Origin, evolution, phylogeny and taxonomy of Pulex irritans

For Review Only 1Origin, evolution, phylogeny and taxonomy of Pulex irritans 2(Siphonaptera:Pulicidae). 3 4ANTONIO ZURITA1, ROCÍO CALLEJÓN1, ÁNGELA M. GARCÍA-SÁNCHEZ1, 5MARA URDAPILLETA2, MARCELA LARESCHI3 & CRISTINA CUTILLAS1 61Department of Microbiology and Parasitology. Faculty of Pharmacy. University of 7Seville. Profesor García González 2, 41012 Seville, Spain. 8 2National Institute of Tropical Medicine (INMET). Neuquén y Jujuy s/n, 3370 Puerto 9Iguazú - Misiones, Argentina. 10 3Center of Parasitology and Vectors Studies (CEPAVE) (CONICET CCT La Plata11 UNPL). Bv 120 s/n e/ 60 y 64, 1900 La plata, Argentina. 12 13 14 15 * Corresponding author: 16 Dr. Cristina Cutillas 17 Department of Microbiology and Parasitology. Faculty of Pharmacy. University of 18 Seville. Prof. García González 2, 41012 Seville, Spain. 19 Phone: +34954556773 20 e-mail: [email protected] Page 1 of 49 Medical and Veterinary Entomology For Review Only 21 Abstract 22 Within Pulicidae family, the so-called human flea, Pulex irritans Linnaeus, 1758 has 23 been the most studied species together with C. felis Bouché, 1835, since they have a 24 cosmopolitan distribution together with the fact that these species are closely related 25 with humans. The main aim of this work was to carry out a comparative morphometric 26 and molecular study of two different populations of P. irritans (Spain and Argentina). 27 For this purpose, the ITS1, ITS2 of the rDNA and partial cytochrome-c oxidase (cox1) 28 and cytochrome b (cytb) mtDNA gene of these taxa were sequenced. Furthermore, we 29 assessed the taxonomy, origin, evolution and phylogeny of P. irritans. In our study, 30 morphometric data did not show significant differences between P. irritans specimens 31 from Spain and Argentina, even when these two populations were collected from 32 different hosts; however, we found a considerable degree of molecular divergence 33 between both populations based on nuclear and mitochondrial markers. Thus, we could 34 suggest that P. irritans, in contrast to other generalist fleas, maintain a certain degree of 35 morphological similarity, at least between Western Palearctic and Neotropical areas. 36 Furthermore, we provided the existence of two well defined geographical genetic 37 lineages within P. irritans species suggesting the existence of two cryptic species which 38 could be discriminated by PCR-linked RFLP. Page 2 of 49Medical and Veterinary Entomology For Review Only 39 Introduction 40 Pulicidae (Order Siphonaptera) has remained as the most studied family of fleas over 41 the world since most fleas of medical or veterinary importance, such as Ctenocephalides 42 felis, Ctenocephalides canis, Pulex irritans or Xenopsylla cheopis, are members of this 43 family. Currently, Pulicidae consists of four tribes, 21 genera, and 167 species (Whiting 44 et al., 2008). Some authors (Lewis, 1998) considered Pulicidae as including Tungidae; 45 however, Whiting et al. (2008) placed this family as a monophyletic group and 46 phylogenetically distant from Tungidae. Recently, Krasnov et al. (2015) placed 47 Pulicidae family on the basis of the flea phylogeny together with Leptopsyllidae. These 48 authors suggested that the mainly Palearctic distribution and origin of Pulicidae and 49 Leptopsyllidae and the mainly Nearctic distribution of the most derived family 50 Ceratophyllidae, indicates an eastward earlier (pre-glaciation) migrations from 51 Palearctic to Nearctic zones through the Bering Land Bridge. On the other hand, 52 Pulicidae exhibits an interesting diversity of host specificity patterns and ecological 53 habits (Whiting et al., 2008). Within this family, the so-called human flea, P. irritans 54 Linnaeus, 1758 (the earliest flea species described) has been the most studied species 55 together with C. felis Bouché, 1835, since they have a cosmopolitan distribution 56 together with the fact that these species are closely related with humans. P. irritans 57 parasitizes a wide variety of hosts, including rodents or birds (Graham et al., 2016); 58 however, it generally parasitizes large wild and domestic mammals, particularly 59 carnivores, livestock and humans (Gratz, 1999). Furthermore, specific host associations 60 vary between geographic regions (Hopla, 1980; Lewis, 1998). Thus, in the last years 61 several authors have assessed the role of this species as a vector of several diseases 62 carrying out epidemiological studies based on the detection and prevalence of certain 63 pathogens such as Yersinia pestis, Rickettsia felis or Bartonella sp. in P. irritans Page 3 of 49 Medical and Veterinary Entomology For Review Only 64 collected from different hosts and different geographical areas (Belthoff et al., 2015; 65 Fontalvo et al., 2017; Palomar et al., 2017). According to Yssouf et al. (2014) the rapid 66 and reliable identification of fleas at species level is an essential component of the fight 67 against flea borne diseases in order to establish epidemiological relationship between 68 flea species and their borne zoonotic agents. Thus, before to assess the prevalence of 69 certain pathogens in fleas it is needed to know accurately which species we are studying 70 in order to establish appropriate prevention and control strategies. 71 During the last fifteen years, molecular approaches have contributed to significant data 72 about the diagnostic determination of genus and species of fleas (Dittmar & Whiting, 73 2003; Vobis et al., 2004; Gamerschlag et al., 2008; Whiting et al., 2008; Marrugal et 74 al., 2013; Zurita et al., 2016). Nevertheless, the specific differentiation of fleas is 75 generally based on a variety of morphological criteria such as the shape and structure of 76 their complex genitalia or the distribution of setae, spines and ctenidia (Linardi & 77 Santos, 2012; López Berrizbeitia et al., 2016; Hastriter et al., 2017). The combination of 78 molecular and morphometric data has allowed to find synonymies or cryptic species on 79 fleas (Zurita et al., 2015; Zurita et al., 2018). These studies revealed the existence of 80 certain genetic plasticity in the Order Siphonaptera which should be taken into account 81 in order to carry out further taxonomic studies. 82 The main aim of this work was to carry out a comparative morphometric and molecular 83 study of two different populations of P. irritans (Spain and Argentina). For this 84 purpose, the ITS1, ITS2 of the rDNA and partial cytochrome-c oxidase (cox1) and 85 cytochrome b (cytb) mtDNA gene of these taxa were sequenced. The taxonomy, origin, 86 evolution and phylogeny of P. irritans are discussed. 