed with bioinformatics methods. Subsequently the proteins were expressed and purified, and inoculated into Kunming mice for immune protection analysis. The amino acid sequence similarities between RMS-3, L7LRK7 and L7LTU1 were up to 90% in Rhipicephalus sanguineus. The recombinant RMS-3 + L7LRK7 + L7LTU1 showed anticoagulant reaction function and could inhibit the activity of CD4+ lymphocytes, when inoculated into Kunming mice. Additionally, After the immunized mice were challenged with Rhipicephalus sanguineus, the percentage of larvae and nymphs that were fully engorged dropped to 40.87% (P 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P

Abstract Anaplasmosis is a highly prevalent tick-borne intracellular bacterial disease that affects various host species globally, particularly ruminants in tropical and subtropical regions. However, information regarding the distribution and epidemiology of anaplasmosis in small and large ruminants on Hainan Isalnd is limited. To address this knowledge gap, the present study aimed to assess the occurrence of Anaplasma spp. infections in goats (N = 731) and cattle (N = 176) blood samples using nested PCR and conventional PCR based assays. The results revealed an overall prevalence of 30.1% in goats and 14.8% in cattle. The infection rates of A. bovis, A. phagocytophilum, A. ovis and A. capra in goat samples were 22.7%, 13.8%, 2.0% and 3.4%, respectively, while the infection rates of A. bovis, A. phagocytophilum and A. marginale in cattle samples were 11.4%, 6.3% and 5.7%, respectively. A. bovis exhibited the highest prevalence among the Anaplasma spp. in both goat and cattle samples. In addition, the most frequent co-infection was the one with A. phagocytophilum and A. bovis. It was found that the age, sex and feeding habits of cattle and goats were considered to be important risk factors. Evaluation of the risk factor relating to the rearing system showed that the infection rate for the free-range goats and cattle was significantly higher when compared with stall-feeding system. This study represents one of the largest investigations on the distribution, prevalence, and risk factors associated with Anaplasma infection in ruminants on Hainan Island, highlighting a higher circulation of the infection in the region than previously anticipated. Further reasesrch is necessary to investigate tick vectors, reservoir animals, and the zoonotic potential of the Anaplasma spp. in this endemic region of Hainan Island.

Abstract Background The behaviors and ontogeny of Aedes aegypti are closely related to the spread of diseases caused by dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV) viruses. During the life cycle, Ae. aegypti undergoes drastic morphological, metabolic, and functional changes triggered by gene regulation and other molecular mechanisms. Some essential regulatory factors that regulate insect ontogeny have been revealed in other species, but their roles are still poorly investigated in the mosquito. Results Our study identified 6 gene modules and their intramodular hub genes that were highly associated with the ontogeny of Ae. aegypti in the constructed network. Those modules were found to be enriched in functional roles related to cuticle development, ATP generation, digestion, immunity, pupation control, lectins, and spermatogenesis. Additionally, digestion-related pathways were activated in the larvae and adult females but suppressed in the pupae. The integrated protein‒protein network also identified cilium-related genes. In addition, we verified that the 6 intramodular hub genes encoding proteins such as EcKinase regulating larval molt were only expressed in the larval stage. Quantitative RT‒PCR of the intramodular hub genes gave similar results as the RNA-Seq expression profile, and most hub genes were ontogeny-specifically expressed. Conclusions The constructed gene coexpression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. Ultimately, these findings will be key in identifying potential molecular targets for disease control.

