Host-associated differences in morphometric traits of parasitic larvae Hirsutiella zachvatkini (Actinotrichida: Trombiculidae)

Host-associated differences in morphometric traits of parasitic larvae Hirsutiella zachvatkini (Actinotrichida: Trombiculidae) Hanna Moniuszko 0 Grzegorz Zalesny 0 Joanna M akol 0 0 Institute of Biology, Department of Invertebrate Systematics and Ecology, Wrocaw University of Environmental and Life Sciences , Koz_uchowska 5b, 51-631 Wrocaw , Poland Examination of host-associated variation in the chigger mite Hirsutiella zachvatkini (Schluger) revealed morphological differences among larvae infesting sympatric hosts: Apodemus agrarius, Apodemus flavicollis and Myodes glareolus. The analysis included 61 variables of larvae obtained from their gnathosoma, idiosoma and legs (measurements and counts). Statistically significant differences were observed for metric characters of the legs as opposed to the scutum. In view of the conspecificity of the mites, supported by comparison of COI gene products obtained from larvae and laboratory-reared deutonymphs, the observed variation is attributed to phenotypic plasticity. The knowledge of larval morphology, including intraspecific variation of metric characters, supported by molecular and host range data, places H. zachvatkini among the most comprehensively defined members of Trombiculidae. Chigger mites - Phenotypic plasticity Trombiculidae sensu Goff (1999) comprise ca. 3000 species, with the vast majority (about 90 %) known exclusively from larvae. Morphology-based methods of species identification, fragmentary knowledge of phenotypic plasticity, scarcity of distributional data, and descriptions based on larvae, make it difficult to evaluate the actual number of species. The difficulties in species delimitation stem also from incomplete knowledge of host spectra and possible host-driven intra-population differences. & Joanna Makol Despite observed morphological differences among Psoroptes skin mites (Astigmata: Psoroptidae), Pegler et al. (2005) found no molecular evidence of species-level diversity and thus refuted the earlier concept of distinct specific identity of the parasites from different host species. Data on host-associated differences among trombiculids are very scarce. Menezes et al. (2011) failed to find any significant morphological differences between groups of Eutrombicula alfreddugesi (Oudemans), which infested different species of lizards, whereas Kuo et al. (2011) observed differences in the degree of engorgement (inferred from idiosoma length and width) of Leptotrombidium imphalum Vercammen-Grandjean and Langston within and among its three host species. Hirsutiella zachvatkini, widely distributed in Europe and Asia, is regarded as one of the most common chigger species. Its presumably wide host spectrum includes rodents, insectivores, lagomorphs and birds (Kudryashova 1998). Active postlarval forms of H. zachvatkini have been re-described by Daniel (1961). Data on metric and meristic characters of larvae have been provided by Stekolnikov (2001a), who has also dealt with chaetotactic anomalies and intraspecific variation of Hirsutiella spp. (Stekolnikov 2001b, 2003), and also by Imaz et al. (2005), however the host-induced variability was not explicitly examined. Here we provide the results of morphometric and molecular analyses of larvae of H. zachvatkini, collected from striped field mouse, Apodemus agrarius (Pallas) (Muridae), yellow-necked mouse, Apodemus flavicollis (Melchior) and bank vole, Myodes glareolus (Schreber) (Cricetidae). Our study aims at answering the question of potential differences between mites infesting different host species. Materials and methods Ectoparasitic larvae (total: 133 specimens) were collected from A. agrarius (46 larvae/11 host specimens), A. flavicollis (45/10) and M. glareolus (42/9). The hosts were captured in Sherman traps (permissions 66/2012, 27/2013 and 41/2013 issued by the Second Local Commission for Animal Experiments) in a deciduous forest stand in the Syco w Municipal Park (51 17022.67200N, 17 42039.76600E), Poland, between September 2012 and April 2014. The larvae were preserved in 96 % ethanol. A molecular analysis, aiming at evaluating the differences between the examined specimens, was carried out on three larvae and three deutonymphs (reared from the most engorged larvae). Each pair (larva ? deutonymph which developed from engorged larva) originated from a different host species. Total genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen). The mites were transferred from 96 % ethanol to ATL lysis buffer with Proteinase K and incubated overnight at 56 C. After digestion, the lysis buffer containing nucleic acids was transferred to a new Eppendorf tube and stored for DNA isolation according to the manufacturers protocol. Amplification of the DNA barcode region (cytochrome c oxidase 1 subunit) was performed using degenerate primers: bcdF04 (50CATTTTCHACTAAYCATAARGATATTGG30) and bcdR04 (50TATAAACYTCD GGATGNCCAAAAAA30) (Dabert et al. 2010) with the following thermocycling conditions: 95 C/3 mininitial denaturation; 95 C/30 s, 48 C/30 s, 72 C/45 s40 cycles; 72 C/7 minfinal extension. The PCR reaction (25 ll) was performed using the following PCR mix: 4 ll of genomic DNA, 10 mM TrisHCl, 50 mM KCl, 1.5 mM MgCl2, 200 lM of each dNTP, 150 pmol of each primer and 2 units of Taq polymerase (EurX). The amplification product was purified using QIAquick PCR purification kit n H 3 a 3 e 4 3 n 1 8 2 2 0 0 3 1 7 7 9 4 5 4 0 4 3 0 4 3 6 o s M 8 8 4 2 2 1 1 1 2 4 3 5 6 3 5 7 5 5 7 7 u a = i t r ad (n ra e y g tiv tud sa .x ta s u a 9 2 it t m 6 m 6 9 6 9 aun seen edo 4= .in 10 11 48 26 28 91 91 20 31 47 35 60 67 37 57 79 57 59 84 89 q r p 4 9 5 3 3 3 1 1 9 1 1 6 1 7 2 1 4 0 P A n M 7 6 3 1 1 8 8 1 2 2 1 5 6 2 5 6 4 4 6 7 r e e i a m T F G T T od so ta L W h a a a a a S S S S M L L W n G G C P P P P P O io IL IW D D V V H A A P A d I ,r f l s f ) ,s e o ra se o i n 7 t t x a 6 n a S le ( a e .2 an th M 0 ch id ev eenb ,tum lev ,aT try tro wrfeo tw scu teh iab em l i a Wb eb f n t m lp I o e sy .x 982 ap ,a num cen ing tew iT fo a .0 rsom tsa ra eb ,un ied 11 .m 4 aT io fV id rm e e s = in .42 ,P id ,)m iro can g e n M 0 e u PWtse its eon lad IL tcu , o d ,G no m . 41 om soo lsa tew l-o rag ife ) n14= .ianxmM ..021203 ttfsahdognho tii;()sdonoud liirssaoodom itscaeednbttrseeeeponw itrraednnom ,trseeanbbFisubd,lieegndx i d b a a e n 3 w w f We ) h p e .2 lc r , an (S rco exd III a 5 a o A c L M 0 Wl eb tea its ae t G ia d lil rin( aT sed 24 ..axm .10306 tsanhoom laopp,tfaeDtltr-seaeo ,i)-sPPL lfseevo caxox,lfeohg ,tIdmm yoM n= inM .910 g dO lsa po axy lee :sC tgn II f e m ra n 7 le ,u Sied o s u t a 7 s f t Wti e .2 to eng ,eV -m se o cu ) sa M 0 ly l a ro ab sse fs (m p a te te n a o a f ap lp s n e b in T o a l a e (Qiagen) and sequenced on both strands (Genomed, Poland). The sequences of H. zachvatkini isolated from analyzed host species were identical, thus only one, obtained from deutonymph that developed from larva parasitising the b (...truncated)