A total of 2272 fleas were collected from 13 micromammal species, with an overall prevalence of 46.7% (n = 706). The overall mean abundance was 1.5 fleas per host and the overall mean intensity was 3.2 fleas per parasitized host (Table 1). Excluding the species in which < 20 individuals were sampled, the micromammals that presented the highest prevalence of fleas were Loxodontomys micropus (Austral greater mouse, 87.5%) and Octodon degus (Fence degu, 78.3%), and the lowest prevalence was found in R. rattus (29.2%). The abundance and mean intensity were higher in O. degus (Table 1). The marsupial Thylamys elegans (Llaca mouse-opossum) had a prevalence of fleas of 51.4%. All of the flea species found in T. elegans corresponded to species that were also found in rodents (Table 3).
Of all collected fleas, 1315 flea specimens were analyzed, corresponding to 27 species from 15 genera and 8 families (Table 4). The most abundant flea species were Sphinctopsylla ares (n = 211) and Neotyphloceras chilensis (n = 202; Table 4). The rodents that presented the greatest flea richness were Abrothrix olivacea (olive grass mouse, 17 spp.), R. rattus (14 spp.), A. hirta (long-haired grass mouse, 11 spp.), and Oligoryzomys longicaudatus (long-tailed pygmy rice rat, 11 spp.; Table 3). Natural areas were where the largest number of flea species (n = 25) and specimens were collected (n = 784), followed by villages (18 species, 349 specimens) and cities (18 species, 181 specimens). Agastopsylla boxi, Ctenoparia jordani, C. topali, Ectinorus cocyti and Plocopsylla lewisi were exclusive to natural areas (national parks and national reserves). Conversely, Xenopsylla cheopis was only found in one city (Iquique). Neotyphloceras chilensis and S. ares were the dominant species in natural areas (N. chilensis (n = 119); S. ares (n = 151)), and villages (N. chilensis (n = 83); S. ares (n = 50)), while Nosopsyllus fasciatus (n = 37), and C. inopinata (n = 25) were the most frequently collected in cities. Leptopsylla segnis, N. fasciatus, and X. cheopis are synanthropic rodent fleas [26], and were more abundant in cities than in villages and natural areas.
Rickettsiae prevalence on fleas
Fifteen flea species were found to be Rickettsia-positive for the short fragment (401 bp) of the gltA gene, 9 for the long fragment (830 bp) of the gltA gene, 10 for the rpoB gene, and 4 for the sca5 gene (Table 4). The highest prevalence (13.2%) was detected with the gltA 401-bp gene, followed by the rpoB (5.9%), gltA 830-bp (5.0%) and sca5 (0.5%) genes (Table 4). Among the flea species in which more than 20 individuals were analyzed, the prevalence varied between 0–35.1%. The Neotyphloceras spp. had the highest prevalence of Rickettsia (gltA 401-bp = 29.4%, gltA 830-bp = 9.56%, and rpoB = 11.25%; Table 4). The four fragments (gltA 401-bp, gltA 830-bp, rpoB and sca5) showed significant differences in the prevalence of detected Rickettsia (χ2 = 193.207, df = 3, P < 0.001), exception for gltA 830-bp and rpoB, which did not show significant differences (χ2 = 1.934, df = 1, P = 0.164). No association was found between the number of fleas analyzed and the prevalence of Rickettsia detected for any of the genes analyzed (rpoB: ρ = 0.4267, P = 0.12; gltA: ρ = 0.3757, P = 0.18; sca5: ρ = 0.3272, P = 0.35).
According to the GLM analysis, the prevalence of Rickettsia infection was significantly higher in the semi-arid region (27.8%). In addition, the overall prevalence was significantly higher in the winter (20.6%) than in the summer (5.3%). The prevalence of Rickettsia was higher in natural areas (15.9%), and cities exhibited a marginally significant lower prevalence (4.97%) compared to the other two location types (village: 11.2%; Table 5). Comparisons between bioclimatic regions showed that in the arid region, the prevalence of Rickettsia was higher in the natural areas and in the winter. While in the semi-arid region, the highest prevalence occurred in the winter (73.7%), and the highest prevalence of Rickettsia was detected in the natural areas (77.8%), differentiating from the cities (14.0%). In the sub-humid region, there was no effect of the factors on the prevalence of Rickettsia, whereas in the hyper-humid region, we detected Rickettsia (5.49%) only in the natural areas.