iGABAR, a member of the Cys-loop ligand-gated ion channel superfamily, is a significant target of the insecticide ivermectin (IVM). GRD is the potential subunit of the insect iGABAR. However, little information about GRD in Ae. aegypti has been reported. In this study, we involved cloning and characterizing the iGABAR subunit GRD of Ae. aegypti (Ae-GRD). Sequence analysis indicated that Ae-GRD, as part of the cysteine-loop ligand-gated ion channel family, is similar to other insect GRD. RNA interference (RNAi) was employed to explore IVM resistance in Ae. aegypti, resulting in a significant reduction in Ae-GRD expression (p < 0.05), and the mortality of Ae. aegypti adults with Ae-GRD knockdown was significantly decreased after exposure to ivermectin. Bioinformatics prediction identified miR-71-5p as a potential regulator of Ae-GRD. In vitro, dual-luciferase reporter assays confirmed that Ae-GRD expression was regulated by miR-71-5p. Microinjection of miR-71-5p mimics upregulated miR-71-5p expression and downregulated Ae-GRD gene expression, reducing mortality by 34.52% following IVM treatment. Conversely, microinjection of a miR-71-5p inhibitor decreased miR-71-5p expression but did not affect the susceptibility to IVM despite increased Ae-GRD expression (p < 0.05). In conclusion, Ae-GRD, as one of the iGABA receptor subunits, is a potential target of ivermectin. It may influence ivermectin resistance by modulating the GABA signaling pathway. The inhibition of Ae-GRD expression by miR-71-5p decreased ivermectin resistance and consequently lowered the mortality rate of Ae. aegypti mosquitoes. This finding provides empirical evidence of the relationship between Ae-GRD and its miRNA in modulating insecticide resistance, offering novel perspectives for mosquito control strategies.

Rhipicephalus sanguineus, the brown dog tick, is the most widespread tick in the world and a predominant vector of multiple pathogens affecting wild and domestic animals. There is an increasing interest in understanding the role of tick microbiome in pathogen acquisition and transmission as well as in environment–vector interfaces. Several studies suggested that the tick microbial communities are under the influence of several factors including the tick species, dietary bloodmeal, and physiological stress. Compared with insects, very little of the microbial community is known to contribute to the nutrition of the host. Therefore, it is of significance to elucidate the regulation of the microbial community of Rh. Sanguineus under starvation stress. Starvation stress was induced in wild-type adults (1 month, 2 months, 4 months, 6 months) and the microbial composition and diversity were analyzed before and after blood feeding. After the evaluation, it was found that the microbial community composition of Rh. sanguineus changed significantly with starvation stress. The dominant symbiotic bacteria Coxiella spp. of Rh. sanguineus gradually decreased with the prolongation of starvation stress. We also demonstrated that the starvation tolerance of Rh. sanguineus was as long as 6 months. Next, Coxiella-like endosymbionts were quantitatively analyzed by fluorescence quantitative PCR. We found a pronounced tissue tropism in the Malpighian tubule and female gonad, and less in the midgut and salivary gland organs. Finally, the blood-fed nymphs were injected with ofloxacin within 24 h. The nymphs were allowed to develop into adults. It was found that the adult blood-sucking rate, adult weight after blood meal, fecundity (egg hatching rate), and feeding period of the newly hatched larvae were all affected to varying degrees, indicating that the removal of most symbiotic bacteria had an irreversible effect on it.

Abstract Detection of chemical cues via chemosensory receptor proteins are essential for most animals, and underlies critical behaviors, including location and discrimination of food resources, identification of sexual partners and avoidance of predators. The current knowledge of how chemical cues are detected is based primarily on data acquired from studies on insects, while our understanding of the molecular basis for chemoreception in acari, mites in particular, remains limited. The poultry red mite (PRM), Dermanyssus gallinae, is one of the most important blood-feeding ectoparasites of poultry. PRM are active at night which suck the birds' blood during periods of darkness and hide themselves in all kinds of gaps and cracks during the daytime. The diversity in habitat usage, as well as the demonstrated host finding and avoidance behaviors suggest that PRM relies on their sense of smell to orchestrate complex behavioral decisions. Comparative transcriptome analyses revealed the presence of candidate variant ionotropic receptors, odorant binding proteins, niemann-pick proteins type C2 and sensory neuron membrane proteins. Some of these proteins were highly and differentially expressed in the forelegs of PRM. Rhodopsin-like G protein-coupled receptors were also identified, while insect-specific odorant receptors and odorant co-receptors were not detected. Furthermore, using scanning electron microscopy, the tarsomeres of all leg pairs were shown to be equipped with sensilla chaetica with or without tip pores, while wall-pored olfactory sensilla chaetica were restricted to the distal-most tarsomeres of the forelegs. This study is the first to describe the presence of chemosensory genes in any Dermanyssidae family. Our findings make a significant step forward in understanding the chemosensory abilities of D. gallinae.

