Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

Abstract

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and A. americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.

Assessment of tick antioxidant responses to exogenous oxidative stressors and insight into the role of catalase in the reproductive fitness of the Gulf Coast tick, Amblyomma maculatum

“…For both the unfed and partially fed ticks, the dissections were performed in ice‐cold M‐199 buffer (Sigma Aldrich, St Louis, MO, USA) and tracheal material in the individual tissues was rinsed away with the same ice‐cold M‐199 buffer, as described by Villarreal et al . (). The dissected midguts or salivary glands were pooled based on the experimental sample type described above and then stored in either RNAlater (Ambion, Austin, TX, USA) for later mRNA extraction or in protein storage buffer (0.5 M piperazine N, N0‐bis‐2‐ethane sulphonic acid, 20 mM Ethylene glycol tetraacetic acid (EGTA), protease inhibitor cocktail; Roche, Indianapolis, IN, USA; 40% glycerol, pH 6.8) followed by storage at −80 °C.…”

“…Total RNA isolation, cDNA synthesis and qRT‐PCR were performed as described previously (Browning et al ., ; Villarreal et al ., ; Budachetri & Karim, ). Briefly, total RNA was extracted from the tick salivary glands or midguts stored in RNAlater using an Illustra RNAspin Mini RNA isolation kit (GE Healthcare, Piscataway, NJ, USA) following the manufacturer's instructions.…”

Syntaxin 1A modulates the sexual maturity rate and progeny egg size related to phase changes in locusts

“…However, genes related to genetic information processing, including SNARE interactions in vesicular transport, proteasome, DNA replication, RNA polymerase and ribosome, are only upregulated at adult stage (Chen et al, 2010). SNARE proteins, such as vti1A, vti1B, NSF and SNAP-25, have been found to be involved in regulating reproduction in ticks Villarreal et al, 2013). Among these SNARE protein genes, Syx1A has been reported to be up-regulated the most strongly in G locusts (Chen et al, 2010).…”

An insight into the functional role of thioredoxin reductase, a selenoprotein, in maintaining normal native microbiota in the Gulf Coast tick (Amblyomma maculatum)

“…The tick salivary glands were fixed in 1X PBS containing 4% formaldehyde and kept at −4°C until further manipulation, as described elsewhere (Villarreal et al 2013). Briefly, the salivary glands were permeabilized using 0.5% Triton X-100 for 30 min and then blocked in 1X PBS containing 3% bovine serum albumin (BSA) for 1 h at room temperature.…”