Infection with Borrelia afzelii reduces moulting time of Ixodes ricinus ticks


 Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated non-pathogenic microorganisms. Both types of micro-organisms can influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme disease in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a large diversity of non-pathogenic bacteria. The purpose of this study was to test experimentally whether B. afzelii and tick-associated non-pathogenic bacteria influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were washed with bleach, which reduced the abundance of non-pathogenic bacteria in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were allowed to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice. In contrast, we found no evidence that B. afzelii infection or larval dysbiosis influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A power analysis found that our sampling effort had sufficient power (>80%) to detect small effects (difference of 5% – 10%) of either B. afzelii infection or larval dysbiosis. Under the laboratory conditions of our study, B. afzelii appears to be a mostly passive passenger in I. Ricinus ticks.


Introduction
The epidemiology of vector-borne diseases is highly sensitive to the biology of the arthropod vector.
Theoretical models that derive the reproduction number (R 0 ) of vector-borne diseases show that this quantity depends on a number of vector behavioural and life history traits, such as the biting rate, survival, and reproduction [1][2][3] . Infection by vector-borne pathogens can reduce the survival and reproduction of the arthropod vector [4][5][6][7] , and thereby reduce the R 0 of the vector-borne disease.
Conversely, vector-borne pathogens can manipulate their arthropod vectors (e.g., increase the biting rate, shift resources from reproduction to survival) to increase their own transmission, and thereby increase the R 0 of the vector-borne disease [8][9][10][11] . Measuring the effects of vector-borne pathogens on the tness of their arthropod vectors is therefore important for understanding the epidemiology of vector-borne diseases.
Arthropod vectors also form intimate associations with microorganisms that do not cause diseases in vertebrate hosts 12 . These non-pathogenic bacteria can be inherited from the mother (i.e., vertical transmission) or acquired from the external environment 13 . Some are obligate symbionts that provide a critical function, for example providing a vitamin that is missing from a nutrient-poor hematophagous (blood-based) diet 14 . The general importance of arthropod-symbiont associations can be demonstrated by dysbiosis treatments (e.g., antibiotics) that decrease the abundance of the symbiont resulting in dramatic reductions in the tness of the arthropod vector [15][16][17][18][19][20] . These non-pathogenic bacteria can also in uence vector competence, which is the ability of the arthropod vector to acquire, maintain, and transmit vector-borne pathogens over its lifecycle [21][22][23][24][25] . Thus, arthropod-associated bacteria can in uence the R 0 of vector-borne diseases by their effects on both vector tness and vector competence for vectorborne pathogens.
Hard ticks of the genus Ixodes transmit a diversity of tick-borne pathogens such as the spirochete bacteria belonging to the Borrelia burgdorferi sensu lato (sl) complex, which includes the causative agents of Lyme disease 3,26,27 . Some reviews have suggested that B. burgdorferi sl can adaptively manipulate its Ixodes tick vector to enhance its own transmission, and hence the R 0 of Lyme disease 11,28 . Ixodes ticks infected with B. burgdorferi sl differ from their uninfected counterparts in a number of tick phenotypes including questing behaviour, survival rates, body weight, and energy reserves [29][30][31][32][33][34][35] .
However, as most of these studies are correlational in nature, there is a need for more controlled infection experiments to quantify the effects that B. burgdorferi sl pathogens have on the tness of their tick vectors. Ixodes ticks also contain a diversity of non-pathogenic bacteria 36,37 , but their effects on tick tness are even less clear 38,39 .
To investigate whether B. burgdorferi sl and tick-associated non-pathogenic bacteria can in uence the population ecology of Ixodes ticks, it is important to consider the tick lifecycle 1,3 . Ixodes ticks have three motile stages, larva, nymph, and adult female, that must take a blood meal from a vertebrate host in order to graduate to the next step in the lifecycle. Larvae acquire B. burgdorferi sl pathogens after engorging on an infected host, and subsequently moult into infected nymphs. The size of the larval blood meal determines the body size and energy reserves of the resultant nymph, which in uence nymphal survival and the ability to search for a host the following year 28,34 . The density of infected nymphs (DIN) largely determines the risk of infection for vertebrate hosts including humans 40 . Adult female ticks typically feed on non-competent vertebrate hosts, such as deer 41,42 and their main contribution to the R 0 of Lyme disease is via the production of larvae. In summary, the R 0 of B. burgdorferi sl is highly sensitive to the life history traits of immature Ixodes ticks, such as larva-to-nymph moulting success, survival of immature ticks, and body weight of immature ticks 1-3 .
Our research group uses experimental infections with the pathogen B. afzelii, the tick I. ricinus, and rodent reservoir hosts to ask questions about the ecology of this common European Lyme disease system 43 . The purpose of the present study was to test whether B. afzelii infection and tick-associated nonpathogenic bacteria in uence the life history traits of immature Ixodes ricinus ticks under standard laboratory conditions.