87 Page 4 of 49Medical and Veterinary Entomology For Review Only 88 Materials and methods 89 Collection of samples 90 Fifty-five fleas from Seville (southwestern of Spain) were collected off-host from a 91 neglected horse stable and near to a children school. On the other hand, thirty-three fleas 92 isolated from one Andean fox (Lycalopex culpaeus) and three South American gray 93 foxes (Lycalopex griseus) from Santa Cruz (south of Argentina) were obtained through 94 the assistance of colleagues (see Acknowledgements). In addition, two individuals of C. 95 felis collected from dogs (Canis lupus familiaris) from Argentina were molecularly 96 studied only for comparative purposes. 97 Fleas obtained were kept in Eppendorf tubes with 70 % ethanol until required for 98 subsequent identification and sequencing (Table 1). 99 Morphological identification 100 Flea specimens collected by us (Spain) were classified by ourselves whereas those fleas 101 provided by our colleagues (Argentina) were classified firstly by them (see 102 Acknowledgements) and then morphologically compared with our specimens in our 103 laboratory. First of all for morphological analysis, whole specimens were examined 104 under optical microscope, whereas, flea legs were cut off in order to carry out the 105 posterior DNA extraction. Secondly, rest of body preserved in vials with 70 % ethanol 106 were cleared with KOH, dehydrated in a growing series of alcohol, diaphanized in 107 eugenol and mounted on permanent slides in Canada balsam or EUKITT for their 108 detailed examination under optic microscope. Photographs were taken by using a 109 Microscope Olympus BX51 equipped with Photographic Camera Olympus DP71. 110 Diagnostic morphological characters of P. irritans were studied by comparing with 111 figures, keys and descriptions given in Hopkins & Rothschild (1953), Barrera (1955), Page 5 of 49 Medical and Veterinary Entomology For Review Only 112 Smit (1958) and Beaucournu & Launay (1990). After morphological identification, ten 113 specimens from Argentina (seven females and three males) and fourteen specimens 114 from Spain (six males and eigth females) were measured according to fifteen different 115 parameters (Table 2). Descriptive univariate statistics (arithmetic means, standard 116 deviations, and coefficient of variation) for all parameters were determined for two 117 populations (Spain and Argentina) using IBM® SPSS® Statistics program version 118 24.0.0.0 (Pardo & Ruiz, 2002). Furthermore, morphometric data was explored using 119 multivariate analysis in five measurements (HL, HW, PROL, MESL, and METL) 120 (Table 2) by the principal component analysis (PCA), a technique for summarizing most 121 of the variation in a multivariate dataset in few dimensions (Rohlf & Marcus, 1993; 122 Klingenberg, 1996; Dujardin & Le Pont, 2004). The analyses were carried out using the 123 BAC v.2 software (Dujardin, 2002; Valero et al., 2009). 124 Molecular study 125 Total DNA was extracted from fleas using flea legs by the DNeasy Blood and Tissue 126 Kit (Qiagen) according to the manufacturer’s protocol. Then, genomic DNA was 127 checked using an electrophoresis in 0.8 % agarose gel electrophoresis infused with 128 SYBR Safe. 129 All molecular markers sequenced in this study (ITS1 and ITS2 rDNA, cox1 and cytb 130 mtDNA) were amplified by polymerase chain reaction (PCR) using a thermal cycler 131 (Eppendorf AG). PCR mix, PCR conditions and PCR primers are summarized in Table 132 S1. The ITS1, ITS2, cox1 and cytb partial gene sequences obtained from P. irritans 133 from the two geographical areas were deposited in GenBank database (Table 1). In 134 order to compare with other Pulicidae species, it were sequenced and analysed ITS1 and Page 6 of 49Medical and Veterinary Entomology For Review Only 135 ITS2 rDNA and cox1 and cytb mtDNA partil genes of C. felis isolated from dogs (C. l. 136 familiaris) from La Plata (Argentina). 137 The PCR products were checked on SYBR Safe stained 2 % Tris–Borate–EDTA (TBE) 138 agarose gels. Bands were eluted and purified from the agarose gel by using the QWizard 139 SV Gel and PCR Clean-Up System Kit (Promega). Once purified, the products were 140 sequenced by Stab Vida (Portugal). To obtain a nucleotide sequence alignment file, we 141 used MUSCLE alignment method (Edgar, 2004) by the MEGA program version 5.2 142 (Tamura et al., 2011). The ITS1 rDNA intraindividual variation was determined by 143 sequencing four to seven clones of two specimens from Spain and one specimen from 144 Argentina. The PCR products were eluted from the agarose gel using the WIZARD® 145 SV Gel and PCR Clean-Up System (Promega) and transformation was carried out as 146 cited by Cutillas et al. (2009). Plasmids were purified using a Wizard Plus SV 147 (Promega) and sequenced by Stab Vida (Portugal) with an universal primer (M13). 148 A restriction map of the cox1 sequences of P. irritans from Spain and Argentina was 149 constructed using The Sequence Manipulation Suite (Stothard, 2000; available at 150 http://www.bioinformatics.org/sms2/rest_map.html) in order to identifiy certain 151 endonucleases which could discriminate between both geographical origins by PCR152 linked random-fragment-length polymorphism (RFLP). 153 In order to assess the similarity among all marker sequences of P. irritans obtained in 154 this study and other Pulicidae species, we analyzed the number of base differences per 155 sequence from between sequences studied using no. of differences method of MEGA 5 156 program version 5.2 (Tamura et al., 2011). The program DOTMATCHER from the 157 European Molecular Biology Open Software Suite package (Rice et al., 2000) was used 158 to find repeats within the ITS1 sequences. Page 7 of 49 Medical and Veterinary Entomology For Review Only 159 Phylogenetic trees were inferred using nucleotide data and performed using two 160 methods: Maximum Likelihood (ML) and Bayesian inferences (B). Maximum 161 Likelihood trees were generated using the PHYML package from Guindon & Gascuel 162 (2003), whereas Bayesian inferences were generated using Mr Bayes-3.2.6 (Ronquist & 163 Huelsenbeck 2003). JMODELTEST (Posada 2008) program was used to determinate 164 the best-fit substitution model for the parasite data (ITS2, cox1 and cytb). Models of 165 evolution were chosen for subsequent analyses according to the Akaike Information 166 Criterion (Huelsenbeck & Rannala 1997; Posada & Buckley, 2004). For the study of the 167 dataset containing the concatenation of three markers (ITS2, cox1 and cytb), analyses 168 based on BI were partitioned by gene and models for individual genes within