26th International Scientific-practice Conference HUMAN AND NATURE SAFETY 6 – 7 of May 2020

Laelapidae (: ) from small rodents: molecular identification

Justina Snegiriovaitė, Evelina Kaminskienė, Jana Radzijevskaja, Algimantas Paulauskas Faculty of Natural Sciences, Vytautas Magnus University, Lithuania [email protected] mites (Acari: Mesostigmata) from small rodents: molecular identification

Introduction

The gamasid mites of the family Laelapidae include a large number of parasitic genera and are most commonly found in nests of various small mammals and/or on their bodies.

q The identification of gamasid species is problematic due to their minute size, morphological similarity and the limited number of available morphological characteristics. q Molecular methods are highly valuable for the species identification of microscopic mites.

The aim of this study was molecular characterization of laelapid mites parasitizing small rodents.

2 Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification Materials and methods q Laelapidae mites were collected from different species of small rodents, trapped in different locations in Lithuania during 2015-2016. q After isolation from rodents, laelapid mites were identified by microscopic using identification keys (Bregetova, 1956; Baker, 1999; Mašán & Fendʼa, 2010; Kaminskiene et al., 2017). Six species of Laelapidae mites were morphologically identified: § Laelaps agilis; § Laelaps hilaris; § Myonyssus gigas; § Haemogamasus nidi; § Eulaelaps stabularis; § Hyperlaelaps microti.

3 Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification Materials and methods Molecular analysis q Genomic DNA from mites was extracted using 2.5% ammonia solution. q Nuclear and mitochondrial genome markers were used for species identifications: the 28S region (domains 1-3) of the nuclear rRNA and the mitochondrial cytochrome oxidase subunit I (COI) gene. q DNA fragments amplified during PCR were visualized and analyzed by gel electrophoresis. q Obtained PCR products were excised from the agarose gel, purified and sequenced. q 28S rRNA and COI gene sequences were edited, aligned and subjected for phylogenetic analysis using Mega X software. q Phylogenetic trees were constructed using the Neighbor-joining method (NJ) with bootstrap analysis of 1000 replicates.

4 Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification

Results

PCR analysis of 28S rRNA gene allowed amplification of 7 different species.

Figure 1. Amplification of 28S rRNA gene produced 856 bp of PCR products. M is 100 bp DNA ladder; K- – negative control, K+ - positive control, 1-10 – DNA of Laelapidae mites

COI gene PCR analysis allowed amplification of 5 different mite species.

Figure 2. Amplification of COI gene produced 709 bp of PCR products. M is 50 bp DNA ladder; K+ - positive control; K- – 5 negative control; 1-16 – DNA of Laelapidae mites Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification 28S rRNA q Seven mite species were molecularly characterized based on 28S rRNA sequence. q Analyzed 28S rRNA sequences within each mites species were 100% identical to each other. q Phylogenetic analysis showed high genetic heterogeneity among mite species from Lalelaps genus.

Figure 3. Phylogenetic tree of 28S rRNA sequences of Laelapidae mites. The sequences analyzed in this study are marked: ●Hg. nidi; ●M. gigas; ●L. agilis; ●L. hilaris; ●H. microti; ●E. stabularis; ●L. jettmari. LT – Lithuania; NG – Norway; F - female; M – male. 6 Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification COI q Five mite species were molecularly characterized based on COI gene sequence. q COI gene sequences of L.agilis, M. gigas and H.microti mites were variable. q Five haplotypes were detected among COI sequences of L. agilis, and two haplotypes among M. gigas and H. microti.

Figure 4. Phylogenetic tree of COI gene sequences of Laelapidae mites. The sequences analyzed in this study are marked: ●Hg. nidi; ●M. gigas; ●L. agilis; ●L.hilaris; ● H. microti. LT – Lithuania; F - female; M – male. 7 Laelapidae mites (Acari: Mesostigmata) from small rodents: molecular identification

Conclusions q This study represents first molecular identification of L. agilis, L. hilaris, E. stabularis, M. gigas, Hg. nidi, and H. microti mites based on the 28S rRNA and COI genes sequences. q Our study provides new information on phylogenetic relationship among analysed seven species of Laelapidae mites and other species of this family which sequences are stored in GenBank.

8