Materials And Methods
Background of the study In a previous study, we set out to test whether manipulation of the larval microbiota would affect the probability that I. ricinus would acquire B. afzelii during the larval blood meal 44 . The larval microbiota was reduced 28-fold by washing eggs with bleach, whereas the control eggs were washed with water. These dysbiosed larvae and control larvae were fed on either B. afzelii-infected mice or uninfected control mice and the resultant engorged larvae were allowed to moult into nymphs under laboratory conditions 44 . In addition to the B. afzelii and microbiome dynamics presented in 44 , we measured ve life history traits including (i) engorged larval weight, (ii) unfed nymphal weight, (iii) larva-to-nymph moulting success, (iv) larva-to-nymph moulting time, and (v) immature tick survival. These life history data therefore represent an excellent opportunity to test whether B. afzelii infection and the larval microbiota in uence the tness of immature I. ricinus ticks under laboratory conditions.

Mice, Borrelia afzelii, and Ixodes ricinus ticks
We used Mus musculus BALB/c mice as the rodent host because we have a history of successful experimental infections with this mouse strain [45][46][47] . During the study, the mice were maintained in individual cages and were given food and water ad libitum. The mice were experimentally infected via tick bite with B. afzelii strain NE4049. This strain was originally isolated from an I. ricinus tick in Switzerland and has multilocus sequence type (MLST) 679 and strain identi cation number 1887 in the Borrelia MLST database, and ospC major group A10. We chose to work with strain NE4049 because it is highly infectious to both rodents and I. ricinus ticks 45,46,48 . The adult female I. ricinus ticks used to produce the eggs and larvae for this study were collected from wild roe deer captured in the Sylve d'Argenson forest near Chizé, France. The I. ricinus ticks used to experimentally infect the mice with B. afzelii strain NE4049 came from a laboratory colony established in 1978 at the University of Neuchâtel.
Approval for the use of experimental animals in the study The authors complied with the ARRIVE guidelines. This study followed the Swiss legislation on animal experimentation. The commission that is part of the "Service de la Consommation et des Affaires Vétérinaires (SCAV)" of the canton of Vaud, Switzerland evaluated and approved the ethics of this study. The SCAV of the canton of Neuchâtel, Switzerland issued the animal experimentation permits for the study (NE04-2014) and for the maintenance of the I. ricinus tick colony on vertebrate hosts at the University of Neuchâtel (NE05-2014).
Egg washing treatment of ten eld-collected families of I. ricinus Ten engorged adult female I. ricinus ticks were collected from wild roe deer captured in the Sylve d'Argenson forest near Chizé, France. The ten female ticks were allowed to lay their eggs in the laboratory ( Figure 1). Four weeks after deposition, each of the 10 clutches of eggs was split into two batches. One batch was rinsed with 10% bleach while the other batch was rinsed with distilled water. The purpose of the bleach rinsing treatment was to remove bacteria on the outer surface of the eggshell, whereas the water rinsing treatment functioned as a control. Eggs were placed in plastic petri dishes sealed with para lm and allowed to hatch into larvae. Thus, for each of the 10 eld-collected tick families, we obtained batches of larvae that had hatched either from bleach-rinsed eggs or from water-rinsed eggs (20 batches in total). To determine whether the bleach treatment had reduced the microbiota in the larval ticks, a group of ~400 larvae was frozen for each of the 20 batches at six weeks after hatching.