partitions 169 were those selected by jModeltest. For ML inference, best-fit nucleotide substitution 170 models included general time-reversible model with gamma-distributed rate variation 171 GTR+G (ITS2) and general time-reversible model with gamma-distributed rate 172 variation and a proportion of invariable sites, GTR+I+G (cox1 and cytb). Support for the 173 topology was examined using bootstrapping (heuristic option) (Felsenstein 1985) over 174 1000 replications to assess the relative reliability of clades. The commands used in 175 MrBayes-3.2.6 for BI were nst=6 with gamma rates (ITS2) and nst=6 with invgamma 176 rates (cox1 and cytb). For BI, the standard deviation of split frequencies was used to 177 assess if the number of generations completed was sufficient; the chain was sampled 178 every 500 generations and each dataset was run for 10 million generations. Adequacy of 179 sampling and run convergence were assessed using the effective sample size diagnostic 180 in TRACER program version 1.6 (Rambaut & Drummond, 2007). Trees from the first 181 million generations were discarded based on an assessment of convergence. Burn-in 182 was determined empirically by examination of the log likelihood values of the chains. 183 The Bayesian Posterior Probabilities (BPP) are percentage converted. Page 8 of 49Medical and Veterinary Entomology For Review Only 184 The phylogenetic analyses, based on ITS2, cox1 and cytb mtDNA sequences were 185 carried out using our sequences and those obtained from GenBank database (appendix 186 1). Phylogenetic trees based on ITS2, cox1, cytb mtDNA and concatenated (ITS2, cox1 187 and cytb) sequences were rooted including outgroup species representing members of 188 the Order Mecoptera: Panorpa meridionalis. This choice was based on the the 189 combination of morphological and molecular data obtained in former studies which 190 provided compelling evidences for a sister group relationship between Mecoptera and 191 Siphonaptera (Whiting, 2002; Whiting et al., 2008). ITS1 sequence of P. meridionalis 192 or other species of Mecoptera was not available neither by amplification of different 193 individuals nor in any public database. Thus, no phylogenetic tree with other 194 Siphonaptera species based on ITS1 sequences was constructed, as well as this 195 molecular marker was discarded for the concatenated dataset. The selection of flea taxa 196 for the concatenated phylogenetic tree was limited to flea species whose ITS2, cox1 and 197 cytb sequences were available on GenBank database. 198 NETWORK (v5.0.0.1) was used to create inter-population median-joining networks 199 (Bandelt et al., 1999; available at http://www.fluxus-engineering.com), to visualize the 200 evolutionary relationships between cox1 and cytb haplotypes. This approach has been 201 shown to yield the best-resolved genealogies relative to other rooting and network 202 procedures (Cassens et al., 2003). Page 9 of 49 Medical and Veterinary Entomology For Review Only 347 Because of morphological specializations, highly promiscuous fleas species such as P. 348 irritans or C. felis which occurs on a wide variety of Carnivora, could show high levels 349 of genetic variability especially when we assess populations which parasitize different 350 hosts or they are settled in different geographical areas. Van der Mescht et al. (2015) 351 suggested that the host specificity might influence the level of intraspecific genetic 352 divergence since more generalist parasite species will show a higher level of 353 intraspecific genetic variation enabling them to infest a broader host range. This fact has 354 been recently demonstrated by Hornok et al. (2018) who found high mitochondrial 355 sequence divergence in some synanthropic flea species such as C. felis or P. irritans. 356 In the present study, morphometric data showed slight differences between P. irritans 357 specimens from Spain and Argentina. This result was corroborated by PCA appearing 358 the Spanish adults of P. irritans slightly bigger. This fact could be explained according 359 to the different geographical origins and/or different hosts. This is in agreement with 360 Medvedev (1998) who characterized the human flea as a monotypic taxon, being 361 inefficient traditional and classic morphological methods in separating its population 362 groups. In our study, morphometric results did not correspond with molecular and 363 phylogenetic ones since these showed a high degree of nucleotide divergence between 364 individuals from both geographical origins. Our results are in agreement with Hornok et 365 al. (2018) who did not observe morphological differences among P. irritans specimens 366 isolated from humans and wild carnivores (badger, jackal and fox) from Hungary and 367 Croatia; however, these authors found a considerable degree of molecular divergence 368 between both populations based on mitochondrial markers. These results disagree with 369 Krasnov et al. (2015) who supported the idea that the process of host selection by fleas 370 is determined by reciprocal relationships between host traits and flea traits. Thus, flea 371 species with similar traits, independently of their phylogenetic affinities, were found on Page 16 of 49Medical and Veterinary Entomology For Review Only 372 the same host species more often than expected by chance from the entire pool of flea 373 species. In this sense, future morphometric studies of P. irritans from different hosts 374 and continents would be necessary. 375 The Internal Transcribed Spacer 1 and 2 ribosomal DNA (ITS1 and ITS2) have been 376 shown to be two of the best molecular markers to analyze genetic relationships at the 377 species level in arthropods (Monje et al., 2013; Zagoskin et al., 2014). 378 At the present work, we observed that ITS2 sequences of P. irritans were markedly 379 shorter than ITS1 sequences, which has already been noticed in other flea species such 380 as C. felis, Stenoponia tripectinata tripectinata, C. canis and N. fasciatus (Vobis et al., 381 2004, Zurita et al., 2015; 2016 and 2018). Furthermore, the ITS1 rDNA regions 382 revealed a considerable length variation between both geographical population caused 383 by a long repetitive region of 86 bp length that appeared twice and once in specimens 384 collected from Spain. Internal repeats in the ITS spacers are usual and have been 385 frequently described. This fact have already noticed in fleas by Gamerschlag et al. 386 (2008) who reported the existence of length differences between the ITS1 rDNA of the 387 African and the South American T. penetrans populations caused by the number of 388 repeats of a repetitive region of 99 bp. Furthermore, these authors detected repetitive 389 sequences within the ITS1 rDNA region of other flea species such as C. felis, 390 Echidnophaga gallinacea, P. irritans, Spilopsyllus cuniculi, and X. cheopis, 391 highlighting that these repetitive elements could serve as a valuable tool for 392 phylogeographic studies. Our study also agrees with Ghavami et al. (2018) who found 393 three repeated units with a length of 98-99 bp and a tandemly repeated sequence within 394 the ITS1 of P. irritans populations isolated from Khodabandeh and Mahneshan (Iran). 