Genetic characterization of Wolbachia from Great Salt Lake brine flies Amanda Truong, Mohammad Sondossi & Jonathan B. Clark Symbiosis ISSN 0334-5114 Symbiosis DOI 10.1007/s13199-016-0446-3 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media Dordrecht. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Symbiosis DOI 10.1007/s13199-016-0446-3 Genetic characterization of Wolbachia from Great Salt Lake brine flies Amanda Truong1 & Mohammad Sondossi 2 & Jonathan B. Clark1 Received: 28 April 2016 /Accepted: 6 September 2016 # Springer Science+Business Media Dordrecht 2016 Abstract Wolbachia are intracellular prokaryotic endosymbi- 1 Introduction onts associated with a wide distribution of arthropod and nem- atode hosts. Their association ranges from parasitism to mu- The genus Wolbachia comprises a group of endosymbiotic tualism, and there is growing evidence that Wolbachia can bacteria found in many species of arthropods and nematodes have dramatic effects on host reproduction, physiology, and (Zug and Hammerstein 2012). Wolbachia have been found to immunity. Although all Wolbachia are currently considered as induce numerous effects on the reproductive mechanisms of single species, W. pipientis, phylogenetic studies reveal about their hosts, including sperm-egg cytoplasmic incompatibility; a dozen monophyletic groups, each designated as a super- killing of male progeny; feminization of males; and partheno- group. This study uses 16S rRNA gene sequences to examine genesis, in which unfertilized eggs develop by default into the genetic diversity of Wolbachia present in three species of female progeny (Stouthamer et al. 1999;Werrenetal.2008). Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, Each of these manipulations has the effect of increasing the and Mosillus bidentatus. The brine fly Wolbachia sequences number of infected females, thereby enhancing the long-term are highly similar, with an average nucleotide sequence diver- survival of the endosymbiont. Wolbachia have complicated gence among the three species of 0.00174. The brine fly associations with their hosts that include mutualism, commen- Wolbachia form a monophyletic group that is affiliated with salism, and parasitism (Hentschel et al. 2000). In addition to a subset of supergroup B, indicating that this supergroup may their influence on host biological, ecological, and evolutionary be more diverse than previously thought. These findings ex- processes, these bacteria have generated much excitement be- pand the phylogenetic diversity of Wolbachia and extend their cause of potential in controlling insect pests and disease vec- host range to taxa adapted to a hypersaline environment. tors, such as those associated with malaria and dengue fever (Bian et al. 2013; Walker et al. 2011). Keywords Wolbachia . Endosymbionts . Hypersaline Molecular phylogenetic analyses reveal as many as 16 environments . Ephydridae . Brine flies monophyletic groups of Wolbachia, designated as super- groups (Glowska et al. 2015). Whereas the host distribution for each supergroup is limited, there is little congruence be- Electronic supplementary material The online version of this article tween host phylogeny and endosymbiont phylogeny. This (doi:10.1007/s13199-016-0446-3) contains supplementary material, suggests that Wolbachia may be transferred fairly frequently which is available to authorized users. between hosts and that Wolbachia infection does not always persist following host speciation events (Aikawa et al. 2009; * Jonathan B. Clark [email protected] Klasson et al. 2009; Woolfit et al. 2009). Within some super- groups, specific genotypes of Wolbachia may be more preva- 1 Department of Zoology, Weber State University, Utah, lent than others, owing to their favorable effects on host fe- Ogden, UT 84408-2505, USA cundity (Serga et al. 2014). In addition, genetic transfer be- 2 Department of Microbiology, Weber State University, Utah, tween Wolbachia and other endosymbionts may also occur Ogden, UT 84408-2505, USA (Nikoh and Nakabachi 2009). Author's personal copy Truong A. et al. To date, Wolbachia have been found in a large number of Purification Kit (Epicentre® Biotechnologies). To provide insect species examined (Hilgenboecker et al. 2008;Zugand some measure of the Wolbachia diversity within a species, Hammerstein 2012), but their presence in many invertebrate DNA from multiple individuals was pooled prior to the final taxa is