Experimental infection of mice with B. afzelii via tick bite
For the main experiment, 40 female, 5-week-old, speci c pathogen-free BALB/c mice were randomly assigned to either the control group or the infection group ( Figure 1). Each mouse in the control group (n = 20) was infested with 5 uninfected I. ricinus nymphs, whereas each mouse in the infected group (n = 20) was infested with 5 nymphs infected with B. afzelii strain NE4049. In this way, each of the 40 mice had a similar immune experience with I. ricinus nymphs. These nymphs came from the University of Neuchâtel laboratory colony and the creation of these nymphs is explained in section 1 of the electronic supplementary material (ESM). Five weeks after the nymphal challenge, an ear tissue biopsy and a blood sample were taken from each of the 40 mice to determine their infection status. The ear tissue biopsy was tested for the presence of B. afzelii using qPCR. The blood sample was tested for the presence of B. afzelii-speci c antibodies using a commercial ELISA. These tests con rmed that the 20 mice in the infected group were infected with B. afzelii, whereas the 20 mice in the control group remained uninfected.
Feeding I. ricinus larvae on B. afzelii-infected mice and uninfected control mice The larvae for each of the 20 batches were split into two groups of ~100 larvae. One group was fed on an uninfected control mouse whereas the other group was fed on a B. afzelii-infected mouse ( Figure 1). For each of the 40 mice, we collected a maximum of 60 engorged larvae; each engorged larva was placed in an individual Eppendorf tube and was allowed to moult into a nymph. Four weeks after the larva-tonymph moult, 10 nymphs were randomly selected from each mouse and were frozen at -80°C. In summary, we froze ~400 nymphs (10 families*2 egg washing treatments* 2 mouse infection statuses*10 nymphs/mouse) for future molecular analyses of B. afzelii infection.
Life history traits of immature I. ricinus ticks For each mouse, we randomly selected a maximum of 20 engorged larval ticks and weighed them within 2 days of their collection (n = 742 engorged larvae). Four weeks after the larva-to-nymph moult, we weighed the same group of nymphs that had been weighed as engorged larval ticks (n = 742 -89 = 653 unfed nymphs, as 89 engorged larvae did not moult into nymphs). To increase the sample size, we weighed an additional 10 unfed nymphs for each of the 40 mice (n = 414 unfed nymphs). The engorged larvae and the unfed nymphs were weighed to a precision of 0.1 mg using an Ultra Microbalance (UMT 5 Comparator, Mettler Toledo, Greifensee, Switzerland).
To determine the larva-to-nymph moulting success, larva-to-nymph moulting time, and immature tick survival, the moulting status and alive status of the engorged larvae (n = 1739) were checked every 2 or 3 days over the larva-to-nymph moulting period (a period of 16 days from 8 August to 24 August 2016).
The moulting status and alive status of all the engorged larvae were checked one last time (13 September 2016), which was 74 days after the larvae were fed on the mice (1 July 2016).
In summary, we collected 5 life history traits for these immature I. ricinus ticks: (i) engorged larval weight within 2 days of drop-off (n = 742), (ii) unfed nymphal weight at 4 weeks after the larva-to-nymph moult (n = 1067), (iii) larva-to-nymph moulting success over 74 days following the start of the larval blood meal (n = 1739), (iv) larva-to-nymph moulting time over 55 days following the start of the larval blood meal (n = 1529), and (v) immature tick survival over 74 days following the start of the larval blood meal (n = 1739). The numbers of engorged larval ticks that were recovered from each of the 40 mice were also recorded.

Molecular methods for I. ricinus larval ticks
The larval ticks that had been frozen for molecular analysis were split into 40 sub-groups with ~200 larval ticks per sub-group (10 families x 2 egg washing treatments x 2 replicates). DNA extraction of these 40 sub-groups of larvae was done using a DNeasy Blood & Tissue spin-column kit (QIAGEN) and following the manufacturer's instructions. The DNA of each sub-group was eluted into 100 μl of distilled water, and the DNA concentration was measured using a Nanodrop. For qPCR, the DNA concentration of each sub-group was adjusted to 5 ng/μl. Two qPCR assays were performed independently for each DNA extraction: tick calreticulin and bacterial 16S rRNA. Each qPCR assay contained 3 μl of template for a total of 15 ng of DNA. The molecular methods are described in detail in section 2 of the ESM.

Molecular methods for I. ricinus nymphal ticks
For each of the 40 mice, a sample of 7 -8 unfed nymphs (that had fed as larvae on the mice) were randomly selected and tested for B. afzelii infection. Nymphs were crushed using the TissueLyser II by shaking them with a stainless-steel bead (1.4 mm in diameter) at a frequency of 30 Hz for 1 min. Total DNA was extracted for each tick using the DNeasy Blood & Tissue 96-well extraction kit (QIAGEN) and following the manufacturer's instructions. The DNA of each tick was eluted into 65 μl of distilled water, and the DNA concentration was measured for each of the ~300 nymphs using a Nanodrop. For qPCR, the DNA concentration of each tick was adjusted to 5 ng/μl. Three qPCR assays were performed independently for each DNA extraction: tick calreticulin, bacterial 16S rRNA, and Borrelia agellin. Each qPCR assay contained 3 μl of template for a total of 15 ng of DNA. The molecular methods are described in detail in section 2 of the ESM.

ELISA to test whether mice developed IgG antibodies against B. afzelii
We used the SERION® ELISA classic Borrelia burgdorferi IgG/IgM immunoassay to detect the presence of IgG antibodies against B. afzelii. Mouse sera was diluted 1:100 in blocking solution that was composed of 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The ELISA plate was incubated with the mouse sera for 45 min at room temperature. The plate was washed three times with a solution of 0.1% TWEEN in PBS for 5 min. The plate was incubated with a goat anti-mouse IgG horseradish peroxidase conjugate diluted 1:5000 in the blocking solution for 45 min at room temperature. The plate was washed three times with PBS-TWEEN as described above. The nal step was to add 100 μl of tetramethylbenzidine (TMB) to each well. The absorbance at a wavelength of 652 nm was measured every two minutes for one hour to determine the strength of the IgG antibody response against B. afzelii. We used the area under the curve function, auc(), in R to calculate the total absorbance over the 60 min of the reaction.