395 Thus, these authors suggested that the different number and size of repetitive units in 396 ITS1 may be the sign of developed traits establishing plesiomorphic characters among Page 17 of 49 Medical and Veterinary Entomology For Review Only 397 different populations. In P. irritans, the number of these units depends on ecological 398 conditions (McKern et al., 2008; Gamerschlag et al. 2008); furthermore, in contrast to 399 other authors such as Vobis et al. (2004), Gamerschlag et al. (2008), Marrugal et al. 400 (2013) and Zurita et al. (2015) who not observed great ITS sequences differences 401 among several populations belonging to the same flea species, we found high values of 402 intraspecific variation between both geographical populations in P. irritans, especially 403 in ITS1 sequences (Intraspecific similarity ranged from 95.9 % to 96.3 %). The 404 existence of two genetic lineages (Spain and Argentina) was corroborated by ITS2 405 phylogenetic tree, thus both populations clustered separately based on their 406 geographical origin. Additionally, when ITS1 sequences of different specimens of P. 407 irritans isolated from different geographical areas were compared the highest values of 408 nucleotide divergence were observed in specimens from Nearctic and Neotropical areas 409 (United States and Argentina), whereas almost no differences were observed among 410 individuals from Palearctic and Afrotropical regions (Spain and Cameroon) (Table 3). 411 These data might suggest a possible American origin for this flea species since ancestral 412 populations usually exhibit higher genetic diversity values compared to recent 413 populations that have expanded into novel territories (Savolainen et al., 2002). 414 Cox1 and cytb markers have been used in flea studies in the last fifteen years with 415 several purposes. In order to assess the phylogeographic structure of certain populations 416 (Dittmar & Whiting, 2003), to study the phylogenetic diversity of some species 417 (Lawrence et al., 2014), to carry out a molecular characterization of certain species 418 (Zurita et al., 2015; Zurita et al., 2016) or even for the reconstruction of ancestral host 419 affiliation and biogeographic history of fleas (Zhu et al., 2015). At the present study, we 420 amplified cox1 partial gene. The obtained sequences showed a low value of intraspecific 421 similarity within P. irritans from Spain and Argentina (91.5 % - 92 %), in contrast to Page 18 of 49Medical and Veterinary Entomology For Review Only 422 the high values of similarity observed for specimens from the same population (> 99 423 %). Zurita et al. (2016) observed values of similarity around 97 % between two 424 congeneric species of fleas (C. felis and C. canis) collected from different geographical 425 areas. Recently, Hornok et al. (2018) based on cox1 sequences of P. irritans from 426 different hosts, observed two diverged mitochondrial lineages between Croatia and 427 Hungary. Therefore, these authors claimed about the necessity to carry out 428 supplementary studies using a large scale sampling of P. irritans from different hosts 429 and geographical areas to conclude in this context. In our study, the comparative study 430 of cox1 sequences of P. irritans isolated from different geographical region showed the 431 lowest values of nucleotide divergence among Palearctic and Australian specimens, 432 whereas, likewise ITS1 analysis, the highest values of nucleotide divergence were 433 observed when these specimens were compared with Argentinean population 434 (Neotropical) (Table 4). These results would support the idea that this species had a 435 South American origin. Historically, DNA barcoding studies on insects and 436 invertebrates have shown maximum intraspecific variation ranging from 3 to 3.9 % 437 (Carew et al. 2007). This high degree of polymorphism for intraspecific analysis could 438 be explained attending to wide geographical localities where the samples were 439 collected. Indeed, in certain groups, such as amphibians, when several individuals of the 440 same species come from distant geographical regions, intraspecific variation can exceed 441 the interspecific variation observed between species of the same genus, making it 442 difficult for the delimitation of species with only the sequence of cox1 (Vences et al. 443 2005). However, in our study, cytb sequence analysis confirmed the existence of two 444 highly divergent mitochondrial lineages within P. irritans (Spain and Argentina) 445 reforced by ribosomal results. Although we observed a high percentage of 446 intrapopulation similarity in both geographical origins (bit lower in Argentina) based on Page 19 of 49 Medical and Veterinary Entomology For Review Only 447 cytb sequences, we noticed greater nucleotide variability in Argentina than in Spain 448 with the existence of a higher number of haplotypes in the South American area. In this 449 case intraspecific similarity observed (90 % to 92.2 %) were similar or even lower than 450 those observed between two different congeneric species such as C. felis and C. canis 451 (90.6 %) or Xenopsylla skrjabini and Xenopsylla conformis (92.5 %) (Table 5). These 452 high degrees of mtDNA intraspecific variability could be explained by the fact that 453 generalist flea shows considerably more intraspecific genetic variation than host454 specific flea species. For a generalist parasite, greater levels of genetic variability can 455 provide evolutionary potential for local host race formation (Gómez-Díaz et al., 2007). 456 Previous examples have been reported for ticks and lice parasitizing sympatric hosts 457 (McCoy et al. 2001; Johnson et al. 2002). For this reason, we analyzed the cox1 and 458 cytb intraspecific similarity between Spaniard and Argentinean populations of another 459 generalist flea like C. felis, but, surprisingly, we did not observe differences between 460 them. Cox1, cytb and concatenated phylogenetic trees reinforced the idea of the 461 existence of two geographical genetic lineages within P. irritans. Thus, cox1 462 phylogenetic tree showed specimens collected from Palearctic and Australian region 463 (Spain, Croatia, China, Hungary and New Zealand) clustered together in the same clade 464 and separated from individuals collected from Neotropical region (Argentina). 