Tick salivary protein Cystatin: structure, anti-inflammation and molecular mechanism.

Ticks are blood-sucking ectoparasites that secrete immunomodulatory substances in saliva to hosts during engorging. Cystatins, a tick salivary protein and natural inhibitor of Cathepsins, are attracting growing interest globally because of the immunosuppressive activities and the feasibility as an antigen for developing anti-tick vaccines. This review outlines the classification and the structure of tick Cystatins, and focuses on the anti-inflammatory effects and molecular mechanisms. Tick Cystatins can be divided into four families based on structures and cystatin 1 and cystatin 2 are the most abundant. They are injected into hosts during blood feeding and effectively mitigate the host inflammatory response. Mechanically, tick Cystatins exert anti-inflammatory properties through the inhibition of TLR-NF-κb, JAK-STAT and p38 MAPK signaling pathways. Further investigations are crucial to confirm the reduction of inflammation in other cell types like neutrophils and mast cells, and fully elucidate the underlying mechanism (like the structural mechanism) to make Cystatin a potential candidate for the development of novel anti-inflammation agents.

Protein profiling of hemolymph in Haemaphysalis flava ticks.

BACKGROUND: Tick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava. METHODS: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ). RESULTS: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-ß, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-ß. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ. CONCLUSIONS: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.

Metagenomics of the midgut microbiome of Rhipicephalus microplus from China.

BACKGROUND: Ticks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods. METHODS: Rhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species' sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison. RESULTS: The dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcus pneumonia infection, human granulocytic anaplasmosis, Shigella sonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer. CONCLUSIONS: Our study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick's midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases.

Variation of mitochondrial minichromosome composition in Hoplopleura lice (Phthiraptera: Hoplopleuridae) from rats.

BACKGROUND: The family Hoplopleuridae contains at least 183 species of blood-sucking lice, which widely parasitize both mice and rats. Fragmented mitochondrial (mt) genomes have been reported in two rat lice (Hoplopleura kitti and H. akanezumi) from this family, but some minichromosomes were unidentified in their mt genomes. METHODS: We sequenced the mt genome of the rat louse Hoplopleura sp. with an Illumina platform and compared its mt genome organization with H. kitti and H. akanezumi. RESULTS: Fragmented mt genome of the rat louse Hoplopleura sp. contains 37 genes which are on 12 circular mt minichromosomes. Each mt minichromosome is 1.8-2.7 kb long and contains 1-5 genes and one large non-coding region. The gene content and arrangement of mt minichromosomes of Hoplopleura sp. (n = 3) and H. kitti (n = 3) are different from those in H. akanezumi (n = 3). Phylogenetic analyses based on the deduced amino acid sequences of the eight protein-coding genes showed that the Hoplopleura sp. was more closely related to H. akanezumi than to H. kitti, and then they formed a monophyletic group. CONCLUSIONS: Comparison among the three rat lice revealed variation in the composition of mt minichromosomes within the genus Hoplopleura. Hoplopleura sp. is the first species from the family Hoplopleuridae for which a complete fragmented mt genome has been sequenced. The new data provide useful genetic markers for studying the population genetics, molecular systematics and phylogenetics of blood-sucking lice.