Polar Biology (2019) 42:357–370 https://doi.org/10.1007/s00300-018-2427-x ORIGINAL PAPER Sex‑ and developmental‑specifc transcriptomic analyses of the Antarctic mite, Alaskozetes antarcticus, reveal transcriptional shifts underlying oribatid mite reproduction Hannah E. Meibers1 · Geofrey Finch1 · Robert T. Gregg1 · Sierra Glenn1 · Keavash D. Assani1 · Emily C. Jennings1 · Benjamin Davies1 · Andrew J. Rosendale1 · Christopher J. Holmes1 · J. D. Gantz2 · Drew E. Spacht3 · Richard E. Lee Jr.2 · David L. Denlinger3 · Matthew T. Weirauch4,5 · Joshua B. Benoit1 Received: 13 March 2018 / Revised: 9 October 2018 / Accepted: 22 October 2018 / Published online: 7 November 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract The oribatid mite Alaskozetes antarcticus, one of the most abundant terrestrial invertebrates in Antarctica, survives extreme temperature fuctuation and desiccation, and thrives in the short growing season characteristic of this polar environment. Several aspects of the mite’s ecology and physiology are well studied, but little is known about its reproduction. In this study, we utilize sex- and development-specifc next-generation RNA-sequencing (RNA-seq) analyses to identify diferentially regu- lated transcripts underlying reproduction of A. antarcticus. Pairwise comparisons between males, females, and tritonymphs revealed more than 4000 enriched transcripts based on diferent transcriptional levels among sexes and developmental stages. More than 500 of these enriched transcripts were diferentially upregulated over 1000-fold. Many of the highly enriched and sex-specifc transcripts were previously uncharacterized or have no known orthology. Of the transcripts identifed, gene ontology-based analyses linked the transcriptional distinctions to diferences in reproduction, chemosensation, and stress response. Our comparative approach allowed us to determine sexually dimorphic transcript expression in A. antarcticus. We anticipate that this study will provide a baseline to better understand the mechanisms that underlie reproduction in both polar and non-polar oribatid mites. Keywords RNA-seq · Reproduction · Testis-specifc serine/threonine protein kinases · Mite · Antarctic reproductive biology Hannah E. Meibers and Geofrey Finch have contributed equally. Introduction Electronic supplementary material The online version of this article (doi:https​://doi.org/10.1007/s0030​0-018-2427-x) contains The Antarctic oribatid mite Alaskozetes antarcticus is among supplementary material, which is available to authorized users. the most common terrestrial invertebrates in the sub-Antarc- * Joshua B. Benoit tic and maritime Antarctica. It is one of the largest terrestrial [email protected] arthropods in Antarctica, measuring approximately 1 mm 1 in length and weighing 200–300 μg. As an herbivore and Department of Biological Sciences, University of Cincinnati, detritivore, A. antarcticus feeds on organic debris including Cincinnati, OH, USA 2 penguin guano, algae, and lichens (Strong 1967; Goddard Department of Biology, Miami University, Oxford, OH, USA 1977, 1980, 1982; Block and Convey 1995). Large aggrega- 3 Departments of Entomology and Evolution, Ecology tions, with hundreds to thousands of individuals, contain all and Organismal Biology, The Ohio State University, developmental stages, with adults comprising approximately Columbus, OH, USA 4 30% of the individuals present (Block and Convey 1995). Center for Autoimmune Genomics and Etiology Many studies of this mite have focused on environmental and Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children’s Hospital Medical Center, stress tolerance (Young and Block 1980; Block and Convey Cincinnati, OH, USA 1995; Benoit et al. 2008; Everatt et al. 2013). 5 Department of Pediatrics, University of Cincinnati College Most studies on acarine reproduction have focused on of Medicine, Cincinnati, OH, USA ticks due to their medical importance and their large size Vol.:(0123456789)1 3 358 Polar Biology (2019) 42:357–370 in comparison to other mites. Reproduction in females has sex-specifc analysis of this Antarctic oribatid mite, along been examined more thoroughly than in males, and most with comparative analyses of putative sex-specifc gene sets work on females has focused on hormonal regulation and among multiple mite species. We anticipate that this study the production of vitellogenin (Roe et al. 2008; Cabrera et al. will provide the groundwork for future studies focusing on 2009). Male-associated studies have examined specifc fac- Antarctic and oribatid mite reproduction. tors that increase in ticks following blood feeding and those that impact female blood feeding (Weiss and Kaufman 2001, 2004; Roe et al. 2008). For non-tick acarines, studies