465 Furthermore, these results show no significant host dependency since specimens 466 collected from Palearctic and Australian areas were isolated from different hosts (see 467 appendix 1). Likewise, cox1, cytb and concatenated phylogenetic trees showed two well 468 supported subclades within P. irritans based on geographical origins (Spain and 469 Argentina). 470 The phylogenetic analysis carried out on the basis on ribosomal and mitochondrial 471 DNA molecular markers suggests the existence of two genetic lineages (Argentina, Page 20 of 49Medical and Veterinary Entomology For Review Only 472 South America and Spain, Europe) of P. irritans populations and the minimum 473 spanning network showed all the cytb haplotypes from Argentina clustered together and 474 with star-like pattern around H2 haplotype. Based on coalescent theory (Slatkin & 475 Hudson, 1991) this star topology showed that P. irritans populations had experienced a 476 significant population expansion. At the centre of the network is haplotype 2, which 477 takes over the highest proportion in the population. This suggests that the haplotype 2 478 should be the ancestral haplotype. This higher genetic diversity in Argentina would 479 reinforce the idea suggested by Buckland & Sadler (1989) that P. irritans, in contrast 480 with other human ectoparasites, could have a South American origin reaching Western 481 Palearctic area through Beringian and Asiatic routes, at any time during the Postglacial. 482 This fact together with a reproductive isolation, could have originated the existence of 483 two cryptic species within P. irritans. In spite of that, to confirm a possible South 484 American origin of this species, more taxonomic, phylogenetic and phylogeographic 485 studies of P. irritans parasitizing different hosts from different geographical areas are 486 needed. 487 In conclusion, the present study provides for the first time, comparative morphometric 488 and molecular data of P. irritans collected from Spain and Argentina (Palearctic and 489 Neotropical areas). On the basis on morphometric results, we found slight differences 490 between both populations. Although we only assessed two populations of P. irritans in 491 this study, our results could suggest the hypothesis that this flea species, in contrast to 492 other generalist fleas, maintain a certain degree of morphological similarity, at least 493 between Western Palearctic and Neotropical areas. Furthermore, based on molecular 494 and phylogenetic data obtained in this work we provided the existence of two well 495 defined geographical genetic lineages within P. irritans species suggesting the existence 496 of two cryptic species which could be discriminated by PCR-linked RFLP. Page 21 of 49 Medical and Veterinary Entomology For Review Only 497 Acknowledgement 498 The present work was supported by a grant of the V Plan Propio de Investigación of the 499 University of Seville, Spain. 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(2007) Lack of 560 host-dependent genetic structure in ectoparasites of Calonectris shearwaters. Molecular 561 Ecology, 16, 5204–15. Page 24 of 49Medical and Veterinary Entomology For Review Only 562 Graham, C.B., Eisen, R.J. & Belthoff, J.R. (2016) Detecting Burrowing Owl 563 Bloodmeals in Pulex irritans (Siphonaptera: Pulicidae). Journal of Medical 564 Entomology, 53, 446–50. 565 Gratz, N.G. (1999) Rodent reservoirs and flea vectors of natural foci of plague, pp. 63– 566 96. In D. T. Dennis, K. L. Gage, N. Gratz, J. D. Poland, and E. Tikhomirov (eds.), 567 Plague manual: Epidemiology, distribution, surveillance and control. World Health 568 Organization, Geneva, Switzerland. 569 Guindon, S. & Gascuel, O. (2003) A simple, fast, and accurate algorithm to estimate 570 large phylogenies by maximum likelihood. Systematic Biology, 52, 696–704. 571 Hastriter, M.W., Miller, K.B., Svenson, G.J., Martin, G.J. & Whiting, M.F. (2017) New 572 record of a phoretic flea associated with earwigs (Dermaptera, Arixeniidae) and a 573 redescription of the bat flea Lagaropsylla signata (Siphonaptera, Ischnopsyllidae). 574 ZooKeys, 657, 67–69. 575 Hopkins, G.H.E. & Rothschild, M. (1953) An Illustrated Catalogue of the Rothschild 576 Collection of Fleas in the British Museum (Nat. Hist.). Vol. I. Tungidae and Pulicidae. 577 Cambridge University Press, Cambridge, UK. 578 Hopla, C. E. (1980) A study of the host associations and zoogeography of Pulex, pp. 579 185–207. In R. Traub and H. Starcke (eds.), In Proceedings of the International 580 Conference on Fleas Lewis, R. E., J. H. Lewis, & C. Maser. (1988) The fleas of the 581 Pacific Northwest. Oregon State University Press, Corvallis, OR.n Fleas, 1977, 582 Peterborough, UK. A.A. Balkema, Rotterdam. 583 Hornok, S., Beck, R., Farkas, R., Grima, A., Otranto, D., Kontschán, J., Takács, N., 584 Horváth, G., Szőke, K., Szekeres, S., Majoros, G., Juhász, A., Salant, H., HofmannPage 25 of 49 Medical and Veterinary Entomology For Review Only 716 Fig. 2. Factor map corresponding to Pulex irritans adults from Spain and South 717 America. Samples are projected onto the first (PC1, 71 %) and second (PC2, 14 %) 718 principal components. Each group is represented by its perimeter. 719 Fig. 3. Alignment of the parcial ITS1 rDNA sequences of Pulex irritans from Spain and 720 Argentina. In red and green bold type the 86 bp repeat unit. Vertical box in blue bold 721 type indicates the polymorphic sites. Gaps generated by alignment (marked by a dash). 722 Fig. 4. Phylogenetic tree of Pulex irritans from different geographical origins (see Table 723 1) based on partial cytochrome c-oxidase 1 (cox1) gene of mitochondrial DNA 724 sequences using the Bayesian (B) and Maximum Likelihood (ML) methods and 725 Bayesian topology. The percentage of replicate trees in which the associated taxa 726 clustered together in the bootstrap test (1,000 replicates) is shown on the branches 727 (B/ML). Bootstrap values lower than 60 % are not shown. The Bayesian Posterior 728 Probabilities (BPP) is percentage converted. 729 Fig. 5. Phylogenetic tree of Pulex irritans from different geographical origins (see Table 730 1) based on partial cytochrome b (cytb) gene of mitochondrial DNA using the Bayesian 731 (B) and Maximum Likelihood (ML) methods and Bayesian topology. The percentage of 732 replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 733 replicates) is shown on the branches (B/ML). Bootstrap values lower than 60 % are not 734 shown. The Bayesian Posterior Probabilities (BPP) is percentage converted. 