Statistical Methods
Statistical analyses were performed using RStudio Version 1.2.5042. The lm() and glm() functions in the base package were used to create the Linear Models (LMs) and the Generalized Linear Models (GLMs), respectively. The lmer() and glmer() functions in the lme4 package were used to create the Linear Mixed effects Models (LMMs), and the Generalized Linear Mixed effects Models (GLMMs), respectively. The corrplot() function in the corrplot package was used to graph the correlation matrix of the life history traits of the immature I. ricinus ticks. Effects with p-values < 0.050 were considered statistically signi cant. After Bonferroni correction for 5 life history traits, p-values < 0.010 were considered statistically signi cant.
Effect of egg bleaching on the microbiota of I. ricinus larvae For each of the 40 sub-groups of larvae, we divided the 16S rRNA gene copy number of the batches of I. ricinus larvae by their calreticulin gene copy number. These 16S rRNA to calreticulin ratios were then log10-transformed to improve the normality of the data. To test whether the egg bleaching treatment reduced the microbiota of the resultant larvae, the log10-transformed 16S rRNA to calreticulin ratios were analysed using linear mixed effects models (LMMs). Egg washing treatment (2 levels: water and bleach) and tick family were modelled as xed and random factors, respectively.

Effect of B. afzelii infection and egg washing treatment on the life history traits of immature I. ricinus ticks
The response variables were the ve life history traits of the immature I. ricinus ticks: (i) engorged larval weight, (ii) unfed nymphal weight, (ii) larva-to-nymph moulting success, (iv) larva-to-nymph moulting time, and (v) immature tick survival. We used mixed effects models to analyze these response variables to account for the fact that ticks that fed on the same mouse are not statistically independent. The log10transformed engorged larval weight, log10-transformed unfed nymphal weight, and larva-to-nymph moulting time were treated as normally distributed response variables that were analyzed using LMMs. The larva-to-nymph moulting success and immature tick survival are binomial response variables that were analyzed using GLMMs. Egg washing treatment (2 levels: bleach and water) and mouse infection status (2 levels: infected and control) were modelled as xed effects. Mouse ID and tick family were modelled as random effects, with mouse ID nested in tick family. If the interaction between egg washing treatment and mouse infection status was not signi cant, the model was re-run without the interaction.

Effect of tick family and mouse ID on the life history traits of immature I. ricinus ticks
To test whether there were signi cant differences among the 10 tick families, the 5 life history traits were modelled using LMMs with normal errors or GLMMs with binomial errors with tick family as a xed effect and mouse ID as a random effect. To test whether there were signi cant differences among the 40 mice, the 5 life history traits were modelled as LMs or GLMs with mouse ID as a xed effect. As before, the weights of the engorged larvae and the weights of the unfed nymphs were log10-transformed.

Correlations between the life history traits of I. ricinus
For this correlation analysis, there were six variables: the number of engorged larvae that were recovered from each mouse and the ve life history traits of the immature I. ricinus ticks. The ve life history traits of the immature ticks included (i) engorged larval weight (mg), (ii) unfed nymphal weight (mg), (iii) larvato-nymph moulting success (%), (iv) larva-to-nymph moulting time (days), and (v) immature tick survival (%). For each of these six variables, the mean was calculated for each of the 40 mice. The pairwise correlations were calculated for each of the 15 pairs of variables across the 40 mice.

Results
Effect of egg bleaching on the microbiota of I. ricinus larvae The I. ricinus larvae that hatched from the eggs washed with bleach had a signi cantly lower bacterial load than the control larvae that hatched from the eggs washed with water ( Figure 2; c 2 = 25.359, df = 1, p = 0.0000005). The log10(16S rRNA/ calreticulin) ratio in the larval ticks of the control group (mean ratio = 6.61, 95% CI = 1.78-24.61) was 28 times higher than the bleach treatment group (mean ratio = 0.24, 95% CI = 0.06-0.88). The bleach treatment was therefore highly effective at reducing the microbiota in the larval ticks ( Figure 2).

B. afzelii induced a strong IgG antibody response in the infected mice
One of the control mice died during the study, so that the realized sample sizes were 19 uninfected control mice and 20 B. afzelii-infected mice. The ability of the ELISA to detect B. afzelii infection in the mice was unambiguous (Figure 3) because there was no overlap in absorbance between the control group (range = 637 to 1,692 absorbance units (AU)) versus the infected group (range = 10,756 AU to 12,572 AU). According to the ELISA, all of the 20 mice challenged with infected nymphs became infected with B. afzelii strain NE4049 and all of the 19 mice challenged with uninfected nymphs remained uninfected (Figure 3). The mean absorbance level of the infected mice (11,749 AU) was signi cantly higher (12.9x) compared to the control mice (912 AU; independent samples t-test: t = 43.88, df = 37, p < 0.000001).