Anaplasmosis is a serious infection which is transmitted by ticks and mosquitos. There are very few reports and studies that have been carried out to understand the prevalence, distribution, and epidemiological profile of Anaplasma spp. infection in dogs in Hainan province/island. In the present study, we have tried to understand the prevalence, distribution, and occurrence of Anaplasma spp. infections in dogs (n = 1051) in Hainan Island/Province to establish a surveillance-based study. The confirmed positive samples by Polymerase chain reaction (PCR) were subjected to capillary sequencing for further strain-specific confirmation, followed by the construction of phylogenetic trees to determine their genetic relations. Various statistical tools were used to analyze related risk factors. There were three species of Anaplasma detected from the Hainan region; namely, A. phagocytophilum, A. bovis, and A. platys. The overall prevalence of Anaplasma is 9.7% (102/1051). A. phagocytopihum was prevalent in 1.0% of dogs (11/1051), A. bovis was found in 2.7% of dogs (28/1051), and A. platys in 6.0% of dogs (63/1051). Our surveillance-based study conducted to understand the occurrence and distribution pattern of Anaplasma spp. in Hainan will help in designing effective control measures along with management strategies so as to treat and control the infection in the area.

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

Abstract Background Non-human primates are often infected with human-pathogenic Cryptosporidium hominis subtypes, but rarely with Cryptosporidium parvum. In this study, 1452 fecal specimens were collected from farmed crab-eating macaques (Macaca fascicularis) in Hainan, China during the period April 2016 to January 2018. These specimens were analyzed for Cryptosporidium species and subtypes by using PCR and sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively. Results Altogether, Cryptosporidium was detected using 18S rRNA-based PCR in 132 (9.1%) sampled animals, with significantly higher prevalence in females (12.5% or 75/599 versus 6.1% or 43/706), younger animals (10.7% or 118/1102 in monkeys 1–3-years-old versus 4.0% or 14/350 in those over 3-years-old) and animals with diarrhea (12.6% or 46/365 versus 7.9% or 86/1087). Four Cryptosporidium species were identified, namely C. hominis, C. parvum, Cryptosporidium muris and Cryptosporidium ubiquitum in 86, 30, 15 and 1 animal, respectively. The identified C. parvum, C. hominis and C. ubiquitum were further subtyped by using gp60 PCR. Among them, C. parvum belonged to subtypes in two known subtype families, namely IIoA14G1 (in 18 animals) and IIdA19G1 (in 2 animals). In contrast, C. hominis mostly belonged to two new subtype families Im and In, which are genetically related to Ia and Id, respectively. The C. hominis subtypes identified included ImA18 (in 38 animals), InA14 (in six animals), InA26 (in six animals), InA17 (in one animal) and IiA17 (in three animals). The C. ubiquitum isolates belonged to subtype family XIId. By subtype, ImA18 and IIoA14G1 were detected in animals with diarrhea whereas the remaining ones were mostly found in asymptomatic animals. Compared with C. parvum and C. muris, higher oocyst shedding intensity was observed in animals infected with C. hominis, especially those infected with the Im subtype family. Conclusions Data from the study suggest that crab-eating macaques are infected with diverse C. parvum and C. hominis subtypes. The C. parvum IIo subtype family previously seen in rodents in China has apparently expanded its host range.

