De novo assembled transcriptomics assisted label-free quantitative proteomics analysis reveals sex-specific proteins in the intestinal tissue of Haemaphysalis qinghaiensis.

The hard tick Haemaphysalis qinghaiensis is the vector of a wide variety of infectious agents, such as spirochetes and other bacteria as well as viruses in the western plateau of China. Tick midgut is the key tissue involved in the host-pathogen-vector interface. Multiple midgut proteins are related to key functions in blood digestion, tick survival, and tick-borne pathogen transmission. However, information on the sex-specific proteins expressed in the midgut tissue of H. qinghaiensis for which the genome has not been sequenced is limited. Hence, we assembled and characterized the transcriptome of the H. qinghaiensis midgut and identified the differentially expressed genes (DEGs) in female and male ticks. The sequencing of the mRNA for this nonmodel species is essential for producing a protein database for mass spectrometry-based identification. Here, we combined high-throughput parallel sequencing and label-free quantitative proteomics analysis to extensively characterize the tick midgut using massive RNA sequencing and mass spectrometry, which allowed the detection of genes and proteins. A total of 279,186 transcripts were annotated into 125,790 coding sequences (CDSs), which were manually curated into 96 different gene families. A total of 12,837 DEGs between the two sexes were found by RNA-seq analysis. Of these, 5401 were upregulated genes, while 7436 were downregulated genes. The most common molecular functions were those related to the endocrine system, translation, signal transduction, transport, and catabolism. Meanwhile, the most common biological processes were related to cellular processes, metabolic processes, cellular anatomical entities, and cargo receptor activities. An analysis of the label-free protein quantitation dataset showed 272 upregulated proteins and 46 downregulated proteins when the fold-change was >2.0 (LC-MS/MS). Association analysis of the transcriptome and proteome with GO functional enrichment showed that the majority of the genes (proteins) were those related to catalytic activity, binding, cellular processes, metabolic processes, and responses to stimuli. This study aims to elucidate the digestive physiology of H. qinghaiensis as well as its physiological sexual dimorphism. This will allow the identification of protein candidates with physiological importance that could be used as targets to control the vector as well as the transmission of tick-borne pathogens to humans and animals.

The diurnal salivary glands transcriptome of Dermacentor nuttalli from the first four days of blood feeding.

The ixodid tick Dermacentor nuttalli is distributed from southern Siberia to North China and is a vector of many pathogens. This species can have severe impacts on animal husbandry and human health. To date, the control of D. nuttalli is limited to the use of acaricides such as organophosphorus, synthetic pyrethroids and amidine pesticides. There are no environmentally friendly or reliable prevention and control measures, and little is known regarding key antigens involved in blood feeding. Salivary glands are major tissues involved in the blood feeding and pathogen transmission of ticks. Therefore, this study focused on salivary glands tissue to identify the dominant antigens of D. nuttalli involved in tick feeding. For this, high-throughput RNA sequencing (RNA-seq) was used for analysis. The transcriptome of female D. nuttalli ticks was assembled and characterized, and differentially expressed genes (DEGs) were identified in the salivary glands of ticks that had not fed (0 h) and of ticks after 24, 48, 72 and 96 h of feeding. There were 22,802,784, 22,275,013, 26,629,453, 24,982,389, and 22,596,230 high-quality clean reads obtained from salivary glands tissues at the five different blood feeding time points. The total number of annotated unigenes was 100,347. The differences in gene expression between different time points were compared, and functional enrichment was performed. Quantitative reverse transcription PCR (RT‒qPCR) was used to validate the RNA-seq results, the results of which showed that the differences in expressed transcripts presented similar trends. Among the identified DEGs, the most numerous were those with catalytic and binding activities and those involved in diverse metabolic pathways and cellular processes. The expression patterns of homologous and family-member proteins throughout the blood feeding period exhibited significant differences, strongly suggesting that the transcriptome composition is highly dynamic and likely subjected to important variation throughout the life cycle. Studies of gene sequences in D. nuttalli will greatly increase the information on tick protective antigens, which could potentially function as effective vaccine candidates or drug targets for the development of environmentally friendly acaricides.