735 Fig. 6. A minimum spanning network constructed using 20 haplotypes of mitochondrial 736 cytb partial gene sequences of Pulex irritans. The sizes of the circles are proportional to 737 the number of haplotypes represented and the numbers correspond to the mutational 738 steps observed between haplotypes. H1 (2): P. irritans from Argentina; H2 (3): P. 739 irritans from Argentina; H3 (1): P. irritans from Argentina; H4 (1) P. irritans from Page 32 of 49Medical and Veterinary Entomology For Review Only 740 Argentina; H5 (1): P. irritans from Argentina; H6 (1): P. irritans from Argentina; H7 741 (1): P. irritans from Argentina; H8 (10): P. irritans from Spain; H9 (1): P. irritans from 742 Spain. 743 Fig. 7. Phylogenetic tree of Pulex irritans from different geographical origins (see Table 744 1) based on concatenated Internal Transcribed Spacer 2 (ITS2), partial cytochrome c745 oxidase 1 (cox1) and cytochrome b (cytb) gene of mitochondrial DNA inferred using the 746 Bayesian (B) and Maximum Likelihood (ML) methods and Bayesian topology. The 747 percentage of replicate trees in which the associated taxa clustered together in the 748 bootstrap test (1,000 replicates) is shown on the branches. The Bayesian Posterior 749 Probabilities (BPP) are percentage converted. 750 Fig. S1. Phylogenetic tree of Pulex irritans from different geographical origins (see 751 Table 1) based on the Internal Transcribed Spacer 2 (ITS2) sequences using the 752 Bayesian (B) and Maximum Likelihood (ML) methods and Bayesian topology. The 753 percentage of replicate trees in which the associated taxa clustered together in the 754 bootstrap test (1,000 replicates) is shown on the branches (B/ML). Bootstrap values 755 lower than 60 % are not shown. The Bayesian Posterior Probabilities (BPP) is 756 percentage converted. Page 33 of 49 Medical and Veterinary Entomology For Review Only ITS1 Location/Country/Sample ID Species/Gender Host Number of fleas Base pairs (bp) Accession number Seville/Spain/PI1 P. irritans /1♂ - 1 962 LT797452 Seville/Spain/PI1 (Clone 3) P. irritans - - 968 LT853871 Seville/Spain/PI1 (Clone 1-2, 4) P. irritans - - 968 LT853872 Seville/Spain/PI2 P. irritans /1♀ - 1 876 LT797453 Seville/Spain/PI4 P. irritans /1♀ - 1 876 LT797454 Seville/Spain/PI4 (Clone 2) P. irritans - - 882 LT853866 Seville/Spain/PI4 (Clone 3) P. irritans - - 882 LT853867 Seville/Spain/PI4 (Clone 5) P. irritans - - 882 LT853869 Seville/Spain/PI4 (Clone 6-7) P. irritans - - 881 LT853868 Seville/Spain/PI4 (Clone 4, 8) P. irritans - - 882 LT853870 Seville/Spain/PI5 P. irritans /1♀ - 1 876 LT797455 Seville/Spain/PI6 P. irritans /1♂ - 1 962 LT797456 Seville/Spain/PI7 P. irritans /1♀ - 1 876 LT797457 Seville/Spain/PI8 P. irritans /1♀ - 1 876 LT797458 Seville/Spain/PI10 P. irritans /1♂ - 1 876 LT797459 Seville/Spain/PI11 P. irritans /1♀ - 1 876 LT797460 Seville/Spain/PI12 P. irritans /1♂ - 1 962 LT797461 Seville/Spain/PI13 P. irritans /1♀ - 1 876 LT797462 Seville/Spain/PI14 P. irritans /1♂ - 1 876 LT797463 Santa Cruz/Argentina/ PI26-35 P. irritans/6♂ 5♀ L. culpaeus and L. griseus 10 796 LT797464 Santa Cruz/Argentina/PI32 (Clone 1) P. irritans L. culpaeus and L. griseus - 796 LT853873 Santa Cruz/Argentina/PI32 (Clone 3) P. irritans L. culpaeus and L. griseus - 796 LT853874 Santa Cruz/Argentina/PI32 (Clone 2, 6, 8) P. irritans L. culpaeus and L. griseus - 796 LT853875 La Plata/Argentina/668, 670 C. felis/2♀ Canis lupus familiaris 2 668 LT853877 ITS2 Location/Country/Sample ID Species/Gender Host Number of fleas Base pairs (bp) Accession number Seville/Spain/PI1 P. irritans /1♂ - 1 322 LT797448 Seville/Spain/PI6 P. irritans /1♂ - 1 322 LT797449 Seville/Spain/ PI2,4,5,7,8,10-14 P. irritans/3♂ 7♀ - 10 322 LT797450 Santa Cruz/Argentina/ PI26-35 P. irritans/6♂ 5♀ L. culpaeus and L. griseus 10 324 LT797451 La Plata/Argentina/668, 670 C. felis/2♀ Canis lupus familiaris 2 327 LT853876 Cox1 Location/Country/ID Species/Gender Host Number of fleas Base pairs (bp) Accession number/Haplo type Seville/Spain/PI2 P. irritans /1♀ - 1 658 LT797468/H4 Seville/Spain/PI6 P. irritans /1♂ - 1 658 LT797469/H5 Seville/Spain/PI1, 4-5, 7-8, 10-14 P. irritans/4♂ 6♀ - 10 658 LT797470/H6 Santa Cruz/Argentina/ PI30 P. irritans /1♀ L. culpaeus 1 658 LT797465/H1 Santa Cruz/Argentina/ PI33 P. irritans /1♂ L. griseus 1 658 LT797466/H2 Santa Cruz/Argentina/ PI26, 27, 35 P. irritans /3♀ L. culpaeus 3 658 LT797467/H3 La Plata/Argentina/668 C. felis/2♀ Canis lupus familiaris 2 601 LT853879 Cytb Location/Country/ID Species/Gender Host Number of fleas Base pairs (bp) Accession number/Haplo type Seville/Spain/PI2 P. irritans /1♀ - 1 374 LT797473/H9 Seville/Spain/PI1, PI4-8, PI10-11, PI13-14 P. irritans/4♂ 6♀ - 10 374 LT797474/H8 Santa Cruz/Argentina/PI26, PI32 P. irritans /2♀ L. culpaeus 2 374 LT797475/H1 Santa Cruz/Argentina/PI27, PI29, PI35 P. irritans /3♀ L. culpaeus 3 374 LT797476/H2 Santa Cruz/Argentina/ PI28 P. irritans /1♀ L. culpaeus 1 374 LT797477/H3 Santa Cruz/Argentina/ PI30 P. irritans /1♀ L. culpaeus 1 374 LT797478/H4 Santa Cruz/Argentina/ PI31 P. irritans /1♀ L. culpaeus 1 374 LT797479/H5 Santa Cruz/Argentina/ PI33 P. irritans /1♂ L. griseus 1 374 LT797480/H6 Santa Cruz/Argentina/ PI34 P. irritans /1♀ L. culpaeus 1 374 LT797481/H7 La Plata/Argentina/668, 670 C. felis/2♀ Canis lupus familiaris 2 374 LT853878 Table 1. GenBank accession numbers of ITS1, ITS2 and partial cytb, cox1 gene sequences of individuals of P. irritans and C. felis obtained in this study. Page 34 of 49Medical and Veterinary Entomology For Review Only Pulex irritans (Spain) Pulex irritans (Argentina) MIN MAX X SD CV MIN MAX X SD CV TLF(mm) 1.8 2.1 1.9 0.1 5 1.7 2.5 2.1 0.2 9 TLM(mm) 1.3 2.1 1.8 0.3 17 1.4 1.8 1.6 0.2 12 TWF(mm) 1.1 1.2 1.1 0.1 9 1.0 1.3 1.1 0.1 9 TWM(mm) 0.8 1.2 1.0 0.2 20 0.7 0.9 0.8 0.1 12 HLF(µm) 398 597 458 40 8 352 469 416 38 9 HLM(µm) 380 498 418 47 11 322 387 354 46 13 HWF(µm) 234 293 259 25 9 205 264 237 22 9 HWM(µm) 205 293 250 32 12 176 234 205 41 20 EL(µm) 82 147 119 21 17 70 147 110 26 23 EW(µm) 53 59 57 3 5 47 64 57 6 10 CRL(µm) 26 40 34 5 14 28 33 30 4 13 DASL(µm) 82 110 97 10 10 80 94 87 10 11 PROL(µm) 87 152 111 16 14 70 137 100 24 24 MESL(µm) 106 167 135 19 14 88 152 124 25 20 METL(µm) 134 182 162 15 9 100 182 145 24 16 Table 2. Morphometric analysis of ten specimens of P. irritans from Argentina (seven females and three males) and fourteen specimens of P. irritans from Spain (six males and eight females) assessed in this study. Individuals