Prevalence of B. afzelii infection in unfed I. ricinus nymphs
A total of 289 unfed nymphs were tested for B. afzelii via qPCR. Of the 289 nymphs, 106 nymphs had fed as larvae on uninfected control mice and 183 nymphs had fed as larvae on infected mice. The prevalence of B. afzelii infection in the nymphs that fed as larvae on the infected mice (71.0% = 130/183) was signi cantly higher (25.1x) compared to the nymphs that fed as larvae on the uninfected control mice Relationship between engorged larval weight and unfed nymphal weight The engorged larval weights and unfed nymphal weights were both measured for a sample of 653 immature I. ricinus ticks. Weights were taken for an additional 89 engorged larvae and 414 unfed nymphs, so that the total sample sizes were 742 engorged larvae and 1067 unfed nymphs, respectively. According to the simple linear regression model, the log10-transformed engorged larval weight was a highly signi cant predictor of the log10-transformed unfed nymphal weight, and the former explained 72.71% of the variation in the latter (Figure 4; regression: F 1, 651 = 1738, p < 0.000001; correlation: r = 0.854, t = 41.859, df = 652, p < 0.000001). Given that the engorged larvae and unfed nymphs were weighed independently and more than 10 weeks apart, the strong relationship between these two variables indicates that our immature tick weight measurements are highly reliable. For this paired sample of 653 immature ticks, the mean weight of the engorged larvae (438.7 mg) was 2.4x greater than the weight of the resultant unfed nymphs (180.8 mg), and this difference was highly signi cant (paired ttest: t = -229.53, df = 652, p < 0.000001). These results show that the engorged larvae lost a lot of weight during the larva-to-nymph moulting process (i.e., due to water loss and the digestion and excretion of faecal material). We found no evidence that B. afzelii infection in the mouse in uenced the e ciency with which the engorged larval weight was converted into unfed nymphal weight (see section 5 of the ESM).
A previous study had shown that I. scapularis larvae that took larger blood meals had a higher probability to acquire B. burgdorferi sensu stricto (ss) 49 . For the subset of I. ricinus larvae that fed on the 20 infected mice, we tested whether the size of the larval blood meal in uenced the probability that the larvae acquired B. afzelii, but we found no evidence for this hypothesis (see section 6 of the ESM).
Larva-to-nymph survival, moulting success, and moulting time Of the 1739 engorged larvae monitored, the mean larva-to-nymph moulting success over 74 days was 91.9% (1598/1739), and the mean immature tick survival over 74 days was 84.5% (1470/1739). Of the 141 larvae that did not moult, 134 died before reaching the moult, and the other 7 had not moulted after 74 days. Of the 1739 engorged larval ticks, the moulting time (days) was recorded for 1529 individuals. The median larva-to-nymph moulting time was 40 days (range = 38 days to 54 days).

Effect of B. afzelii infection and egg washing treatment on the life history traits of immature I. ricinus ticks
We found no signi cant effects of B. afzelii infection in mice, egg washing treatment, or their interaction on the engorged larval weights or unfed nymphal weights (Table 1; see section 7 of the ESM). The results remained the same when the explanatory variable of mouse infection status was replaced with tick infection status (see section 8 of the ESM). No signi cant effects of B. afzelii infection in mice, egg washing treatment, or their interaction were found on larva-to-nymph moulting success, larva-to-nymph moulting time, or immature tick survival (Table 1); the only exception was the effect of B. afzelii infection in mice on the larva-to-nymph moulting time ( Figure 5; Table 1; c 2 = 16.510, df = 1, p < 0.0001). The mean larva-to-nymph moulting time for the ticks that fed on the infected mice (41.4 days) was 1.0 day faster (2.4% faster) than the ticks that fed on the uninfected control mice (42.4 days). The effect of B. afzelii infection remained signi cant after using a non-parametric independent two-samples Wilcoxon test (W = 359542, p < 0.00001; see section 9 of the ESM) and after Bonferroni correction for multiple comparisons (i.e., p = 0.050/5 = 0.010).

