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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) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, and 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, in comparison with the control group. These data indicate that RmS-3 + L7LRK7 + L7LTU1 has good immune protection and has the potential to be developed into a vaccine against the larvae and nymphs of R. sanguineus .

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.

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.

3,4-Dihydroxyphenylacetaldehyde synthase (DHPAAS) catalyzes oxygen-dependent conversion of 3,4-dihydroxyphenylalanine (dopa) to 3,4-dihydroxyphenylacetaldehyde (DHPAA), a likely cross-linking agent precursor of the insect cuticle. In the current study, extensive in vivo experiments in Aedes aegypti show that DHPAAS is essential for abdominal integrity, egg development and cuticle structure formation. Solid-state 13 C nuclear magnetic resonance analysis of the Ae. aegypti cuticle molecular structure shows chemical shifts of 115 to 145 ppm, suggesting the presence of catechols derived from DHPAA. The crystal structure of insect DHPAAS was then solved, revealing an active site that is divergent from that of the homologous enzyme dopa decarboxylase. In the DHPAAS crystal structure, stabilization of the flexible 320–350 region accompanies the positioning of the 350–360 loop relatively close to the catalytic Asn192 residue while the conserved active site residue Phe103 adopts an open conformation away from the active center; these distinct features participate in the formation of a specific hydrophobic tunnel which potentially facilitates delivery of oxygen to pyridoxal 5’-phosphate in the conversion of dopa to DHPAA. This study investigates the role of 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) in mosquito cuticle formation, a key factor in their development and survival. The findings reveal that DHPAAS is essential for abdominal integrity and egg development, with unique structural features suggesting a novel oxygen delivery mechanism.

The Hainan gibbon (Nomascus hainanus) is one of the most endangered primates globally, threatened by habitat destruction, genetic diversity loss, and ecological competition. In this study, given the critical role of the gut microbiota in host immune regulation and nutrient metabolism, we investigated the composition of and age-related variations in the gut microbiota in Hainan gibbons. Using 16S rRNA sequencing, we systematically investigated the gut microbial diversity of Hainan gibbons. We collected 41 fecal samples from Hainan Tropical Rainforest National Park, covering three age groups: juveniles (4–6 years), subadults (7–10 years), and elderly animals (≥13 years). This study found that microbiota composition changed significantly with age. Juveniles had higher microbial diversity and complexity, while subadults showed an increased abundance of Fibrobacter and Prevotella in their microbial communities, along with a Tax4Fun-predicted enrichment of functional genes related to energy metabolism, cell motility, and nervous system functions. LEfSe analysis identified statistically significant microbial taxa among different age groups, with Bacteroidota and Firmicutes being the dominant phyla across all groups with varying proportions. These results highlight the critical role of the gut microbiota in the health and adaptability of Hainan gibbons, offering insights for conservation strategies. The findings of this study are significant for understanding the changes in gut microbiota and their ecological functions across different life stages of endangered primates.

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.

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.

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.

The midgut microbiota of Aedes aegypti is crucial for the mosquito’s development, nutrition, and immunity. However, its communities are also distinctively influenced by the colonization of different microorganisms, influencing its susceptibility to pathogens and transmission capacity. In this study, we investigated the effects of infections with Escherichia coli, Staphylococcus aureus, and Beauveria bassiana on the midgut microbial composition of Ae. aegypti. These microorganisms were inoculated into the midguts of third-instar larvae using a soaking method. Midgut samples were then analyzed through high-throughput 16S rDNA sequencing to assess bacterial load and microbiota composition of fourth-instar larvae and female adult mosquitoes. The results reveal that E. coli-colonized fourth-instar larvae (CO_4W) exhibited 20 unique genera, whereas the S. aureus-colonized group (S_4W) had operational taxonomic units assigned to 194 bacterial taxa, including a notable decrease in Elizabethkingia. In addition, B. bassiana infection led to a significant reduction of Elizabethkingia meningoseptica in larvae, decreasing from 42.9% in the control group (CK_4W) to 0.9% in the B. bassiana-infected group (B_4W). Distinct microbial profiles were also compared between adult mosquitoes and fourth-instar larvae. Significant abundance changes were found in Firmicutes, Bacteroidota, and Proteobacteria among different groups. Metabolic pathway predictions using PICRUSt suggested that microorganism invasion enriched the pathways involved in carbohydrate metabolism and amino acid metabolism. This enrichment suggests that the microbiota may undergo specific adaptive responses to pathogen presence. Overall, our results provide new insights into the relationship between the invasion of microorganisms and midgut bacterial communities in mosquitoes.

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 cox 1 gene and phylogenetic analysis. We have also used the DNA sequences of the cox 1 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 cox 1 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.