Lizard feeding enhances Ixodes pacificus vector competency

A vector’s susceptibility and ability to transmit a pathogen— termed vector competency—determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. While rodents and birds are reservoirs for Bb and infect juvenile ticks, lizards are Bb-refractory. Despite I. pacificus feeding on a range of hosts, it is undetermined how larval host bloodmeal identity affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb-infected mice. We found that lizard-fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse-fed larvae. We also conducted the first RNA-seq analysis on whole-bodied I. pacificus and found significant upregulation of antioxidants and antimicrobial peptides in the lizard-fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in disease transmission and upends prior notions about the role of lizards in Lyme disease transmission.


Introduction 66
Vector competency-the ability of a vector to successfully acquire and transmit a 67 pathogen-and the factors that modulate it are increasingly the focus of efforts to 68 control the emergence and spread of vector-borne zoonotic diseases [1][2][3][4][5]. Five C3H/HeJ mice were used to feed nymphs (Jackson Laboratory, Bar Harbor, 149 Maine). Three of the C3H/HeJ mice were inoculated with Bb leaving the remaining two 150 mice as uninfected controls. Nymphs that fed as larvae on either lizards or mice were 151 then placed on either Bb-infected or uninfected C3H/HeJ mice for nymphal feeding. 152 Host-to-tick acquisition experiments were conducted in three separate trials. The first 153 trial was conducted at Indiana University, where lizard-fed and mouse-fed nymphs were 154 fed on C3H/HeJ mice infected with Bb at a concentration of 10 5 spirochetes/mL (1,000 155 total spirochetes). The second and third trials were conducted in the animal facilities at 156 San Francisco State University where lizard-fed and mouse-fed ticks were 157 subsequentially fed on C3H/HeJ mice infected with 10 7 and 10 6 spirochetes/mL 158 (100,000 and 10,000 spirochetes total), respectively.

Statistical analyses 175
To determine if larval bloodmeal host was a predictor of nymphal pathogen 176 acquisition, we used a generalized mixed-effect model (GLMM) with a binomial error 177 distribution. We used larval host bloodmeal (lizard or mouse feeding) as a fixed effect 178 and trial as a random effect to account for experimental variation between trials. 179 Analyses were performed using the glmm package (v. 1 Ticks from the third pathogen transmission experiment were used for 183 transcriptome analysis. Unfed nymphs and engorged nymphs were divided into six either fed on a lizard or a mouse as a larva. Then, molted nymphs from either larval 186 bloodmeals either remained unfed, fed on an uninfected mouse, or fed on a Bb-187 inoculated mouse (Fig. 1). Each experimental group will hereinafter be described with 188 the following abbreviations: unfed nymphs are referred to as "UF" and fed nymphs are 189 referred to by whether they were fed on a Bb-positive "+Bb," or Bb-negative "-Bb" 190 C3H/HeJ mouse. Additionally, the larval bloodmeal (lizard or mouse) in each 191 experimental group is indicated in the subscript following the abbreviation. 192 Groups one and two, which represent our unfed nymphs, "UF lizard " and "UF mouse " 193 ( Fig. 1), were set aside to examine the effect of the lizard or mouse larval bloodmeal on 194 I. pacificus gene expression. The remaining four groups were engorged nymphal ticks. 195 Uninfected control groups three and four, "-Bb lizard " and "-Bb mouse " were nymphs that fed 196 on uninfected C3H/HeJ mice during their nymphal bloodmeal (Fig. 1). Groups five and 197 six, "+Bb lizard " and "+Bb mouse " were nymphs that fed on Bb infected C3H/HeJ (Fig. 1). 198 We prepared three replicates from each of the six experimental tick feeding 199 conditions ( Fig. 1)