Effects of tick family and mouse ID on the life history traits of immature I. ricinus ticks
We found signi cant effects of mouse ID and tick family on all ve life history traits, with the exception of the effect of tick family on larva-to-nymph moulting success (for details see section 10 of the ESM). For the engorged larval weight (log10-transformed), tick family, mouse ID, and the residuals accounted for 8.0%, 5.8%, and 86.3% of the variance, respectively. Similarly, for the unfed nymphal weight (log10transformed), tick family, mouse ID, and the residuals accounted for 7.9%, 4.0% and 88.1% of the variance, respectively. Thus, the majority of the variance in immature tick weight occurred at the level of the individual ticks and remains unexplained.
Correlations between the life history traits of immature I. ricinus ticks There were ve signi cant correlations between the six variables, which included the the number of engorged larvae that were recovered from each mouse and the ve life history traits of the immature I. ricinus ticks ( Figure 6). There was a negative correlation between the number of engorged larvae collected and survival of immature ticks (r = 0.438, p = 0.005). The remaining 4 pairwise correlations were positive: (i) survival of immature ticks and larva-to-nymph moulting success (r = 0.782, p < 0.0001), (ii) larva-to-nymph moulting time and engorged larval weight (r = 0.575, p = 0.0002), (iii) larva-to-nymph moulting time and unfed nymphal weight (r = 0.471, p = 0.0025), and (iv) engorged larval weight and unfed nymphal weight (r = 0.895, p < 0.0001; see also Figure 4). Power analysis to determine the minimal detectable effect size For each of the ve life history traits of the immature I. ricinus ticks, we conducted a retrospective power analysis to determine the minimal effect size that our sampling effort could have detected (see section 11 of the ESM). This power analysis showed that our sampling effort had a power >80% to detect a treatment effect (of B. afzelii infection or egg bleaching) that either increased or decreased tick phenotype by 10% for each of the ve life history traits. For moulting time, engorged larval weight, and unfed nymphal weight, our sampling effort had a power >80% to detect a treatment effect (of B. afzelii infection or egg bleaching) that either increased or decreased tick phenotype by ~5%. In summary, our sampling effort was more than su cient to detect small positive or negative effects of B. afzelii infection or egg bleaching on tick phenotype.

