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Host gill attachment enables blood-feeding by the salmon louse (Lepeophtheirus salmonis) chalimus larvae, and alters parasite development and transcriptome

Blood-feeding is a common strategy among parasitizing arthropods, including the ectoparasitic salmon louse (Lepeophtheirus salmonis), feeding off its salmon host’s skin and blood. Blood is rich in nutrients, among these iron and heme. These are essential molecules for the louse, yet their oxidative properties render them toxic to cells if not handled properly. Blood-feeding might therefore alter parasite gene expression. We infected Atlantic salmon with salmon louse copepodids and sampled the lice in four different experiments at day 10 and 18 post infestation. Parasite development and presence of host blood in their intestines were determined. We find that lice start feeding on blood when becoming mobile preadults if sitting on the fish body, however they may initiate in blood-feeding at the chalimus I stage if attached to gills. Lice attached to gills develop at a slower rate. Lice of similar instar age from gills versus lice from skin epidermis were sampled in three different experiments, and gene expression was analyzed by RNA-sequencing. By differential expression analysis, we found 355 transcripts elevated in lice sampled from gills and 202 transcripts elevated in lice sampled from skin consistent in all experiments. Genes annotated with “peptidase activity” are among the ones elevated in lice sampled from gills, while in the other group genes annotated with “phosphorylation” and “phosphatase” are most predominant. Transcripts elevated in lice sampled from gills are often genes relatively highly expressed in the louse intestine compared with other tissues, while this was not the case for transcripts found elevated in lice sampled from skin. In both groups, more than half the transcripts were also higher expressed after attachment. In conclusion, blood-feeding results in an alteration in gene expression, and a premature onset of blood-feeding likely causes the parasite to develop at a slower pace.


INTRODUCTION
Distribution and characteristics of lice 180 Figure 1 depicts the distribution of different developmental stages and instar ages of lice on the 181 host at 10 and 18 dpi. At 10 dpi, most lice are attached to the fins, but there is also a high 182 proportion of lice on the body and gills. At 18 dpi, however, the highest proportion of lice is 183 found on the body of the salmon. Different stages are distributed differently between sites. At 184 10 dpi, there are chalimus II larvae (mainly males) on the body and fins, but none on gills. Here, 185 we rather find chalimus I (higher proportion of middle than old) larvae. At 18 dpi, there are 186 preadult I females on the body and fins, but not on the gills. On gills, we rather find a higher 187 proportion of old chalimus II females at 18 dpi. There is a higher proportion of preadult I male 188 lice found on the body compared with the fins. On the fins, most of the preadult I lice are young 189 females. 190 Upon termination of the two experiments at 18 dpi, all lice were removed from the salmon, and 191 their settlement site, developmental stage, the presence of a frontal filament, and the visibility 192 of an intestine filled with blood were assessed (Table 1). On the body and fins, most lice were 193 at the preadult I stage, while on the gills, most lice were in the chalimus II stage. The majority 194 of the preadult I lice were located on the host body, and the minority was located on the gills. 195 The preadult I lice on the gills, however, were more often secured by their frontal filament. Of 196 the preadult I lice still attached by the filament, only the ones on the gills had a blood-filled 197 intestine (Fig. 2c). None of the lice on the fins had a blood-filled intestine, and on the host body, 198 only the mobile lice had apparently fed on blood. Additionally, chalimus I (Fig. 2a) and 199 chalimus II (Fig. 2b) larvae attached to the gills had fed on blood, whereas lice of the same ages 200 on the fins and body had no visible blood in the intestine. 201

Effect of gill settlement on gene expression in chalimus larvae 202