Host-to-tick Bb acquisition experiment 240
The effect of larval host bloodmeal on I. pacificus nymphal vector competency 241 was examined in a host-to-tick pathogen acquisition experiment where replete larval I. 242 pacificus were obtained from mice or lizards and then subsequently fed on Bb-infected 243 C3H/HeJ mice (Fig. 1). A total of 36 lizard-fed (+Bb lizard ) and 46 mouse-fed (+Bb mouse ) I. 244 pacificus nymphs were used across three experimental trials (Fig. S2). 245 I. pacificus nymphs that fed on Bb-inoculated C3H/HeJ mice were significantly 246 more likely to become infected if they previously fed on lizards as larvae than if they fed 247 on mice (߯² (1, N = 82) = 7.8266, p =.0051). During their nymphal bloodmeal, 64% of 248 lizard-fed ticks (N= 23/36) became infected with Bb compared to 30% of the mouse-fed 249 ticks (N=14/46; Fig. 2). Even after accounting for trial as a random effect, our GLMM 250 analyses found that the lizard larval bloodmeal is a significant, positive predictor of Bb 251 acquisition in I. pacificus (Table 1) Table S2. 265 To visualize overall differences in gene expression profiles across the 266 experimental groups, we created a heatmap and a PCA plot of our 18 replicates. The 267 heatmap, generated from sample-to-sample distances, is based on read counts for all 268 genes and showed that tick engorgement status (unfed vs. engorged) induced 269 significant changes in I. pacificus gene expression (Fig. 3a). Additionally, the PCA plot 270 indicated significant distinction of overall gene expression between unfed ticks of either 271 bloodmeal type, UF lizard and UF mouse (Fig. 3b). The engorged experimental groups 272 (groups three to six) had similar gene expression profiles and did not distinctly cluster 273 together by experimental condition (Fig. 3b). 274 To investigate the mechanism through which host blood alters tick vector 277 competency, we took a global transcriptomic approach to identify key genes or 278 pathways modulated by mouse or lizard hosts. Differential gene expression analyses 279 focused on several pairwise comparisons to examine transcriptomic differences 280 between 1) the lizard versus mouse bloodmeal in the unfed group 2) unfed versus fed 281 ticks, and 3) bloodmeal identity distinctions between Bb exposed groups. 282 The comparison between our unfed nymphs (UF lizard vs. UF mouse ), demonstrated 283 that the lizard bloodmeal induced distinct transcriptomic changes in I. pacificus with 468 284 significantly differentially expressed genes (DEGs). While many of the DEGs remain 285 undescribed, some of the highest upregulated genes induced by the lizard bloodmeal in 286 the unfed group included antioxidants and antimicrobial peptides (Fig. 4a). The 287 antioxidant glutathione peroxidase was the most significant DEG and was upregulated 288 48.5-fold after the lizard bloodmeal compared to mouse bloodmeal. Other tick 289 antioxidants that were upregulated after the lizard bloodmeal include peroxidase 290 (upregulated 21-fold) and glutathione-S-transferase (upregulated four-fold; Fig. 4a). We 291 also found several DEGs that are related to the regulation of antimicrobial peptides but 292 have never been described in I. pacificus ticks, such as acanthoscurrin-1, 293 acanthoscurrin-2-like, micropulsin and micropulsin isoform, which were upregulated by 294 27.9, 104, 4, and 22.6-fold, respectively (Fig. 4a). 295 To analyze the DEGs between engorged and un-engorged ticks, we combined 296 the two unfed groups (UF lizard & UF mouse ) as our reference 'unfed' group and compared 297 this group to all of the 'engorged' nymphs (i.e. -Bb lizard , -Bb mouse , +Bb lizard , & +Bb mouse ).
significant DEGs with a majority of difference in expression being upregulated genes in 300 the engorged groups (Fig. 4b). Of the top 100 most significant DEGs, 25% were related 301 to cuticle formation. Other notable genes that were differentially expressed include the 302 antioxidant and detoxifying genes, glutathione peroxidase and sulfotransferase, which 303 were both upregulated 4096-fold in the engorged group. Over a hundred DEGs between 304 unfed and engorged ticks remain uncharacterized. 305 Despite significant differences in pathogen acquisition success between the 306 +Bb lizard and +Bb mouse in pathogen transmission experiments, only 25 genes were 307 differentially expressed between the two groups (Fig. 4c). The two most significantly 308 DEGs included exonuclease V-like (upregulated 32-fold) and 4-coumarate -CoA ligase 309 (downregulated 8-fold) in +Bb lizard (Fig. 4c)   pacificus gene expression (Fig. 3). Unfed nymphs clustered significantly by larval induced distinct transcriptomic alterations in I. pacificus. We analyzed gene expression 369 profiles in unfed nymphs right after they molted from larvae to nymph. Our analysis 370 suggests that the effect of the larval bloodmeal on I. pacificus gene expression is 371 carried through the transstadial molt and is present prior to the initiation of the nymphal 372 bloodmeal. Among the unfed ticks, bloodmeal history drove divergence of 468 373 significantly expressed genes between un-engorged lizard and mouse fed ticks. The 374 most significant DEG between unfed ticks with different bloodmeal histories was 375 glutathione peroxidase in the lizard-fed group (Fig. 5a). Glutathione peroxidase is an 376 important anti-oxidative enzyme, that works by reducing H 2 O 2 and detoxifying OH 377 radicals and prevents oxidative stress and cell damage in the tick [48,49]. Two other 378 known anti-oxidative enzymes, peroxidase and glutathione S-transferase were 379 significantly upregulated after the lizard bloodmeal (Fig. 5b&c). A nutritional 380 dependence on blood has required ticks to evolve and produce anti-oxidants to digest 381 an inherently toxic meal containing high levels of iron and pro-oxidant levels [49]. 382 Notably, glutathione peroxidase is homologous to SALP25d, a tick antioxidant produced 383 in the salivary glands that has been shown to promote the transmission of Bb from tick 384 to host and protects Bb from harmful hydroxyl radicals in vitro [16]. The upregulation of 385 glutathione peroxidase in lizard-fed ticks has the potential to directly benefit Bb 386 colonization from host to tick during the nymphal bloodmeal by increasing antioxidant 387 concentration and protecting Bb from the harmful oxidative components of blood. 388 There was also a strong signal of microbial defense signals in unfed tick 389 comparison. The antimicrobial peptides (AMPs) acanthoscurrin-1, acanthoscurrin-2, nymphs with prior lizard bloodmeals relative to prior mouse bloodmeals (UF lizard ; Fig. 5d-392 g). Acanthoscurrin is a glycine-rich cationic AMP, known to be expressed in the 393 hemocytes of tarantula spiders, Acanthoscurria gomesiana, and has activity against the To further characterize the physiological changes that occur during I. pacificus 412 host feeding, we analyzed unfed versus fed ticks, 24 hours after ticks completed their 413 transcriptional changes to the physical structure of the tick [46] (Fig. 3b). The greatest 415 number of DEGs was between fed and unfed ticks (Fig. 4). Genes related to cuticle 416 formation, antioxidant production, and detoxification were all significantly upregulated in 417 fed ticks and are consistent with structural reformation that occurs during the 418 engorgement process when ticks must rapidly synthesize a new cuticle over the course 419 of taking a large bloodmeal [14]. Glutathione peroxidase and sulfotransferase were 420 highly upregulated during engorgement and are critical for detoxifying the massive host 421 bloodmeal and protect ticks from harmful oxidative stress inherent in blood feeding [47]. Gene expression of I. pacificus is heavily shaped by engorgement status and 426 bloodmeal history in unfed ticks but among the engorged nymphs (groups 5&6; Fig. 1