Discussion
Effects of B. afzelii on the life history traits of I. ricinus: Our study found that the Lyme disease pathogen B. afzelii reduced the larva-to-nymph moulting time of its principal tick vector I. ricinus by 2.4% (i.e., 1.0 day) under laboratory conditions. In contrast, we found no evidence that infection with B. afzelii in uenced the other four life history traits of immature I. ricinus ticks under laboratory conditions. Similarly, we found no evidence that reduction of the larval microbiota via the egg bleaching treatment in uenced these same life history traits. The ve life history traits investigated in this study describe the transition of engorged larvae to unfed nymphs over the rst 2.5 months of the tick life cycle, and they are expected to in uence the population ecology of I. ricinus and hence the R 0 of B. afzelii [1][2][3] . The sample size differed among the ve life history traits and ranged from 742 to 1739 immature ticks that had fed as larvae on 40 different mice. Our power analyses demonstrated that our sampling effort was more than su cient to detect small positive or negative effects (changes of 5% to 10%) of either B. afzelii infection or the egg bleaching treatment on each of the ve life history traits. Thus, under the laboratory conditions used in this study, neither B. afzelii infection nor reduction of the larval microbiome had major effects (i.e., >10% difference) on the tness of immature I. ricinus ticks.
Importance of controlled experimental infections: Our study shows the importance of using experimental infections to test whether infection with B. burgdorferi sl in uences the life history traits of Ixodes ticks. A number of studies that reported effects of B. burgdorferi sl on Ixodes phenotype (e.g., behaviour, body size, fat content, survival) used ticks that had been naturally infected in the eld 28,[30][31][32][33][34] . This approach cannot control for other confounding factors that in uence tick phenotype, such as tick age, vertebrate host species that provided the larval blood meal, quality of the host blood meal, presence of other microbes, etc. For example, vertebrate host species differ in their ability to transmit B. burgdorferi to feeding larval ticks [50][51][52][53] , and in blood meal quality, which in uences variation in larva-to-nymph moulting success 54 . Together, these two phenomena can produce signi cant associations between tick infection status and tick life history traits, which are then misinterpreted as demonstrating that the former causes variation in the latter.
Effect of B. burgdorferi on larva-to-nymph moulting time of Ixodes ticks: Our study found that B. afzelii infection in the mouse reduced the larva-to-nymph moulting time of the engorged larvae by 2.4% (i.e., 1.0 days) under laboratory conditions. Although the effect size was modest, it was highly signi cant (p < 0.0001). Faster larva-to-nymph development rates enhance the tness of both I. ricinus and B. afzelii 1-3 . In nature, larvae acquire their blood meal during the summer and then moult into unfed nymphs; most of these nymphs overwinter and quest the following spring, but some nymphs will quest later that same fall 55 . The decision of whether to quest that same fall or the following spring depends on the timing of the larval blood meal and the larva-to-nymph development rates 55 . If B. afzelii can speed up larva-to-nymph development so that nymphs can quest that same fall rather than the following spring, it would increase the R 0 of B. afzelii and Lyme disease [1][2][3] . Future studies should investigate whether B. afzelii speeds up the larva-to-nymph moulting time under ecologically relevant conditions. Effect of B. burgdorferi on moulting success and survival of immature Ixodes ticks: We found no effect of B. afzelii infection on larvae-to-nymph moulting success or immature tick survival. The engorged larvae were kept under favourable environmental conditions (high humidity and at room temperature), which may be a limitation if the effects of B. afzelii infection on tick phenotype are more visible under harsher environmental conditions. Studies using eld-collected I. ricinus ticks found that nymphs infected with B. burgdorferi sl have higher survival under stressful conditions of humidity and temperature compared to uninfected nymphs 32 , and that B. burgdorferi sl-infected nymphs survive better under cold temperatures and high-frequency temperature variations 33 . A study on another tick-borne bacterium, Anaplasma phagocytophilum found that infection reduced the molting success and survival of immature I. scapularis ticks under laboratory conditions 5 . In contrast, another study found that A. phagocytophilum increased the expression of a tick antifreeze glycoprotein, which enhanced survival of engorged larvae under cold temperature conditions 56 . These contradictory results demonstrate that the environmental conditions may in uence the effect that tick-borne pathogens have on the life history traits of their tick vectors.
Effects of B. afzelii on body size of immature I. ricinus ticks: We found no effect of B. afzelii infection on the body size of immature I. ricinus ticks. Our results contradict previous studies that found that immature Ixodes ticks infected with B. burgdorferi sl were larger than uninfected ticks 34,35,49 . A study on eld-collected I. ricinus nymphs found that infected individuals had a larger body size and higher energy reserves compared to uninfected individuals 34 . Similarly, an experimental study with bank voles, B. afzelii, and I. ricinus found that nymphs that had acquired B. afzelii during the larval blood meal from infected animals were heavier than the corresponding uninfected nymphs 35 . This result led the authors to suggest that B. afzelii manipulates the larvae to take larger blood meals 35 . Another experimental study with mice, B. burgdorferi ss, and I. scapularis reversed this chain of causality and found that larvae that take a larger blood meal from infected mice are more likely to acquire B. burgdorferi ss 49 . In the rst scenario, the pathogen is the cause of the difference in the size of the larval blood meal between infected and uninfected vectors, whereas in the second scenario, the variation in the size of the larval blood meal causes the variation in infection status. Our study found no evidence for either scenario.
Scope of selection for manipulation: Hematophagous (blood-feeding) insects and ticks differ dramatically in their biology (e.g., motility, host seeking, number and duration of blood meals, etc.) and the scope of any potential manipulation by a pathogen is therefore expected to differ between the two types of vectors 1 . In mosquito-malaria systems, the malaria parasite manipulates the mosquito vector to increase its biting rate, which enhances the parasite transmission at the expense of mosquito tness [57][58][59] . In contrast, in hard ticks of the genus Ixodes, each stage takes one blood meal from a single host, and there is no evidence that tick-borne pathogens can manipulate their tick vectors to take multiple blood meals. Following their acquisition during the larval blood meal, the interests of the tick-borne pathogen and the immature tick are largely the same, as both require the engorged larva to moult into a nymph, survive, and take a nymphal blood meal in order to complete their life cycles. One exception is when the infected Ixodes nymph chooses a vertebrate host that is not competent to harbor the tick-borne pathogen 29 . Another exception is that if there are any energetic costs of sexual development in the nymph (i.e., male and female nymphs are different and can be distinguished after the nymphal blood meal), the tickborne pathogens would prefer the reallocation of all these energetic resources towards questing and nding a host. In summary, there are more reports of adaptive manipulation for insect-borne pathogens compared to tick-borne pathogens 8 and one potential explanation is that the life cycle of hard ticks is not very conducive to manipulation because each stage is strongly selected to complete a single blood meal on a single host.
Effect of microbiome dysbiosis on life history traits of immature I. ricinus ticks: The bleach treatment of the eggs reduced the abundance of the bacterial microbiota (as measured by the 16S rRNA gene copy number) in the I. ricinus larvae by a factor of 28 44 . In insects, smearing bacteria over the surface of newly deposited eggs is a common extracellular route of transferring symbionts from mothers to their offspring 60 . In a previous study on the same sample of ticks, analysis of the 16S rRNA gene diversity suggested that the egg bleaching treatment also changed the composition of the larval microbiota; the relative abundance of bacteria presumably associated with the egg surface (e.g., Pseudomonas) decreased, whereas the relative abundance of endosymbiotic bacteria (e.g., Candidatus Midichloria mitochondrii) increased 44 . Despite the strong reduction of the larval microbiota, the egg bleach treatment did not have any effect on the life history traits of the immature Ixodes ticks. This result contradicts a study that manipulated the microbiota of I. scapularis larvae by allowing engorged females to lay their eggs in sterile tubes 25 . In that study, dysbiosed I. scapularis larvae took larger blood meals than control larvae 25 . Our results are similar to studies on I. ricinus and I. paci cus that found that injection of antibiotics into engorged females had no effect on their reproductive tness 38,39 . In other tick species that carry nutritional symbionts, such as Coxiella-like endosymbionts and Francisella-like endosymbionts that provide B vitamins, antibiotic treatments reduced tick tness (e.g., moulting rate, body weight, fecundity, larval survival) and induced physical abnormalities [15][16][17][18][19][20] . Normal tick phenotypes were recovered after combining the antibiotic treatment with an arti cial blood meal that contained the missing B vitamins Page 14/27 19,61,62 . Egg bleaching is not expected to have an impact on these Coxiella-like and Francisella-like endosymbionts because these obligate intracellular bacteria are exclusively transmitted through the egg cytoplasm 19,61,62 . In summary, a successful and dramatic reduction of the microbiota inherited via egg smearing had no meaningful impact on the life history traits of immature I. ricinus ticks.
Density-dependent mortality of immature I. ricinus ticks: We found evidence for density-dependent mortality of immature I. ricinus ticks. Mice were infested with a standardized number of I. ricinus larvae, but differences in larval attachment and/or mouse grooming behaviour 63 led to variation in the number of engorged larval ticks that were recovered among mice. There was a strong negative correlation between the number of engorged larvae that were recovered for each mouse and the probability of immature tick survival. Density-dependent mortality occurs when the host develops anti-tick immunity so that the tness of engorged larvae decreases over successive infestations [64][65][66] . However, the mice were only infested once in the present study, and the mechanism underlying this negative density dependence is therefore unclear. Nevertheless, density-dependent mortality of immature ticks on vertebrate hosts is an important mechanism regulating Ixodes tick populations [67][68][69] . We also found strong positive correlations between larva-to-nymph moulting time and the weights of the engorged larvae and unfed nymphs. This result suggests that larger larval blood meals take longer to digest, which increases the duration of the larva-to-nymph moult but results in a larger unfed nymph. The effects were relatively modest; for example, two engorged larvae with weights of 400 mg and 500 mg will moult in 40.5 days and 46.3 days, respectively.
Effects of mouse ID and tick family: We found highly signi cant effects of tick family and mouse ID on almost all of the life history traits of the immature I. ricinus ticks. Variance component analysis showed that when tick family and mouse ID are combined, they account for 14% and 12% in the total variance of engorged larval weight and unfed nymphal weight, respectively. The factors underlying differences among tick families include genetics, nutritional condition of the mother tick, quality of the blood meal, microbiome of the mother tick, clutch size, and other factors. Similarly, despite the fact that the BALB/c mice were acquired from a commercial breeder, they still differ in body size, nutritional condition, skin microbiota, stress levels, and other factors. Regardless of the reason for the phenotypic variation among larvae from different families and that feed on different mice, studies that do not account for these sources of variation can produce biased results. For the statistical analysis of these data, it is essential to use mixed effects models that estimate and thereby control the variance in tick phenotype that is due to tick family and mouse ID.