In order to determine the effect of gill settlement on the gene expression in chalimus larvae, 203 RNA-sequencing of pooled individuals was performed. All counts per million (CPM) values 204 can be found in Supplementary Table S1. The overall gene expression of the individual samples 205 in comparison with chalimus I and chalimus II larvae of different instar age (data taken from 206 Eichner et al. 2018) is shown in a correspondence analysis (CA) plot (Fig. 4). 207 All lice from this study sampled at 10 dpi are clustering together with chalimus I larvae sampled 208 directly before molting as well as molting ones (old, molt) from Eichner et al.  Supplementary Table  217 S2. 5878 genes were differentially expressed in at least one of the experiments (Supplementary  218   Table S3). 219 Most DE genes were found in Experiment 1, 10 dpi (2188 or 2015 for gill and skins samples 220 respectively). In Experiment 2, 10 dpi only, 1112 or 1081 transcripts respectively were found, 221 of which 79% or 68% respectively are overlapping with the ones found in Ex1. DE genes found 222 at 18 dpi are less overlapping with DE genes found in Ex1 at 10 dpi. Only 43% or 32% 223 respectively of the genes found here are overlapping with genes from the respective groups in 224 Ex1, 10 dpi and 35% or 28% respectively are overlapping with Ex2, 10 dpi (Fig. 3) Table S6 and S7), while 59 are  227   significant different, but regulation directions are differing between experiments (31 elevated  228   in lice from gills at 10 dpi but lower at 18 dpi, 24 the other way around and 4 are differing  229 between the two experiments sampled at day 10) (Supplementary Table S8). Transcripts solely 230 regulated at either 10 dpi or 18 dpi are listed in Supplementary Table S4 and S5, respectively. 231 We found a high number (49) of genes annotated with Pfam domain PF00040: "Fibronectin 232 type II domain" among the DE genes. Mostly, these are elevated in lice sampled from gills at 233 10 dpi. However, a smaller number of PF00040 is within under the DE genes which are elevated 234 in lice sampled from skin than from gills. Additionally, the Pfam domain PF00089: "Trypsin", 235 are mostly found in the group of lice sampled from gills. 236 The sizes and overlaps of gene sets that were DE in each experiment separated by expression 237 pattern (elevated in lice sampled from gills or elevated in lice sampled from skin respectively) 238 are depicted together with most representative GO terms in Figure 3 Venn diagrams. All 239 significantly enriched GO terms are listed in Supplementary Table S9 where summarized GO  240 annotations belonging to biological process are visualized in a TreeMap (REVIGO). "Peptidase 241 activity" is an enriched GO term in lice sampled from gills across all groups (except the ones 242 exclusively found in Ex2, 10 dpi), and in particular "serine type endopeptidase activity", 243 whereas "serine-type endopeptidase inhibitor activity" is enriched in lice sampled from skin. 244 Notably, "glycolysis" as well as "oxidoreductase activity" are GO terms highly enriched in 245 genes elevated in lice sampled from gills. GO terms containing "phosphorylation" as well as 246 "phosphatase" are found enriched in nearly all groups in genes elevated in lice sampled from 247 skin (also here, the exception is the ones exclusively found in Ex2, 10 dpi).

Equal gene expression changes throughout all three experiments 249
To determine which genes may be important in relation to the blood meal in general, 250  (Fig. 6). Often, genes in the group found elevated in lice sampled from gills fall under 266 the enriched GO group "catalytic activity" (all are shown in Fig. 6a). Remarkably strong 267 enriched (factor 30) DE genes elevated in lice from skin, are genes belonging to "extracellular 268 matrix". However, only five genes are in that group. More than 30 genes were found in GO 269 categories "catalytic activity", "hydrolase activity", "binding" and "ion binding" (Fig. 6b). All 270 enriched GOslim terms, numbers of genes found in each category, and enrichment factor for 271 the two groups are shown in Figure 6. 272 42% of the 355 genes elevated in lice sampled from gills have a fold-change more than two 273 compared to skin. When also taking the p-value into account we find 21%, which are strongly 274 regulated (average fold-change over two and average padj-value ≤ 0.005). In this group, the 275 strongest elevated transcript is most similar to a nematode astacin (EMLSAT00000010457). 276 Among the other strong ones regulated are three transcripts with FNII domains, another 277 transcript with an astacin domain, as well as nine transcripts with trypsin domains, as well as a 278 chemosensory protein (EMLSAT00000005105), yet also many transcripts with no annotation 279 or known protein domains. There are several transcripts with similarity to various proteases that 280 are up-regulated in lice from gills. All genes are listed in Supplementary Table S6. 