Serotonin (5-HT) plays a vital role in many physiological processes in insects, regulating physiological activities such as growth and movement through multiple 5-HT receptors (5-HTRs), which were potential targets for some new insecticides. However, the specific function of individual 5-HTRs in Ae. aegypti is still unclear. In this study, we investigated the function of the 5-HT7A receptor during Ae. aegypti development. 5-HTR7A transcripts were detected at all stages of development by real-time PCR. The results indicated that the gene expression was highest in the limbs (p < 0.01). We also generated 5-HTR7A mutant mosquitoes using CRISPR-mediated gene editing. The mutants had an abnormal phenotype at the larval stage, including an aberrant head-to-chest ratio and decreased motor activity. The mutant pupae developed abnormally, and most died (56.67%) (p < 0.0001). Using external stimuli to larvae and pupae with abnormal phenotypes, we found the mutant G1 and G2 generations responded to external stimuli in a longer time than the wild-type (WT) mosquitoes, and most of the mutants were 2 to 3 s slower than the WTs to respond to external stimuli (p < 0.01). Due to higher mortality, mutant larvae and pupae had fewer numbers than the WTs. The egg hatching rate of mutant G1 and G2 generations was lower than that of the WTs (p < 0.01). The expression level of 5-HTR7A in the mutants decreased by about 65% compared with the control group using real-time PCR (p < 0.05). In all, the 5-HT7A receptor plays an important role in the metamorphosis, development and motor function of Aedes aegypti.

Mosquitoes transmit a number of diseases in animals and humans, including Dengue, Chikungunya and Zika viruses that affect millions of people each year. Controlling the disease-transmitting mosquitoes has proven to be a successful strategy to reduce the viruses transmission. Polyamines are required for the life cycle of the RNA viruses, Chikungunya virus and Zika virus, and a depletion of spermidine and spermine in the host via induction of spermine N-acetyltransferase restricts their replication. Spermine N-acetyltransferase is a key catabolic enzyme in the polyamine pathway, however there is no information of the enzyme identification in any insects. Aliphatic polyamines play a fundamental role in tissue growth and development in organisms. They are acetylated by spermidine/spermine N1-acetyltransferase (SAT). In this study we provided a molecular and biochemical identification of SAT from Aedes aegypti mosquitoes. Screening of purified recombinant proteins against polyamines established that aaNAT5b, named previously based on sequence similarity with identified aaNAT1 in insects, is active to spermine and spermidine. A crystal structure was determined and used in molecular docking in this study. Key residues were identified to be involved in spermine binding using molecular docking and simulation. In addition, SAT transcript was down regulated by blood feeding using a real time PCR test. Based on its substrate profile and transcriptional levels after blood feeding, together with previous reports for polyamines required in arboviruses replication, SAT might be potentially used as a target to control arboviruses with human interference.

Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.

Insect phenoloxidase, presented as an inactive precursor prophenoloxidase (PPO) in hemolymph, catalyzes melanin formation, which is involved in wound healing, pathogen killing, reversible oxygen collection during insect respiration, and cuticle and eggshell formation. Mosquitoes possess 9 to 16 PPO members across different genera, a number that is more than that found in other dipteran insects. However, the reasons for the redundancy of these PPOs and whether they have distinct biochemical properties and physiological functions remain unclear. Phylogenetic analysis confirmed that Aedes aegypti PPO6 (Aea-PPO6) is an ortholog to PPOs in other insect species, classified as the classical insect type, while other Aea-PPOs are unique to Diptera, herein referred to as the dipteran type here. We characterized two Aea-PPO members, Aea-PPO6, the classical insect type, and Aea-PPO10, a dipteran type, which exhibit distinct substrate specificities. By resolving Aea-PPO6' s crystal structure and creating a chimera protein (Aea-PPO6-cm) with Motif 1 (217GDGPDSVVR225) from Aea-PPO10, we identified the motif that determines PPO substrate specificity. In vivo, loss of Aea-PPO6 led to larval lethality, while Aea-PPO10 was involved in development, pigmentation, and immunity. Our results enhance the understanding of the functional diversification of mosquito PPOs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Spinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and is efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) ∝6 subunit have been identified that are associated with levels of spinosad resistance, but the molecular characterization of the nAChR gene family and a causative association between nAChR ∝6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. RESULTS: In this study, we identified 10 candidate nAChR subunits in Ae. Aegypti, nAChR∝1–∝9 and nAChR𝝱1, showing similarly orthologous relationships with Anopheles gambiae. With the application of the CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 50 end of the Aae∝6 (Ae. aegypti nAChR ∝6) gene in a homozygous mutant strain (Aae∝6-KO). The mutation produced two successive pre-mature stop codons, resulting in loss of function in the target receptor. The Aae∝6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wildtype. A recessive mode of inheritance for spinosad resistance was found in the Aae∝6-KO strain. CONCLUSION: CRISPR/Cas9 introduced truncated Aae∝6 receptor in Ae. aegypti resulted in an increased level of resistance to spinosad, suggesting that the conserved nAChR ∝6 subunit is the target for spinosad insecticide. [ABSTRACT FROM AUTHOR]