Conclusion
In summary, we found that infection with B. afzelii reduced the larva-to-nymph moulting time of engorged I. ricinus larvae, but the effect was modest (2.4% reduction). In contrast, we found no evidence that infection with B. afzelii in uenced the other four life history traits of immature I. ricinus ticks including engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. Although the egg bleaching treatment was highly effective at reducing the abundance of nonpathogenic bacteria in I.  Figure 1 Experimental design. Engorged female I. ricinus ticks (n = 10) were collected from roe deer captured in the Chizé forest, France and laid their eggs in the laboratory. Each of the 10 egg clutches was split into two batches and rinsed with either 10% bleach (n = 10 batches) or distilled water (n = 10 batches) and hatched into larvae. Larvae for each of the 20 batches (10 tick families x 2 egg washing treatments) were split into two subgroups of ~100 larvae (total of 40 subgroups) that were fed on either an uninfected control mouse (n = 20) or a B. afzelii-infected mouse (n = 20). For each of the 40 mice, up to 60 engorged larvae were collected and placed in individual Eppendorf tubes to moult into nymphs. The larva-to-nymph moulting success and survival of these engorged larvae (n = 1739) was surveyed over a period of 74 days. For each of the 40 mice, up to 20 engorged larvae were randomly selected to be weighed as engorged larvae and again as unfed nymphs (4 weeks after the larva-to-nymph moult). A subset of ~400 nymphs was frozen at -80°C for future molecular analyses. A subset of ~300 of these nymphs was tested for B. afzelii infection using qPCR.

Figure 2
The egg bleaching treatment reduced the microbiota of the I. ricinus larvae. The abundance of the tickassociated non-pathogenic bacteria is given by the log10-transformed ratio of the bacterial 16S rRNA gene copy number to the tick calreticulin gene copy number. This ratio was 28 times higher for the I.
ricinus larvae that hatched from the eggs washed with water compared to the larvae that hatched from the egg washed with bleach, and this difference was statistically signi cant (p = 0.0000005). The

Supplementary Files
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