281 In the group of genes elevated in lice sampled from skin, we find fewer genes highly 282 differentially expressed than in lice sampled from gills. Only 16% have a fold-change of two 283 or higher compared to samples from gills. Most up-regulated in this group is a transcript with 284 no predicted annotation or Pfam domains (EMLSAT00000009920). Among the strongest 285 elevated genes in all three experiments (average fold-change over two and p-value ≤ 0.005) in 286 lice sampled from skin are several transcripts with predicted FNII domains. Moreover, we find 287 several genes with no annotation or known protein domains. All genes are listed in 288 Supplementary Table S7. Only 17 (8%) of the transcripts in the DE gene group elevated in lice sampled from skin are 307 also higher expressed in the intestine than in other investigated tissues (12% lowest of all tissues 308 investigated) (LiceBase). Only two were much higher (more than 100 times) expressed in 309 intestine than other tissues (LiceBase). However, the ten of these also found on the oligo 310 microarray were not highest expressed in the intestine, except one (EMLSAT00000008355) 311 In this study, we have investigated the biology of blood-feeding in the marine ectoparasitic 317 salmon louse with a special focus on gene expression of immobile lice situated on host gills. 318 We chose immobile lice, because this allowed us to focus on those individuals that had stayed 319 at one location at least since extruding the frontal filament in the late copepodid stage. Being attached to the gills allowed the lice to initiate blood-feeding prior to becoming mobile. Two 321 parallel experiments terminated at 10 days post infestation, and one experiment terminated at 322 18 days post infestation were included in the RNA-seq and subsequent gene expression 323 analyses. 324 At 10 dpi, the amount of lice is rather evenly distributed between the investigated body parts 325 (32 or 29% on body, 43 or 39% on fins and 24 or 30% on gills in Ex1 and Ex2 respectively). 326 The favored site at 18 dpi is the body with 66 or 61% in Ex1 or Ex2 respectively (15 or 14% 327 on fins and 17 or 23% on gills). Lice at day 10 are in chalimus I or chalimus II stage and attached 328 by the frontal filament. At day 18, we found 75 or 74% preadult lice in Ex1 or Ex2 respectively. this as well indicates that the lice tend to leave the fins for other host feeding areas when 336 becoming mobile. A preadult I louse that is still attached to its host by its frontal filament has 337 recently molted from the chalimus II stage, and has stayed at that feeding site since attachment. 338 There are no preadult I lice with a visible blood-filled intestine on the fins, whereas we find this 339 on the gills and the body. Interestingly, of the lice still on their filament, only those on gills 340 have apparently fed on blood. Moreover, already in the chalimus I stage, we find lice with feeding is initiated from the mobile preadult I stage and onwards in the development of the 345 salmon louse occurring under natural conditions. 346 Development of lice on the gills was delayed, compared to development of lice on body or fins. 347 At 10 dpi, no chalimus II lice were found and a higher proportion of chalimus I lice was of less 348 developed instar age on gills. Comparing only the attached chalimus at 18 dpi (25% of all lice), 349 48 or 59% in Ex1 or 2 respectively are found on gills. In addition, on gills, there is a higher 350 proportion of male chalimus II lice, which develop faster than females. Developing on host gills 351 caused the salmon lice to develop slower than those developing on other locations. There have 352 been contradicting results about this in the past (Johnson, 1993;Johnson and Albright, 1992), 353 however here we have determined instar ages, and not only the developmental stages, which 354 adds more confidence to our results. 