Microbial communities and symbionts in the hard tick Haemaphysalis longicornis (Acari: Ixodidae) from north China

Abstract

Background: Close relationships between ticks and microbial communities are important for tick fitness and pathogen colonization and transmission. Haemaphysalis longicornis, distributed widely in China, can carry and transmit various pathogens and pose serious damages to public health and economics. However, little is known about the broader array of microbial communities and symbionts in H. longicornis under natural conditions. In the present study, we investigated the composition of bacterial communities associated with H. longicornis and evaluated the putative symbionts.

Methods: The eubacterial 16S rRNA gene clone libraries of H. longicornis were constructed and analyzed by restriction fragment length polymorphism (RFLP) and DNA sequencing. In addition, diagnostic PCR was performed to assess the prevalence, vertical transmission and infection sites of the symbionts in H. longicornis.

Results: Vertically-transmitted symbionts, potential pathogens and allochthonous nonpathogenic bacteria were identified from the field-collected H. longicornis. Three types of symbionts (Coxiella-like, Arsenophonus-like and Rickettsia-like symbionts) were identified in a single host simultaneously. A series of analyses revealed the vertical transmission, prevalence, and infection sites of these symbionts. However, only Coxiella-like bacteria were transmitted stably in the laboratory-reared ticks. In addition, we identified a novel Coxiella-like agent with 95.31% sequence similarity to the taxon described previously.

Conclusions: The present study demonstrated that natural H. longicornis harboured a diverse array of microbial communities. Three types of symbionts were identified in a single host simultaneously. Moreover, high prevalence, vertical transmission and the infection sites supported an obligate symbiotic association between Coxiella symbiont and its host. The role of Coxiella symbiont in the host fitness and the interaction among microbial communities remained to be elucidated. Our investigation of microbial communities in the ticks revealed the complexity of ecological interactions between host and microbe and provided insight for the biological control of ticks.

Figure 1

Detection of vertical transmission of CLS-Hl (a), RLS-Hl (b) and ALS-Hl (c) by diagnostic PCR amplification. Lanes 1 to 8: M, DNA ladder; E, eggs from field-collected females; L, larvae; N, nymphs; AF: adult females; AM: adult males; E1: eggs from lab-reared females; N, negative control (distilled water).

Figure 2

Detection of infection sites of CLS-Hl (a), RLS-Hl (b) and ALS-Hl (c) by diagnostic PCR amplification. Lanes 1 to 6: M, DNA ladder; O, ovaries; Mt, Malpighian tubules; Gs, salivary glands; Mg, midguts; N, negative control (distilled water).

Figure 3

Phylogenetic tree of two types of Coxiella-like bacteria based on 16S rRNA gene sequence similarity. The tree was rooted with Bacillus subtilis (GenBank: X60646) and constructed using neighbour-joining method and clustering nodes were also recovered in maximum likelihood method. Numbers at nodes represent the levels of bootstrap support (%) based on neighbour-joining analysis of 1000 replicated data sets. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.