Characterization of the primary endosymbionts in the salivary secretion of the lone star tick (Amblyomma americanum)

The lone star tick (Amblyomma americanum) is an important vector of Francisella tularensis, Ehrlichia chaffeensis, E. ewingii, and Heartland virus, as well as being one of the causal factors of the alpha-gal syndrome. Tick endosymbiotic bacteria are commensally associated with ticks by nutritional complementation, such as providing essential vitamin B. Abundant tick endosymbionts lowering the vectorial capacity of ticks have been reported in some cases. We hypothesized that two of the primary endosymbionts of the lone star tick, Coxiella-like endosymbiont (CLE) and Rickettsia amblyommatis (Ra), are secreted at the time of tick feeding, which likely affects the host immune responses. We tested this hypothesis by examining partially-fed female ticks' salivary secretion (SS) and salivary gland (SG). Both microscopic observation and species-specific quantitative PCR (qPCR) supported the hypothesis. We found that CLE is predominant in SG at 97.1% (33/34) and SS at 58.8% (20/34), while lower frequency and abundance were observed for Ra, with a prevalence of 41.2% (14/34) in SG and 17.6% (6/34) in SS. Microscopic observation also supported the presence of bacteria in SS, with two different morphotypes of bacteria in the tick SS, likely CLE (cocci-form) and Ra (rod-shaped), which were confirmed by in situ hybridization using species-specific probes targeting the 16S rRNA of both endosymbionts. Additionally, we plan to use immunohistochemistry to detect alpha-gal molecules in SG and SS, correlating their presence with secreted endosymbionts. We will further examine the effects of tick endosymbionts on the host immune system during tick feeding and pathogen transmission.