Molecular surveillance and genetic diversity of Anaplasma spp. in cattle (Bos taurus) and goat (Capra aegagrus hircus) from Hainan island/province, China.

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.

De novo transcriptome sequencing of the northern fowl mite, Ornithonyssus sylviarum, shed light on parasitiform poultry mites evolution and its chemoreceptor repertoires.

The northern fowl mite (NFM), Ornithonyssus sylviarum, and the poultry red mite (PRM), Dermanyssus gallinae, are the most serious pests of poultry, both of which have an expanding global prevalence. Research on NFM has been constrained by a lack of genomic and transcriptomic data. Here, we report and analyze the first global transcriptome data across all mite live stages and sexes. A total of 28,999 unigenes were assembled, of which 19,750 (68.10%) were annotated using seven functional databases. The biological function of these unigenes was classified using the GO, KOG, and KEGG databases. To gain insight into the chemosensory receptor-based system of parasitiform mites, we furthermore assessed the gene repertoire of gustatory receptors (GRs) and ionotropic receptors (IRs), both of which encode putative ligand-gated ion channel proteins. While these receptors are well characterized in insect model species, our understanding of chemosensory detection in mites and ticks is in its infancy. To address this paucity of data, we identified 9 IR/iGluRs and 2 GRs genes by analyzing transcriptome data in the NFM, while 9 GRs and 41 IR/iGluRs genes were annotated in the PRM genome. Taken together, the transcriptomic and genomic annotation of these two species provide a valuable reference for studies of parasitiform mites and also help to understand how chemosensory gene family expansion/contraction events may have been reshaped by an obligate parasitic lifestyle compared with their free-living closest relatives. Future studies should include additional species to validate this observation and functional characterization of the identified proteins as a step forward in identifying tools for controlling these poultry pests.

Biochemical Evolution of a Potent Target of Mosquito Larvicide, 3-Hydroxykynurenine Transaminase.

A specific mosquito enzyme, 3-hydroxykynurenine transaminase (HKT), is involved in the processing of toxic metabolic intermediates of the tryptophan metabolic pathway. The HKT enzymatic product, xanthurenic acid, is required for Plasmodium spp. development in the mosquito vectors. Therefore, an inhibitor of HKT may not only be a mosquitocide but also a malaria-transmission blocker. In this work, we present a study investigating the evolution of HKT, which is a lineage-specific duplication of an alanine glyoxylate aminotransferases (AGT) in mosquitoes. Synteny analyses, together with the phylogenetic history of the AGT family, suggests that HKT and the mosquito AGTs are paralogous that were formed via a duplication event in their common ancestor. Furthermore, 41 amino acid sites with significant evidence of positive selection were identified, which could be responsible for biochemical and functional evolution and the stability of conformational stabilization. To get a deeper understanding of the evolution of ligands' capacity and the ligand-binding mechanism of HKT, the sequence and the 3D homology model of the common ancestor of HKT and AGT in mosquitoes, ancestral mosquito AGT (AncMosqAGT), were inferred and built. The homology model along with 3-hydroxykynurenine, kynurenine, and alanine were used in docking experiments to predict the binding capacity and ligand-binding mode of the new substrates related to toxic metabolites detoxification. Our study provides evidence for the dramatic biochemical evolution of the key detoxifying enzyme and provides potential sites that could hinder the detoxification function, which may be used in mosquito larvicide and design.

Seasonal Variation of Midgut Bacterial Diversity in Culexquinquefasciatus Populations in Haikou City, Hainan Province, China.

Culex quinquefasciatus, one of the most significant mosquito vectors in the world, is widespread in most parts of southern China. A variety of diseases including Bancroft's filariasis, West Nile disease, and St. Louis encephalitis could be transmitted by the vector. Mosquitoes have been shown to host diverse bacterial communities that vary depending on environmental factors such as temperature and rainfall. In this work, 16S rDNA sequencing was used to analyze the seasonal variation of midgut bacterial diversity of Cx. Quinquefasciatus in Haikou City, Hainan Province, China. Proteobacteria was the dominant phylum, accounting for 79.7% (autumn), 73% (winter), 80.4% (spring), and 84.5% (summer). The abundance of Bacteroidetes in autumn and winter was higher than in others. Interestingly, Epsilonbacteraeota, which only exists in autumn and winter, was discovered accidentally in the midgut. We speculated that this might participate in the nutritional supply of adult mosquitoes when temperatures drop. Wolbachia is the most abundant in autumn, accounting for 31.6% of bacteria. The content of Pantoea was highest in the summer group, which might be related to the enhancement of the ability of mosquitoes as temperatures increased. Pseudomonas is carried out as the highest level in winter. On the contrary, in spring and summer, the genus in highest abundance is Enterobacter. Acinetobacter enriches in the spring when it turns from cold to hot. By studying the diversity of midgut bacteria of Cx. quinquefasciatus, we can further understand the co-evolution of mosquitoes and their symbiotic microbes. This is necessary to discuss the seasonal variation of microorganisms and ultimately provide a new perspective for the control of Cx. quinquefasciatus to reduce the spread of the diseases which have notably vital practical significance for the effective prevention of Cx. quinquefasciatus.