from both sexes have been pooled in both populations. TLF = total female length, TLM = total male length, TWF = total female width, TWM = total male width, HLF = total length of the female head, HLM = total length of the male head, HWF = total width of the female head, HWM = total width of the male head, EL = total length of the spermatheca, EW = total width of the spermatheca, CRL = total length of the extended region of the crochet, DASL = total length of the dorsal aedeagal sclerite, PROL= total length of the prothorax, MESL = total length of the mesothorax, METL = total length of the metathorax, MAX = maximum, MIN = minimum, SD = standard deviation. X = arithmetic mean, CV = Coefficient of Variation (percentage converted). In black bold type parameters which showed certain differences between both geographical origins. Page 35 of 49 Medical and Veterinary Entomology For Review Only ITS1 P. irritans from Spain LT797452LT797463 P. irritans from Argentina LT797464 P. irritans from United States GQ387496. Host unknown P. irritans from Cameroon EU169198. Human host P. irritans from Iran KX822017. Host unknown P. irritans from Spain LT797452LT797463 99.5-100* P. irritans from Argentina LT797464 95.9-96.3 100* P. irritans from United States GQ387496. Host unknown 94.0-95.4 94.4 - P. irritans from Cameroon EU169198. Human host 99.5-99.8 96.2 95.1 - P. irritans from Iran KX822017. Host unknown 99.6-99.9 96.5 93.9 99.6 - Table 3. Intrapopulation* and intraspecific similarity observed amog all the ITS1 sequences of Pulex irritans from different geographical areas obtained in this work and from Genbank database. Values are given in percentages. Page 36 of 49Medical and Veterinary Entomology For Review Only COX1 PI/Seville (Spain)/ LT797468-70 (H4-H6) PI/Santa Cruz (Argentina)/ LT797465-67 (H1-H3) P. irritans/Spain/ Badger/ KF479246 P. irritans/Spain/ human/ KF479247 P. irritans/New Zealand/dog/ KY048351 P. irritans/Hungary/ fox/ MG668624 P. irritans/Hungary/ badger/ MG668626 P. irritans/Hungary/ jackal/ MG668627 P. irritans/China/ polecat/ MF000666 P. irritans/Croatia/ human/ MG668622 C. felis/ Argentina/ LT853879 C. felis/Spain/ LN827896 C. canis/ Iran/LN827901 E.. gallinacea/ Australia/ JN008921 E.. iberica/ Spain/ KF479239 PI/Seville (Spain)/LT79746870 (H4-H6) *99.699.8 PI/Santa Cruz (Argentina)/ LT797465-67 (H1-H3) 91.4-92.0 *99.299.8 P. irritans/Spain/badger/ KF479246 96.0-96.2 92.8-93.0 - P. irritans/Spain/human/ KF479247 99.5-99.8 91.8-92.2 96.4 - P. irritans/New Zealand/dog KY048351 99.8-100 91.5-91.8 96.0 99.7 - P. irritans/Hungary/fox/ MG668624 96.0-96.2 93.2-93.4 98.6 96.4 96.0 - P. irritans/Hungary/badger/ MG668626 96.0-96.2 93.2-93.4 98.6 96.4 96.0 100 - P. irritans/Hungary/jackal/ MG668627 96.0-96.2 93.2-93.4 98.6 96.4 96.0 100 100 - P. irritans/China/polecat/ MF000666 99.6-99.8 91.4-91.8 96.2 99.4 99.8 96.2 96.2 96.2 - P. irritans/Croatia/human/ MG668622 99.4-99.6 91.2-91.6 95.6 99.2 99.6 95.6 95.6 95.6 99.4 - C. felis/Argentina/ LT853879 85.7-86.0 84.9-85.2 85.9 85.9 85.9 87.3 87.3 87.3 86.5 86.3 - C. felis/Spain/LN827896 85.7-86.0 84.9-85.2 85.9 85.9 85.9 87.3 87.3 87.3 86.5 86.3 100 - C. canis/Iran/LN827901 86.4-86.5 85.2 86.4 86.7 86.4 87.3 87.3 87.3 86.9 86.7 97.7 97.7 - E. gallinacea/ Australia/JN008921 87.9-88.0 87.5 88.9 88.0 87.9 88.5 88.5 88.5 88.3 88.1 85.7 85.7 86.0 - E.. iberica/Spain/KF479239 88.7-88.9 89.2-89.5 89.9 88.9 88.7 89.6 89.6 89.6 88.7 88.9 86.7 86.7 87.2 93.5 - Table 4. Intrapopulation (*), intraspecific and interspecific similarity observed among all the partial cox1 mtDNA gene sequences of Pulex irritans from different geographical areas obtained in this work and other Pulicidae species from GenBank database. Values are given in percentages. (PI = Pulex irritans). Page 37 of 49 Medical and Veterinary Entomology For Review Only CYTB PI/Seville (Spain)/ LT797473-74 (H8-H9) PI/Santa Cruz (Argentina)/ LT797475-76 (H1-H7) A. erinacei/ Spain/ LT604120 A. erinacei/ Corse (France)/ LT627350 C. felis/Spain/ LN897470 C. felis/ Argentina/ LT853878 C. canis/Iran/ LN897471 X. conformis/ KM890723 X. skrjabini/ KM890718 X. cheopis/ Canary Islands/ LT604122 S. cuniculi/ KM890622 S. girardi/ KM890686 E. oschanini/ KM890719 PI/Seville (Spain)/ LT797473-74 (H8-H9) *99.7100 PI/Santa Cruz (Argentina)/ LT797475-81 (H1-H7) 90.9-92.2 *97.899.7 A. erinacei/Spain/ LT604120 84.9-85.2 82.5-83.6 - A. erinacei/Corse (France)/ LT627350 85.5-85.8 82.5-83.6 98.9 - C. felis/Spain/ LN897470 82.0-82.3 80.6-81.2 84.7 85.2 - C. felis/Argentina/ LT853878 82.0-82.3 80.6-81.2 84.7 85.2 100 - C. canis/Iran/ LN897471 82.3 79.8-80.4 85.5 86.0 90.6 90.6 - X. conformis/ KM890723 80.1-80.4 78.2-78.8 82.0 82.5 83.9 83.9 83.6 - X. skrjabini/ KM890718 79.8-80.1 77.7-78.2 81.7 82.5 84.4 84.4 82.8 92.5 - X. cheopis/Canary Islands/ LT604122 75.8 72.6-73.4 79.8 80.1 82.0 82.0 81.2 79.6 80.9 - S. cuniculi/ KM890622 81.5 81.7-82.3 82.3 82.8 83.1 83.1 83.9 82.3 81.7 80.1 - S. girardi/ KM890686 80.6-80.9 78.5-79.8 83.6 84.7 84.9 84.9 84.1 84.9 82.8 80.1 78.5 - E. oschanini/KM890719 84.9-85.2 81.7-82.3 84.1 84.7 83.6 83.6 83.9 83.9 83.1 78.5 82.0 79.3 - Table 5. Intrapopulation (*), intraspecific and interspecific similarity observed among all the partial cytb mtDNA gene sequences of Pulex irritans from different geographical areas obtained in this work and other Pulicidae species from GenBank database. Values are given in percentages. (PI = Pulex irritans). Page 38 of 49Medical and Veterinary Entomology For Review Only ITS1 ITS2 Cytb cox1 PCR Mix PCR Buffer (5X) 10 µl 10 µl 10 µl 10 µl dNTPs (10mM) 2 µl 1 µl 1 µl 1 µl MgCl2 (25 mM) 6 µl 6 µl 4 µl 4 µl Forward Primer (10 M) 5 µl 5 µl 5 µl 5 µl Reverse Primer (10 M) 5 µl 5 µl 5 µl 5 µl Template DNA 5 µl 5 µl 5 µl 5 µl goTaq DNA polymerase 0,5 µl 0,5 µl 0,5 µl 0,5 µl Autoclaved distilled water to 100 µl 50 µl 50 µl 50 µl PCR Primers Forward Primer NC5 (Gasser et al., 1996) senITS2 (Vobis et al., 2004) CytbF (Dittmar &Whiting, 2003) LCO1490 (Folmer et al., 1994) Reverse Primer ITS1rev (Marrugal et al.., 2013) ITS2R (Luchetti et al., 2007) A5F (Dittmar &Whiting, 2003) HCO2198 (Folmer et al., 1994) PCR Conditions Initial Denaturing 94 ºC for 5´ 94 ºC for 5´ 95 ºC for 12´ 96 ºC for 2´ Number of cycles 35 35 30 40 Denaturing 94 ºC for 30´´ 94 ºC for 60´´ 95 ºC for 30´´ 94 ºC for 30´´ Annealing 58 ºC for 30´´ 55 ºC for 60´´ 40 ºC for 30´´ 50 ºC for 30´´ Primer extension 72 ºC for 90´´ 72 ºC for 60´´ 68 ºC for 2´ 72 ºC for 60´´ Final extension 72 ºC for 5´ 72 ºC for 10´ 68 ºC for 7´ 72 ºC for 7´ Table S1. PCR mix, primers and conditions used for each molecular marker sequenced in this study. Page 39 of 49 Medical and Veterinary Entomology