unknown. In particular, there is no identification of 16S rRNA gene amplification reactions. Wolbachia from host species inhabiting extreme environments. Control primers for the mitochondrial cytochrome oxidase Great Salt Lake (GSL), in northern Utah, is the largest saline subunit I gene (COI) and the nuclear rRNA internal tran- lake in the western hemisphere and is characterized by salinity scribed spacer region (ITS-1) were also used to confirm the levels that range from 6 % to full saturation. Among the few identity of each host species. Wolbachia DNA was detected animals that live and reproduce in the lake’s waters are the brine for all individuals from each species, using polymerase chain shrimp, Artemia franciscana, and shore flies (known as brine reaction (PCR) with primers specific for Wolbachia the 16S flies) of the Diptera family Ephydridae. As a group, shore flies rRNA gene. Primer sequences are as follows: are particularly adaptable and are prominent in a number of marginal habitats, including hot springs, oil ponds, highly sa- Wolbachia 16S rRNA (forward): 5′ -TTGT line lakes, and inland alkaline pools and marshes (Foote 1995). AGCCTGCTATGGTATAACT. The family Ephydridae (shore flies) is one of the largest in Wolbachia 16S rRNA (reverse): 5′ -GAAT the order Diptera, with nearly 2000 species described (Wirth AGGTATGATTTTCATGT. et al. 1992). Brine flies are among the most conspicuous in- COI (LepR1): 5′-TAAACTTCTGGATGTCCAAA habitants of Great Salt Lake, the center of a valuable wetland AAATCA. ecosystem in the Great Basin of North America. Few inverte- COI (LepF1): 5′-ATTCAACCAATCATAAAGAT brates possess the historical, cultural, and biological legacy ATTGG. achieved by GSL brine flies and their prominence is well- ITS-1 (7246): 5′-GCTGCGTTCTTCATCGAC. documented in the famous accounts of the western expedi- ITS-1 (7247): 5′-CGTAACAAGGTTTCCGTAGG. tions of John Fremont (1845) and Howard Stansbury (1852). During the summer months, brine flies reach densities estimat- ed at 600 million adults per km shoreline (Felix and Rushforth 2.2 DNA sequencing and phylogenetic analysis 1980). Although Great Salt Lake shorelines are dynamic and transitory, using a conservative average of 4800 km yields an Wolbachia PCR products were visualized by electrophoresis estimate of nearly three trillion adults, and, depending on the and were cloned with the TOPO© Cloning Kit (Invitrogen). time of year, an equal number of larvae and pupae. Purified plasmids were sequenced from both strands using T7 This study uses partial 16S rRNA gene sequences to exam- and SP6 primers. Sequences have been deposited in GenBank ine Wolbachia associated with three species of GSL brine (http://www.ncbi.nlm.nih.gov/) under the accession numbers flies: Ephydra gracilis, Cirrula hians,andMosillus KX139035-KX139040. The brine fly amplicons were bidentatus. To our knowledge, this study represents the first examined for chimeric sequences using Mallard version 1.02 description of Wolbachia from hosts that inhabit an extreme (Ashelford et al. 2006), using a 99 % cutoff value and a log- environment. The brine fly Wolbachia reported here are linear curve for identifying outliers. The reference sequence monophyletic and affiliated with supergroup B, which in- used was the E. coli K-12 16S rRNA (NR_102804). The brine cludes a diverse number of insect hosts. The brine fly fly Wolbachia 16S rRNA gene sequences were aligned with Wolbachia form a distinct lineage that is affiliated with a sub- those from selected supergroups using Clustal Omega set of supergroup B Wolbachia, revealing two well-supported (http://www.ebi.ac.uk/Tools/msa/). Table 1 provides details clades. These results augment the diversity of Wolbachia and of the sequences included in the analyses. suggest that increased sampling may lead to the identification The initial phylogenetic analysis included 900 positions of additional supergroups of this fascinating endosymbiont. within the 16S rRNA gene using parsimony and distance methods as implemented by PAUP 4.0b10. The parsimony analysis used random addition of sequences (ten replicates) 2 Materials and methods and TBR branch-swapping. 684 characters were constant, 61 were parsimony-uninformative, and 155 were parsimony-in- 2.1 DNA isolation and PCR amplification formative. The distance analysis used Neighbor-joining with the Kimura two-parameter sequence correction. Sequences Adult brine flies were collected