The microbiota changes of the brown dog tick, Rhipicephalus sanguineus under starvation stress.

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.

Functional analysis reveals ionotropic GABA receptor subunit RDL is a target site of ivermectin and fluralaner in the yellow fever mosquito, Aedes aegypti.

BACKGROUND: Ionotropic γ-aminobutyric acid (iGABA) receptors are involved in various physiological activities in insects, including sleep, olfactory memory, movement, and resistance to viruses. Ivermectin and fluralaner can disturb the insect nervous system by binding to iGABA receptors, and are therefore an effective means for controlling insect pests. However, the molecular mechanisms underlying the insecticidal effect of both the compounds on Aedes. aegypti remain unexplored. RESULTS: In this study, we investigated the spatiotemporal expression profile of Ae. aegypti RDL (Ae-RDL), a subunit of iGABA receptor. RDL dsRNA suppressed the expression of Ae-RDL mRNA in Ae. aegypti larvae and adult by 60% and 50.67%,  resepectly. However, the physiology of Ae. aegypti larvae was not significantly affected. The mortality of Ae. aegypti larvae and adult females subjected to Ae-RDL knockdown significantly decreased after exposure to ivermectin and fluralaner. Additionally, Ae-RDL was cloned into Xenopus laevis oocytes and characterized using the two-electrode voltage-clamp method. The inward current was induced by GABA binding to the functional Ae-RDL homomeric receptors at a median effective concentration (EC50 ) of 100.4 ± 59.95 µM (n > 3). The significant inhibitory effect of ivermectin and fluralaner on inward current indicated that both insecticides exerted a significant antagonistic effect on Ae-RDL. However, ivermectin also showed strong agonistic as well as weak activation effects on Ae-RDL. These contrasting effects of ivermectin on Ae-RDL depended on ivermectin concentration. CONCLUSION: Our study revealed that Ae-RDL subunit is a target of ivermectin and fluralaner, providing new insights into the insecticidal mechanism of both compounds in Ae. aegypti. © 2022 Society of Chemical Industry.

Structure and Functions of Bacterial Outer Membrane Protein A, A Potential Therapeutic Target for Bacterial Infection.

Outer membrane protein A (OmpA) is a unique outer membrane protein which is abundantly present in the outer membrane of Gram-negative bacteria. OmpA is a transmembrane structural protein with a conserved amino acid sequence among different bacteria. This protein is involved in a number of functions like adhesion, toxicity, invasiveness, and biofilm formation in Gram-negative bacteria. Many studies have proposed that OmpA could be a therapeutic target for bacterial infection. Our review focusses on the studies involving recent development in the structure and functions of OmpA and further discussing its potential as a therapeutic target for bacterial infection.

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti.

BACKGROUND: Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies-CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE-based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. METHODS: Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. RESULTS: We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. CONCLUSIONS: Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.

Understanding Tick Biology and Its Implications in Anti-tick and Transmission Blocking Vaccines Against Tick-Borne Pathogens.

Ticks are obligate blood-feeding ectoparasites that transmit a wide variety of pathogens to animals and humans in many parts of the world. Currently, tick control methods primarily rely on the application of chemical acaricides, which results in the development of resistance among tick populations and environmental contamination. Therefore, an alternative tick control method, such as vaccines have been shown to be a feasible strategy that offers a sustainable, safe, effective, and environment-friendly solution. Nevertheless, novel control methods are hindered by a lack of understanding of tick biology, tick-pathogen-host interface, and identification of effective antigens in the development of vaccines. This review highlights the current knowledge and data on some of the tick-protective antigens that have been identified for the formulation of anti-tick vaccines along with the effects of these vaccines on the control of tick-borne diseases.

Corrigendum: Understanding Tick Biology and Its Implications in Anti-tick and Transmission Blocking Vaccines Against Tick-Borne Pathogens.

[This corrects the article DOI: 10.3389/fvets.2020.00319.].

An efficient high-welfare feeding device for assessing northern fowl mite interventions in vivo: an improved method for the identification of protective antigens/systemic acaricides/repellent effect.

The northern fowl mite (NFM), Ornithonyssus sylviarum, is an obligate hematophagous ectoparasite of domestic and wild birds, and it is an economic pest of laying hen in North America, China, India, Australia, Myanmar, and Brazil. Such an economically important pest remains neglected in many parts of the world, including Asian countries. Therefore, concerted action is required in both basic and applied research directed at the biology and control of this destructive pest. In the present study, we have developed a novel, high-welfare in vivo feeding capsule that would permit pre-screening of new interventions, repellency and deterrence effects of plant-derived products and other semiochemical compounds before proceeding to large-scale field experiments/bioassays, while the minimum number of animals is required to obtain results. Mites were fed on the birds through either a mesh or without a mesh. The average feeding rates of mites was significantly higher when fed directly on chickens, whereas 106 µm nylon mesh was the top-performing mesh when compared with 125 µm aperture nylon mesh. For optimal feeding, the feeding capsules contain NFM and are attached to the skin of the chicken's thigh for 6 h. This is a simple, reproducible, and easy approach and can be adapted to facilitate many aspects of bioassays.

Comparative morphological and transcriptomic analyses reveal chemosensory genes in the poultry red mite, Dermanyssus gallinae.

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.

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

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