Canine and feline heartworm disease caused by Dirofilaria immitis is a serious and sometimes fatal infection transmitted by mosquitos. Little is known about the prevalence or distribution of D. immitis infection in dogs and cats on the island of Hainan island/province or coastal cities of China. The present study examined the occurrence of D. immitis infections in dogs (n ¼ 869) and cats (n ¼ 51) in Hainan island/province and prevalence in dogs from 3 coastal cities (Shenzhen [n ¼ 55], Shanghai [n ¼ 69], and Hangzhou [n ¼ 45]) in southern and eastern China. A commercial antigen detection (AD) test and 2 PCR methods (16S ribosomal RNA and Wolbachia surface protein [WSP] genes) were used to determine the prevalence of D. immitis from animals .6 mo old with no previous history of D. immitis preventive treatment or heartworm infection. Gene sequencing was used to confirm positive PCR samples. The AD test was not used on cat samples. Using the AD test, the prevalence in dogs was 0.5% (4/869) in Hainan island/province, 0% (0/55) in Shenzhen, 1.5% (1/69) in Shanghai, and 0% (0/45) in Hangzhou. Prevalence by 16S rRNA gene PCR was 7.4% (64/869) of dogs from Hainan island/province, 0% (0/55) in dogs from Shenzhen, 1.5% (1/69) in dogs from Shanghai, and 0% (0/45) in dogs from Hangzhou. Prevalence by WSP gene PCR in dogs was 5.3% (46/869) in Hainan island/province, 0% (0/55) in Shenzhen, 1.5% (1/69) in Shanghai, and 0% (0/45) in Hangzhou. Prevalence in the 51 cats from Hainan island/province was 9.8% and 5.9% by 16S rRNA and WSP gene PCR, respectively. The present study demonstrates that canine heartworm exposure occurs in dogs and cats in Hainan island/province and that PCR methods detected a higher prevalence than did the AD method. The 16S rRNA gene PCR detected more positive samples than did the WSP gene PCR in both dogs and cats. The 3 coastal cities had very few dogs that had evidence of D. immitis exposure. [ABSTRACT FROM AUTHOR]

Background: The elevated postprandial triacylglycerol (TG) concentration is associated with elevated coronary artery disease. Oral fatty tolerant test (OFTT) is less performed in the health. This study was to evaluate the effect of sex and body mass index (BMI) on postprandial TG concentration of the low fat meal in healthy young persons. This study included 112 healthy college students (18.8+/-1.6y). Their body height and weight were measured for body mass index (BMI). According to BMI, 27 subjects were in the under-weight subgroup, 60 in the normal weight subgroup and 25 in the over-weight subgroup. After overnight fasting low fat OFTT (27 g fat, 600 kcal) was performed and the plasma TG and glucose concentrations were measured before and at 2, 4 and 6 hour after a fat meal. The area under the curve (AUC) of TG was calculated. Results: The fasting TG levels were similar and the fasting TG levels gradually increased as BMI increased in both sexes. The postprandial TG levels at 2 and 4 h decreased in female, but did not significant change in male. In female, the TG curves of 3 BMI subgroups showed saddle type, but in male the TG curve of the over-weight subgroup had a peak at 2 h, on the other hand the TG curve of under- weight subgroup had a dip at 2 h. Conclusions: Gender and BMI are important influencing factors for TG metabolism after fat meal in the youth. The young male persons with over-BMI have abnormal TG metabolism. [ABSTRACT FROM AUTHOR]