have Materials and methods been limited to morphological-based observations of male and female reproductive processes (Pound and Oliver Jr. Mite collections and RNA extraction 1976; Mother-Wagner and Seitz 1984; Walzl 1992; Norton 1994), impact of Wolbachia on reproduction (Breeuwer Antarctic mites were collected from Humble Island, near 1997; Weeks and Breeuwer 2001), and studies on the basic Palmer Station, Antarctica (64°45′59″S, 64°05′60″W) in reproductive output of specifc mite species (Norton 1994). January 2017 and maintained in the laboratory at 4 °C under Females of A. antarcticus may develop up to 14 eggs at long day length (20-h light:4-h dark), conditions typical of a time but commonly as few as 4–6 eggs can be found when summer at Palmer Station. Mites were provided access to females are dissected (Block 1980; Convey 1994b, c). Egg algae (Prasiola crispa) and other organic debris collected maturation appears to occur only during the adult’s second with the mites. To ensure standardization, mites were held year (Convey 1994a; Block and Convey 1995). A synchro- under these conditions for two weeks before examination. nous burst of egg production occurs early in the austral Males, females, and tritonymphs (fnal juvenile stage) were spring and summer, with low levels and less synchronous separated based on described morphological characteristics egg production persisting into late summer and winter (Con- (Block and Convey 1995). Male and female were denoted vey 1994a; Block and Convey 1995). Female mites collected by the characters of the genital areas. Males have a smaller, from sub-Antarctic islands had a higher reproductive invest- more rounded, genital area with six or more setae. Females ment than those from maritime Antarctica (Convey 1998), have a larger, more oblong, genital area with only two setae suggesting that harsher environments reduce investment in (Block and Convey 1995). Females were examined for the reproduction. Only a single molt can be achieved each year; presence of developing eggs within the body cavity. Select thus, mite maturation requires at least 4–5 years (Convey males from each cohort (Five mites per sample) were placed 1994a, b). Transfer of sperm occurs through an indirect pro- within Petri dishes, which were examined for the presence cess, where males deposit a stalked spermatophore on the of deposited spermatophores (males were only used if exam- substrate and females take up the spermatophore through ined mites deposited spermatophores). Samples were frozen their genital aperture (Norton 1994; Block and Convey at − 70°C until used. Each sample consisted of 40–50 mites. 1995), a strategy that is similar to that of other oribatid mites RNA was extracted by homogenization (BeadBlaster 24, (Søvik 2002; Søvik and Leinaas 2003a, b; Pfngstl 2013), Benchmark Scientifc) in Trizol (Invitrogen), based on the except for those that undergo asexual reproduction (Maraun manufacturer’s protocol and modifcations based on other et al. 2003; Cianciolo and Norton 2006; Brandt et al. 2017). acarid studies (Rosendale et al. 2016). Extracted RNA Though some specifc aspects of reproduction have been was treated with DNase I (Thermo Scientifc) and cleaned examined for A. antarcticus and other oribatid mites, tran- with a GeneJet RNA Cleanup and Concentration Micro Kit scriptional aspects underlying reproduction in these mites (Thermo Scientifc) according to the manufacturer’s proto- have not been examined. cols. RNA concentration and quality were examined with In this study, we utilized RNA-seq to examine the molec- a NanoDrop 2000 (Thermo Scientifc). Two independent ular mechanisms underlying Antarctic mite reproduction. biological replicates were generated for the male, female, Males and females were examined, along with tritonymphs, and tritonymph samples. to establish female-, male-, and tritonymph-specifc tran- Poly(A) libraries were prepared by the DNA Sequenc- script libraries. Although these studies revealed few female- ing and Genotyping Core at Cincinnati Children’s Hospi- specifc transcripts, many male-specifc transcripts were tal Medical Center. RNA was quantifed using a Qubit 3.0 identifed. Transcripts with distinctly sex-specifc expression Fluorometer (Life Technologies). Total RNA (150–300 ng) were validated through polymerase chain reaction (PCR) and was poly(A) selected and reverse transcribed using a TruSeq quantitative PCR. These sex-specifc libraries were also Stranded mRNA Library Preparation Kit (Illumina). An compared to other mites using two methods: 1. examination 8-base molecular barcode was added to allow multiplex- of overlapping diferences in transcript levels between male ing, and following 15 cycles of PCR amplifcation each and female mites,