BLAST analysis and phylogenetic inference
A total of 167 sequences of gltA 401-bp (n = 68), gltA 830-bp (n = 40), rpoB (n = 54) and sca5 (n = 5) genes were analyzed (Table 6). For gltA 401-bp, out of the 68 sequences, 28 isolated from Delostichus phyllotis (n = 1), L. segnis (n = 1), N. crassipina (n = 1), N. pardinasi (n = 3), Neotyphloceras spp. (n = 7), N. fasciatus (n = 3), Plocopsylla sp. (n = 2), S. ares (n = 3), T. rhombus (n = 1) and Tetrapsyllus tantillus (n = 6) were 100% identical to Rickettsia sp. (GenBank: KY705378) obtained from the tick Amblyomma parvitarsum. Another 19 gltA sequences (401-bp) detected in Neotyphloceras spp. (n = 16), Chiliopsylla allophyla (n = 2) and C. inopinata (n = 1) were closely related to Rickettsia sp. MEAM1 (99%; GenBank: CP016305) isolated from whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) (n = 16) and Rickettsia sp. Gr15 (GenBank: KP675966) detected in the tick Hyalomma marginatum (n = 3). Twenty-one sequences amplified from Neotyphloceras spp. (n = 1), S. ares (n = 13) and T. rhombus (n = 6) showed 97–98% identity with Rickettsia sp. (GenBank: U59712) isolated from Adelia bipunctata (Coleoptera: Coccinellidae). One sequence amplified from S. ares showed 93% similarity with uncultured Rickettsia sp. (GenBank: KY433588) detected in a tick.
Two sequences of gltA 830-bp segments showed high identity (99%) to “Candidatus Rickettsia senegalensis” (GenBank: KU499847) previously identified in a cat flea (C. felis). Forty sequences obtained from S. ares (n = 12), T. rhombus (n = 6), Neotyphloceras spp. (n = 19) and C. inopinata (n = 1) shared 97–98% identity with Rickettsia spp. (GenBank: KF646706; KY799066; U76908; and AJ269522) isolated from the insects Nesidiocoris tenuis (Heteroptera: Miridae), Mansonia uniformis (Diptera: Culicidae), Empoasca papayae (Hemiptera: Cicadellidae) and Adalia decempunctata (Coleoptera: Coccinellidae).
Seventeen amplified rpoB sequences in Neotyphloceras spp. shared 93–100% similarity with Rickettsia sp. MEAM1 (GenBank: CP016305) isolated from B. tabaci. Another 24 sequences derived from C. allophyla (n = 2), C. inopinata (n = 1), Neotyphloceras spp. (n = 1), S. ares (n = 14) and T. rhombus (n = 6) showed between 91% and 100% homology with Rickettsia sp. (GenBank: JF966777) of Synosternus pallidus (Siphonaptera: Pulicidae). Nine amplified sequences from Neotyphloceras spp. (n = 9) were 94–96% similar to Rickettsia sp. (GenBank: KX300157) isolated from a bat (Myotis emarginatus). Finally, 4 sequences isolated from Neotyphloceras spp. (n = 3) and T. rhombus (n = 1) showed lower homology with Rickettsia sp. (94%, GenBank: KX300203) isolated from a bat (Eptesicus serotinus).
Three sca5 fragments isolated from C. allophyla (n = 2) and C. inopinata (n = 1) showed homology with Rickettsia felis (94%; GenBank: GQ385243), and 2 fragments detected from S. ares showed low identity to R. hoogstraalii (GenBank: EF629536) (Table 6).
The phylogenetic tree shows two well-differentiated clades with 100% nodal support (Fig. 2). Clade R1 was formed by sequences obtained from Neotyphloceras fleas collected in Las Chinchillas NR (31°30′36″S, 71°05′15″W), Canela Baja (31°23′54″S, 71°27′27″W), and Fray Jorge NP (30°23′S, 71°23′W). Rickettsia. bellii (GenBank: DQ146481) was positioned on a basal branch in this group. The clade R2 was subdivided into two subclades: R2a and R2b. R2a, with 93% nodal support, is related to sequences obtained from T. rhombus and S. ares collected in Los Queules NR, Cobquecura, and Coyhaique NR, comprising a larger area of distribution (latitude: -35° to -45°S) than clade R1. Subclade R2b was formed by sequences obtained from C. inopinata and C. allophyla collected in Los Queules NR and Nonguén NR, respectively. The newly generated sequences were positioned closely to R. hoogstraalii (GenBank: FJ767737) isolated from Haemaphysalis sulcata (tick) in Croatia [27], R. asembonensis detected in C. felis from Peru (GenBank: KY650697) [28] and R. felis isolated from C. felis in Brazil (GenBank: JN375498) [29].