), 427
there was not a strong signal of bloodmeal history or infection status (Fig. 3b). Despite 428 the significant differences in pathogen acquisition between host bloodmeal experimental 429 groups (Fig. 2), only 25 genes with no known pathogen or immune function were 430 differentially expressed between these groups. Comparing these results to our gene 431 expression analysis from unfed nymphs, the strongest divergence in gene expression is 432 present in the unfed ticks. This suggests that the physiological changes induced by the 433 larval bloodmeal has lasting effects into the nymphal stage. 434 We document a strong correlation between host bloodmeal and vector 435 competency, but there were limitations to our study. Naturally low burdens prevented us questing larvae is problematic because it is difficult to verify whether a tick had a 438 previous, incomplete bloodmeal [27]. Our transcriptomic results indicate that bloodmeal 439 history was only significantly different in the unfed nymphal group while fed nymphs 440 were less apparent, which strongly suggests that larval host blood meal identity played 441 a larger role in gene expression than tick source (Fig. 3). 442 Despite these intriguing results, our study highlights the importance of a more 443 complete annotation of the reference transcriptome for Ixodes spp. ticks. A large 444 proportion of DEGs remain uncharacterized indicating additional investigation into tick 445 molecular function and transcriptomics is needed. The lack of differentially expressed 446 genes in our comparison of Bb-exposed nymphs with different bloodmeal histories could 447 be attributed to the timing of RNA sampling (24 hours after completed bloodmeal). 448 Examination of gene expression before, during, and immediately after feeding would 449 improve insight into the mechanism of pathogen colonization into I. pacificus. 450 Additionally, future experiments should focus on understanding the role of antioxidants 451 and the AMPs identified in this study in modifying tick vector competency. We found a 452 strong association between lizard bloodmeal history and antioxidant activity as well as 453 AMP production. These responses coupled with naturally high natural tick burdens and 454 preferential feeding on lizards may suggest an evolutionary benefit to feeding on the 455    bloodmeals on either mice (mouse-fed; +Bb mouse ) or lizards (lizard-fed; +Bb lizard ). Lizard-630 fed larvae were significantly more likely to become infected when subsequently feeding 631 on a B. burgdorferi infected mouse as nymphs than nymphs that previously fed on mice 632 as larvae, with 64% of lizard-fed ticks infected compared to 30% of the mouse-fed ticks.