Developmental Proteomics Reveals the Dynamic Expression Profile of Global Proteins of Haemaphysalis Longicornis (Parthenogenesis)

Background: Ticks are important parasites that cause more diseases than most other animal parasites. Haemaphysalis longicornis is used as an experimental animal model for the study of three-host ticks due to its special life cycle and easy maintenance in the laboratory and in its reproduction. The life cycle of H. longicornis goes through a tightly regulated life cycle to adapt to the changing host and environment, and these stages of transition are also accompanied by proteome changes in the body. Methods: In this study, the aim was to use the isobaric tags for relative and absolute quantification (iTRAQ) technique to systematically describe and analyze the dynamic expression of protein and the molecular basis of the proteome of H. longicornis in seven differential developmental stages (eggs, unfed larvae, fed larvae, unfed nymphs, fed nymphs unfed adults, and fed adults). Results: A total of 2,059 proteins were identified, and their expression profiles were classified at different developmental stages. In addition, it was found that tissue and organ development-related proteins and metabolism-related proteins showed that they were involved in different physiological processes throughout the life cycle through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed proteins (DEPs). More importantly, we found that the upregulated proteins of fed adult ticks were mainly related to yolk absorption, degradation, and ovarian development-related proteins. The abundance of the cuticle proteins in the unfed stages were significantly higher compared with those of the fed ticks in the previous stages. Conclusions: In short, the protein spectrum changes identified in this study provide a reference proteome for future studies of tick functional proteins and provide candidate targets for elucidating tick development and developing new tick control strategies.

The Ovarian Development Genes of Bisexual and Parthenogenetic Haemaphysalis longicornis Evaluated by Transcriptomics and Proteomics

The tick Haemaphysalis longicornis has two reproductive groups: a bisexual group (HLBP) and a parthenogenetic group (HLPP). The comparative molecular regulation of ovarian development in these two groups is unexplored. We conducted transcriptome sequencing and quantitative proteomics on the ovaries of HLBP and HLPP, in different feeding stages, to evaluate the molecular function of genes associated with ovarian development. The ovarian tissues of HLBP and HLPP were divided into three feeding stages (early-fed, partially-fed and engorged). A total of 87,233 genes and 2,833 proteins were annotated in the ovary of H. longicornis in the different feeding stages. The differentially expressed genes (DEGs) of functional pathway analysis indicated that Lysosome, MAPK Signaling Pathway, Phagosome, Regulation of Actin Cytoskeleton, Endocytosis, Apoptosis, Insulin Signaling Pathway, Oxidative Phosphorylation, and Sphingolipid Metabolism were most abundant in the ovary of H. longicornis in the different feeding stages. Comparing the DEGs between HLBP and HLPP revealed that the ABC Transporter, PI3K-Akt Signaling Pathway and cAMP Signaling Pathway were the most enriched and suggested that the functions of signal transduction mechanisms may have changed during ovarian development. The functions of the annotated proteome of ovarian tissues were strongly correlated with the transcriptome annotation results, and these were further validated using quantitative polymerase chain reaction (qPCR). In the HLBP, the expression of cathepsin L, secreted proteins and glycosidase proteins was significantly up-regulated during feeding stages. In the HLPP, the lysozyme, yolk proteins, heat shock protein, glutathione S transferase, myosin and ATP synthase proteins were up-regulated during feeding stages. The significant differences of the gene expression between HLBP and HLPP indicated that variations in the genetic background and molecular function might exist in the two groups. These results provide a foundation for understanding the molecular mechanism and exploring the functions of genes in the ovarian development of different reproductive groups of H. longicornis.

