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. 2020 Apr 22:11:493.
doi: 10.3389/fmicb.2020.00493. eCollection 2020.

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny

Affiliations

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny

Michael Ben-Yosef et al. Front Microbiol. .

Abstract

Obligatory hematophagous arthropods such as lice, bugs, flies, and ticks harbor bacterial endosymbionts that are expected to complement missing essential nutrients in their diet. Genomic and some experimental evidence support this expectation. Hard ticks (Acari: Ixodidae) are associated with several lineages of bacterial symbionts, and very few were experimentally shown to be essential to some aspects of tick's fitness. In order to pinpoint the nature of interactions between hard ticks and their symbionts, we tested the effect of massive elimination of Coxiella-like endosymbionts (CLE) by antibiotics on the development and fitness of the brown dog tick (Rhipicephalus sanguineus). Administration of ofloxacin to engorged (blood fed) nymphs resulted in significant and acute reduction of their CLE loads - an effect that also persisted in subsequent life stages (aposymbiotic ticks). As a result, the post-feeding development of aposymbiotic female (but not male) nymphs was delayed. Additionally, aposymbiotic adult females needed a significantly prolonged feeding period in order to replete (detach from host), and had reduced engorgement weight and a lower capacity to produce eggs. Consequently, their fecundity and fertility were significantly reduced. Eggs produced by aposymbiotic females were free of CLE, and the resulting aposymbiotic larvae were unable to feed successfully. Our findings demonstrate that the observed fitness effects are due to CLE reduction and not due to antibiotic administration. Additionally, we suggest that the contribution of CLE is not mandatory for oocyte development and embryogenesis, but is required during feeding in females, when blood meal processing and tissue buildup are taking place. Presumably, under these extreme physiological demands, CLE contribute to R. sanguineus through supplementing essential micro- and macronutrients. Further nutrient complementary studies are required to support this hypothesis.

Keywords: antibiotic treatment; arthropod symbiosis; hematophagy; reproductive fitness; ticks.

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Figures

FIGURE 1
FIGURE 1
A visual depiction of the tick’s life cycle relative to the experimental procedures. Antibiotics or saline were injected once only to replete nymphs. Development and fitness measurements, symbiont quantifications (qPCR, PCR) and microbiome surveys (NGS) were performed on injected nymphs and subsequent life stages.
FIGURE 2
FIGURE 2
Relative abundance of CLE in different life stages and according to treatment as quantified by qPCR. (A) CLE abundance in replete nymphs (RN), teneral 3-day-old antibiotic-treated or non-treated females (see below for abbreviations) and mature 40-day-old females (MF) (n = 5–6 in each group). Antibiotics were administered in saline (ofloxacin, OFX or tetracycline, TET) or saline:DMSO solution (rifampicin, RIF), and implemented by injection to nymphs at 50 ng/mg weight. Control treatments included no treatment (C1) and saline (C2) or saline:DMSO (C3) injections. (B) CLE abundance in mature 40-day-old, unfed females (MF) and males (MM), feeding females (sampled 7 days after release on a host, FF), and post oviposition females (POF) previously injected as nymphs with ofloxacin (white boxes) or saline (shaded boxes). Different letters above means or asterisks (*), denote significant difference (Tukey HSD or ANOVA comparisons, P < 0.05; n = 5–11 in each group). (C) Diagnostic PCR detecting the 16S rRNA gene of CLE in 9 eggs (upper panel) and 9 larvae (lower panel) produced by females injected as nymphs with saline (C2) or ofloxacin (OFX). A 1kb DNA ladder (L), and two CLE positive (1,2) and negative (no template; 3,4) control assays are included in each panel. The tick’s 18S rRNA gene was consistently amplified and detected in all samples (not shown).
FIGURE 3
FIGURE 3
Effect of ofloxacin (OFX) on nymphal development: (A) Molting dynamics and (B) male and female nymph pre-molt period, following injections of saline (C2, shaded bars) or saline containing antibiotics (OFX, dosed at 50 ng/mg nymph weight, empty bars). Injections were performed 24–48 h after the nymphs had left their hosts. Different letters above means denote significant difference (Tukey HSD comparisons, P < 0.05; n = 44–95 in each group). Females developed faster than males and suffered a small but significant delay in molting following injection of antibiotics. Male development was not affected.
FIGURE 4
FIGURE 4
Fitness parameters of adult females previously injected as nymphs with ofloxacin (OFX) or saline (C2; empty and shaded bars/boxes, respectively): (A) Dynamics of detachment from the host and mean feeding period, (B) correlation between weight and body size (scutal index) and mean engorgement weight of replete females, (C) correlation between egg-mass weight and body size, and mean fecundity of females, (D) treatment-related differences in percent of weight converted to eggs and (E) egg hatching rate. Additionally depicted is (F) feeding success of larvae originating from antibiotic-treated and saline-injected females (number of replete larvae/mg larvae released on a host; see also Figure 2C depicting the detection of CLE in these larvae). Least square means or means (depicted by bar and box-plots, respectively) along with their standard errors are indicated in each figure. Asterisks (*) indicate significant differences among groups. Females: n = 27–30 in each group. Larvae: n = 6 replicates in each group.
FIGURE 5
FIGURE 5
Microbiomes of nymphs: (A) Cluster and (B) Principal Coordinate Analyses based on Bray–Curtis distance measures, depicting similarities between microbiomes of untreated (C1, filled circles), saline-injected (C2, filled triangles), or antibiotic-treated (OFX, empty triangles) replete nymphs, sampled a week after injections (n = 5 in each group). Coupled to the cluster dendrogram is the abundance data matrix for the 15-most prevalent OTUs detected in our dataset. Color intensity is proportional to the relative abundance values in each column. Principal Coordinate scatterplots were ordinated according to the first two principal components representing the highest percentage of explained variability. Each point represents an individual sample. Prior to analyses, sequences were classified into OTUs having 97% sequence similarities (single and doubletons were excluded). A complete list of OTUs composing these microbiomes is available at Supplementary Table S6.
FIGURE 6
FIGURE 6
Microbiomes of females: (A) Cluster and (B) Principal Coordinate Analyses based on Bray–Curtis distance measures, depicting similarities between microbiomes of saline-injected, unfed (C2, filled triangles) or fed (C2-fed, inverted filled triangles) females, and antibiotic-treated, unfed (OFX, empty triangles), or fed (OFX-fed, inverted empty triangles) females (n = 5 in each group). Additionally depicted are field-collected, unfed females (FC, filled diamonds), n = 5. Coupled to the cluster dendrogram is the abundance data matrix for the 15 most prevalent OTUs detected. Color intensity is proportional to the relative abundance values in each column. Principal Coordinate scatterplots were ordinated according to the first two principal components representing the highest percentage of explained variability. Each point represents an individual sample. Prior to analyses, sequences were classified into OTUs having 97% sequence similarities (single and doubletons were excluded). See Supplementary Table S7 for a complete list of OTUs composing these microbiomes.

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