355 We conclude that during the normal development on the body or the fins, the salmon louse does 356 not start to feed on blood until reaching the mobile preadult I stage. By that reasoning, we 357 wanted to compare gene expression of chalimus larvae located on the vascular gills with access 358 to blood with that of chalimus larvae of equal development from the rest of the body. The 359 salmon louse has approximately 13,000 protein encoding genes 360 (http://metazoa.ensembl.org/Lepeophtheirus_salmonis), and we find in our RNA-seq analyses 361 that over 5800 genes had an altered expression in at least one of our experiments. As expected, 362 we found a higher amount of overlapping DE genes in the two experiments sampled at 10 dpi. 363 These are chalimus I larvae, which are soon molting to chalimus II, while lice sampled at 18 364 dpi are chalimus II larvae, shortly prior to molting to preadult I lice. As such, all lice are sampled 365 at a similar instar age. However, phenotype and lifestyle differ in preadult lice and one can 366 expect expression of genes in preparation for this stage in the lice sampled at 18 dpi. The high 367 amount of DE genes exclusively found in Ex1, 10 dpi could be caused by batch differences 368 between Ex1 and Ex2, or could be as a result of more powerful statistics due to a higher amount of parallel samples (8 vs 6 biological parallels of each group in Ex1 and Ex2, respectively). 370 However, we know also that minor differences in processes. There are also more genes with a greater fold change within the group of DE genes 381 elevated in lice sampled from gills (42% over 2-fold change, whereas only 16% in lice sampled 382 from skin), pointing towards a high demand of these gene products when feeding on blood. 383 However, as GO terms can be unspecific or general, the following discussion deals with 384 selected groups of transcripts. 385

Iron and heme 386
Among the regulated transcripts, the iron storage units of ferritin (LsFer1 (RACE sequence) 387 and LsFer2: EMLSAT00000006305) are both elevated in chalimus larvae sampled from gills 388 compared with other settlement sites. We have previously established that these genes are 389 important for the adult female salmon louse blood-feeding and reproductive success, as the 390 parasite had a clear gut and failed to produce viable eggs upon silencing the two genes 391 (Heggland et al., 2019b). Blood contains several iron-proteins, and when initiating blood-392 feeding, the salmon louse needs to obtain a way of storing and detoxifying iron absorbed from 393 the blood. Up-regulating ferritin when ingesting a blood meal is therefore an important defense mechanism for a blood-feeding parasite. The putative heme scavenger receptor, LsHSCARB 395 (EMLSAT00000005382), is elevated in lice on gills at 18 dpi compared to lice on skin. We 396 recently found that upon silencing LsHSCARB by RNA interference, adult female lice had 397 absorbed less heme and produced fewer viable eggs and less offspring (Heggland et al., 2019a).

Putative anti-coagulation 455
Blood coagulation is a key mechanism in maintaining homeostasis in vertebrates if a blood 456 vessel were to rupture. A parasite feeding on vertebrate blood would therefore require 457 mechanisms in order to counteract this to maintain its feeding habit. Anti-coagulation factors 458 targeting host proteins could thus be vital for the successful blood-feeding in the parasitizing 459 arthropod. A thrombin (coagulation factor) inhibitor, hemalin, was found to be important to 460 avoid clotting of the blood meal in the bush tick Haemaphysalis longicornis (Liao et al., 2009). should be further characterized, in order to elucidate a possible role of FNII in blood-feeding. 504 Given the earlier reports that FNII domains in vertebrates may be important for blood clotting, 505 one hypothesis is that proteins with FNII domains only could have an anti-coagulant effect. 506 CONCLUSIONS 507 Blood is a major dietary component for the ectoparasitic salmon louse, which the parasite has 508 access to when attached to a salmonid host. We find that the salmon louse initiates blood-509 feeding during the mobile preadult I stage. However, if the parasite is attached to host gills, it 510 may start feeding on blood already at the chalimus I stage. The premature onset of blood-511 feeding caused lice on gills to develop at a slower pace than lice that were attached to host fins 512 and general body surfaces. Chalimus lice of equivalent age on gills versus other attachment 513 sites were therefore analyzed for gene expression comparisons. Several genes are elevated in 514 lice attached to the gills, and among these, we find e.g. genes of importance for the absorption, several genes with FNII domains. The results of this study raise a number of new gene targets 518 to investigate further in order to elucidate the blood-feeding habit of the infamous salmon louse.