Mapping the Potential Distribution of Ticks in the Western Kanto Region, Japan: Predictions Based on Land-Use, Climate, and Wildlife

Background: Tick distributions have changed rapidly with changes in human activity, land-use patterns, climate, and wildlife distributions over the last few decades. Methods: To estimate potential distributions of ticks, we conducted a tick survey at 134 locations in western Kanto, Japan. We estimated the potential distributions of six tick species (Amblyomma testudinarium Koch, 1844; Haemaphysalis flava Neumann, 1897; Haemaphysalis kitaokai Hoogstraal, 1969; Haemaphysalis longicornis Neumann, 1901; Haemaphysalis megaspinosa Saito, 1969; and Ixodes ovatus Neumann, 1899) using MaxEnt modeling based on climate patterns, land-use patterns, and the distributions of five common wildlife species: sika deer (Cervus nippon Temminck, 1838), wild boar (Sus scrofa Linnaeus, 1758), raccoon (Procyon lotor Linnaeus, 1758), Japanese raccoon dog (Nyctereutes procyonoides Gray, 1834), and masked palm civet (Paguma larvata C.E.H. Smith, 1827)). Results: We collected 24,546 individuals of four genera and 16 tick species. Our models indicated that forest connectivity contributed to the distributions of six tick species and that raccoon distribution contributed to five tick species. Other than that, sika deer distribution contributed to H. kitaokai, and wild boar distribution, bamboo forest, and warm winter climate contributed specifically to A. testudinarium. Conclusions: Based on these results, the dispersal of some tick species toward residential areas and expanded distributions can be explained by the distribution of raccoons and by forest connectivity.

Antiviral RNA interference in disease vector (Asian longhorned) ticks

Disease vectors such as mosquitoes and ticks play a major role in the emergence and re-emergence of human and animal viral pathogens. Compared to mosquitoes, however, much less is known about the antiviral responses of ticks. Here we showed that Asian longhorned ticks (Haemaphysalis longicornis) produced predominantly 22-nucleotide virus-derived siRNAs (vsiRNAs) in response to severe fever with thrombocytopenia syndrome virus (SFTSV, an emerging tick-borne virus), Nodamura virus (NoV), or Sindbis virus (SINV) acquired by blood feeding. Notably, experimental acquisition of NoV and SINV by intrathoracic injection also initiated viral replication and triggered the production of vsiRNAs in H. longicornis. We demonstrated that a mutant NoV deficient in expressing its viral suppressor of RNAi (VSR) replicated to significantly lower levels than wildtype NoV in H. longicornis, but accumulated to higher levels after knockdown of the tick Dicer2-like protein identified by phylogeny comparison. Moreover, the expression of a panel of known animal VSRs in cis from the genome of SINV drastically enhanced the accumulation of the recombinant viruses. This study establishes a novel model for virus-vector-mouse experiments with longhorned ticks and provides the first in vivo evidence for an antiviral function of the RNAi response in ticks. Interestingly, comparing the accumulation levels of SINV recombinants expressing green fluorescent protein or SFTSV proteins identified the viral non-structural protein as a putative VSR. Elucidating the function of ticks’ antiviral RNAi pathway in vivo is critical to understand the virus-host interaction and the control of tick-borne viral pathogens.

Acaricidal properties of 5-methylfurfural identified from Valeriana fauriei and its structural analogues against synanthropic mites and Asian longhorned tick with color alterations

Acaricidal activities and color alterations of 5-methylfurfural derived from Valeriana fauriei essential oil and its structural analogues against Dermatophagoides farinae, D. pteronyssinus, Haemaphysalis longicornis and Tyrophagus putrescentiae were evaluated in the present study. Based on the LD50 values of 5-methylfurfural and its analogues, 4,5-dimethylfurfural showed the highest acaricidal activity (LD50; 9.95, 9.91, and 7.12 μg/cm2), followed by 5-methylfurfural (11.87, 11.00, and 8.59 μg/cm2), furfural (12.94, 13.25, and 10.36 μg/cm2), and V. fauriei essential oil (15.15, 13.64, and 10.14 μg/cm2) against D. farinae, D. pteronyssinus and T. putrescentiae, respectively. However, all tested compounds did not show the acaricidal activities against H. longicornis. Interestingly, the color alterations of the mites and ticks were observed by furfural, 5-methylfurfural, and 4,5-dimethylfurfural from colorless to red brown during the acaricidal experiments. Furthermore, 4,5-dimethylfurfural which exhibited the highest acaricidal activity was formulated as nanoemulsion. The nanoemulsion of 4,5-dimethylfurfural showed higher acaricidal activity than it was emulsified in ethanol. The nanoemulsion was also found to show color changes of the mites and ticks from colorless to red brown. The results suggest that 5-methylfurfural and its analogues could be developed as an effective and easy-to-recognize acaricides to mites and ticks.