For Review Only Figure 1 254x190mm (96 x 96 DPI) Page 40 of 49Medical and Veterinary Entomology For Review Only Figure 2 138x90mm (96 x 96 DPI) Page 41 of 49 Medical and Veterinary Entomology For Review Only Appendix 1 List of taxa used in the analysis, including GenBank accession numbers and host information. Species Family Host Accession number Gen Region Sequence length Pulex irritans Pulicidae Homo sapiens EU169198 ITS1 929 Pulex irritans Pulicidae Homo sapiens GQ387496 ITS1 948 Pulex irritans Pulicidae Unknown KX822017 ITS1 1,208 Ophthalmopsylla kiritschenkoi Leptopsyllidae Unknown GQ161960 ITS2 474 Ophthalmopsylla extrema Leptopsyllidae Unknown GQ161956 ITS2 466 Amphipsylla quadratoides quadratoides Leptopsyllidae Unknown AY072642 ITS2 497 Leptopsylla sp. Leptopsyllidae Unknown EF504221 ITS2 459 Leptopsylla sp. Leptopsyllidae Unknown EF504223 ITS2 449 Neopsylla siboi Ctenophthalmidae Unknown AF353113 ITS2 479 Neopsylla teratura Ctenophthalmidae Unknown AF353122 ITS2 479 Neopsylla stevensi Ctenophthalmidae Unknown AY337033 ITS2 479 Neopsylla specialis Ctenophthalmidae Unknown AF353120 ITS2 479 Xenopsylla cheopis Pulicidae Rattus sp. DQ295061 ITS2 356 Xenopsylla cheopis Pulicidae Rattus sp. DQ295059 ITS2 356 Xenopsylla cheopis Pulicidae Rattus sp. LT604121 ITS2 358 Ctenocephalides felis Pulicidae Canis lupus familiaris LN827903 ITS2 327 Ctenocephalides canis Pulicidae Canis lupus familiaris LN827905 ITS2 327 Ctenocephalides canis Pulicidae Canis lupus familiaris LN864485 ITS2 327 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT703438 ITS2 360 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT604114 ITS2 361 Tunga penetrans Tungidae Homo sapiens DQ844716 ITS2 471 Tunga penetrans Tungidae Homo sapiens DQ844724 ITS2 473 Tunga trimamillata Tungidae Unknown AY425820 ITS2 470 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937042 ITS2 332 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937039 ITS2 332 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937038 ITS2 332 Citellophilus tesquorum dzetysuensis Ceratophyllidae Unknown EU770316 ITS2 332 Citellophilus tesquorum altaicus Ceratophyllidae Unknown EU770312 ITS2 332 Nospsyllus fasciatus Ceratophyllidae Apodemus sylvaticus LT158059 ITS2 318 Nosopsyllus fasciatus Ceratophyllidae Muridae LT158060 ITS2 318 Nosopsyllus barbarus Ceratophyllidae Rattus sp. LN881537 ITS2 318 Panorpa meridionalis Panorpidae - LT604124 ITS2 1,121 Echidnophaga gallinacea Pulicidae Oryctolagus cuniculus JN008921 Cox1 650 Echidnophaga myrmecobii Pulicidae Oryctolagus cuniculus JN008919 Cox1 649 Echidnophaga iberica Pulicidae Oryctolagus cuniculus KF479239 Cox1 658 Echidnophaga sp. Pulicidae Mammal JN008922 Cox1 654 Echidnophaga ambulans ambulans Pulicidae Tachyglossus aculeatus KR363632 Cox1 601 Xenopsylla cunicularis Pulicidae Oryctolagus cuniculus KF479238 Cox1 658 Pulex irritans Pulicidae Meles meles KF479246 Cox1 658 Pulex irritans Pulicidae Homo sapiens KF479247 Cox1 658 Pulex irritans Pulicidae Canis lupus familiaris KY048351 Cox1 658 Pulex irritans Pulicidae Jackal MG668627 Cox1 489 Pulex irritans Pulicidae Badger MG668626 Cox1 489 Pulex irritans Pulicidae Fox MG668624 Cox1 489 Pulex irritans Pulicidae Homo sapiens MG668622 Cox1 489 Pulex irritans Pulicidae Vormela peregusna MF000666 Cox1 672 Spilopsyllus cuniculi Pulicidae Oryctolagus cuniculus KF479236 Cox1 658 Spilopsyllus cuniculi Pulicidae Oryctolagus cuniculus KF479237 Cox1 658 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT604116 Cox1 658 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT604115 Cox1 658 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT627349 Cox1 658 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT703440 Cox1 658 Ctenocephalides felis Pulicidae Canis lupus familiaris LN827896 Cox1 600 Page 48 of 49Medical and Veterinary Entomology For Review Only Ctenocephalides felis Pulicidae Canis lupus familiaris LT853879 Cox1 600 Ctenocephalides felis felis Pulicidae Felis catus KF684891 Cox1 601 Ctenocephalides felis strongylus Pulicidae Canis lupus familiaris KF684876 Cox1 601 Ctenocephalides orientis Pulicidae Canis lupus familiaris KF684871 Cox1 601 Ctenocephalides canis Pulicidae Canis lupus familiaris KP684210 Cox1 658 Ctenocephalides canis Pulicidae Canis lupus familiaris LN827901 Cox1 600 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937072 Cox1 677 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937071 Cox1 677 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LK937073 Cox1 677 Nosopsyllus fasciatus Ceratophyllidae Crocidura russula LT158040 Cox1 658 Nosopsyllus fasciatus Ceratophyllidae Apodemus sylvaticus LT158041 Cox1 658 Nosopsyllus barbarus Ceratophyllidae Rattus sp LN881549 Cox1 658 Nosopsyllus barbarus Ceratophyllidae Rattus sp LN881550 Cox1 658 Panorpa meridionalis Panorpidae - LT604125 Cox1 658 Panorpa meridionalis Panorpidae - LT604126 Cox1 658 Stenoponia tripectinata tripectinata Stenoponiidae Mus musculus LN897473 Cytb 374 Ophthalmopsylla praefecta praefecta Leptopsyllidae Unknown KM890714 Cytb 369 Ctenocephalides felis Pulicidae Canis lupus familiaris LN897470 Cytb 374 Ctenocephalides felis felis Pulicidae Unknown KM890759 Cytb 369 Ctenocephalides canis Pulicidae Canis lupus familiaris LN897471 Cytb 374 Ctenocephalides felis damarensis Pulicidae Unknown KM890641 Cytb 369 Xenopsylla cheopis Pulicidae Rattus sp. LT604122 Cytb 374 Archaeopsylla erinacei erinacei Pulicidae Unknown KM890725 Cytb 369 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT604120 Cytb 374 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT604117 Cytb 374 Archaeopsylla erinacei Pulicidae Erinaceus europaeus LT627350 Cytb 374 Synopsyllus girardi Pulicidae Unknown KM890686 Cytb 369 Xenopsylla conformis conformis Pulicidae Unknown KM890723 Cytb 369 Xenopsylla skjrabini Pulicidae Unknown KM890718 Cytb 369 Xenopsylla ramesis Pulicidae Unknown KM890637 Cytb 342 Echidnophaga oschanini Pulicidae Unknown KM890719 Cytb 369 Spilopsyllus cuniculi Pulicidae Unknown KM890622 Cytb 369 Cediopsylla inaequalis inaequalis Pulicidae Unknown KM890600 Cytb 369 Nosopsyllus barbarus Ceratophyllidae Rattus sp LN897460 Cytb 374 Nosopsyllus barbarus Ceratophyllidae Rattus sp LN897462 Cytb 374 Nosopsyllus fasciatus Ceratophyllidae Muridae LT158049 Cytb 374 Nosopsyllus fasciatus Ceratophyllidae Apodemus sylvaticus LT158043 Cytb 374 Nosopsyllus iranis theodori Ceratophyllidae Gerbillus dasyurus KM890603 Cytb 369 Nosopsyllus laeviceps ellobii Ceratophyllidae Unknown KM890720 Cytb 369 Panorpa meridionalis Panorpidae - LT604127 Cytb 374 Panorpa meridionalis Panorpidae - LT604128 Cytb 374 Page 49 of 49 Medical and Veterinary Entomology