FEMS Microbiology Letters, 363, 2016, fnw147 doi: 10.1093/femsle/fnw147 Advance Access Publication Date: 7 June 2016 Research Letter R E S E A RCH L E T T E R – Environmental Microbiology Endosymbiotic bacteria in honey bees: Arsenophonus spp. are not transmitted transovarially 1,2,∗ 2 3 Orlando Yanez˜ , Laurent Gauthier , Panuwan Chantawannakul Downloaded from and Peter Neumann1,2,3 1Institute of Bee Health, Vetsuisse Faculty, University of Bern, Schwarzenburgstrasse 161, CH-3003 Bern, Switzerland, 2Swiss Bee Research Centre, Agroscope, Bern, Switzerland and 3Bee Protection Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand http://femsle.oxfordjournals.org/ ∗Corresponding author: Institute of Bee Health, Vetsuisse Faculty, University of Bern, Schwarzenburgstrasse 161, CH-3003 Bern, Switzerland. Tel: +41-(0)31-631-57-66; E-mail: [email protected] One sentence summary: Arsenophonus endosymbiotic bacteria are not transmitted transovarially in honey bees. Editor: David Clarke ABSTRACT Intracellular endosymbiotic bacteria are common and can play a crucial role for insect pathology. Therefore, such bacteria by Orlando Yanez on July 5, 2016 could be a potential key to our understanding of major losses of Western honey bees (Apis mellifera) colonies. However, the transmission and potential effects of endosymbiotic bacteria in A. mellifera and other Apis spp. are poorly understood. Here, we explore the prevalence and transmission of the genera Arsenophonus, Wolbachia, Spiroplasma and Rickettsia in Apis spp. Colonies of A. mellifera (N = 33, with 20 eggs from worker brood cells and 100 adult workers each) as well as mated honey bee queens of A. cerana, A. dorsata and A. florea (N = 12 each) were screened using PCR. While Wolbachia, Spiroplasma and Rickettsia were not detected, Arsenophonus spp. were found in 24.2% of A. mellifera colonies and respective queens as well as in queens of A. dorsata (8.3%) and A. florea (8.3%), but not in A. cerana. The absence of Arsenophonus spp. from reproductive organs of A. mellifera queens and surface-sterilized eggs does not support transovarial vertical transmission. Instead, horizontal transmission is most likely. Keywords: Endosymbionts; Arsenophonus; honey bees; Apis mellifera; Apis dorsata; Apis florea INTRODUCTION et al. 2003; Perlman, Hunter and Zchori-Fein 2006; Kozek and Rao 2007;Sem´ etey´ et al. 2007; Bressan et al. 2009;Novakov´ a,´ Hypsaˇ Endosymbiotic bacteria are widespread in arthropods (Hilgen- and Moran 2009; Wilkes et al. 2011). boeker et al. 2008; Duron et al. 2008). Their interactions with Transmission of endosymbiotic bacteria to novel hosts is an hosts are highly variable ranging from obligate (primary) to fac- apparent key element to understand their biological and po- ultative (secondary) symbiosis and from parasitic to mutual- tential benefits for their hosts. The endosymbionts are usually istic symbiosis (Werren, Skinner and Huger 1986; Perotti et al. transmitted vertically. Their spread into the host population can 2007). The bacteria of the genera Wolbachia (Alphaproteobacte- be achieved, in some cases, by manipulating host reproduction, ria, Rickettsiales), Spiroplasma (Mollicutes, Entomoplasmatales), and may cause feminization, cytoplasmic incompatibility and Rickettsia (Alphaproteobacteria, Rickettsiales) and Arsenophonus male killing (Werren, Skinner and Huger 1986; Breeuwer and (Gammaproteobacteria, Enterobacteriales) are, in general, fac- Werren 1990; Werren and O’Neill 1997; Hurst et al. 1999; Hurst ultative endosymbionts and exhibit an extensive host range, and Jiggins 2000; Werren, Baldo and Clark 2008; Engelstadter¨ including arthropods, nematodes, plants and vertebrates (Bove´ Received: 21 December 2015; Accepted: 27 May 2016 C FEMS 2016. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2 FEMS Microbiology Letters, 2016, Vol. 363, No. 14 and Hurst 2009). Transmission may be transovarial, in which the in Chiang Mai and Phatthalung (Thailand), kept in 95% EtOH and bacteria are already present within the eggs (Rollend, Fish and stored at –80◦C until further analyses. Childs 2013) or transovum, in which the bacteria are present on the eggshells (Prado, Rubinoff and Almeida 2006). Endosymbi- Sampling and screening of Apis otic bacteria can also be transmitted horizontally through con- mellifera colonies tact with infected individuals (Thao and Baumann 2004;Gehrer and Vorburger 2012; Ahmed et al. 2013) and from the environ- Mated A. mellifera queens were sampled from colonies that ment (Bright and Bulgheresi 2010), which is thought to enhance were tested positive for either Wolbachia, Spiroplasma, Rick- spread to distantly related host species (Russell and Moran 2005; ettsia or Arsenophonus.ForthescreeningoflocalA. mellifera Gehrer and Vorburger 2012). colonies (N = 33, predominantly A. m. carnica), 20 eggs from In honey bees (Apis spp.), Wolbachia has been detected worker brood cells and 100 adult workers from the middle in workers of Apis mellifera capensis and A. m. scutellata frames were collected at three apiaries in Bern, Switzerland. (Jeyaprakash, Hoy and Allsopp 2003, 2009). Some Wolbachia All egg samples were homogenized with a sterile plastic pes- strains have been characterized for A. m. capensis (Jeyaprakash, tle in 50μl of ChelexR solution (Bio-Rad, Hercules, CA, USA) Hoy and Allsopp 2009), but in general virtually nothing is known for DNA extraction. Samples were incubated at 95◦Cfor20 about effects on host bees. It has been suggested that one of min and centrifuged at 12 000 rpm for 2 min. Twenty fold those strains may be responsible for thelytokous parthenogen- dilutions were used for PCR reactions. DNA was extracted esis (Hoy et al. 2003), but this hypothesis was later rejected (Lat- from the pooled worker samples following standard procedures R torff, Moritz and Fuchs 2005). Two species of Spiroplasma, Spiro- (Evans et al. 2013) using the NucleoSpin Tissue kit (Macherey- Downloaded from plasma apis (Mouches et al. 1983)andS. melliferum (Clark et al. Nagel, Dueren, Germany) following the supplier’s guidelines. 1985), have been characterized, and S. apis maybethecausal PCR was performed using the high-fidelity Kapa HiFi DNA agent of ‘May disease’ (Mouches, Bove´ and Albisetti 1984). Rick- Polymerase Kit (Kapa Biosystems, Woburn, MA, USA) follow- ettsia in honey bees has been associated with milky hemolymph ing the manufacturer’s recommendations. Primers and PCR of infected workers (Wille and Pinter 1961), but later it was conditions were obtained from previous publications (Table S1, shown that the causal agent was filamentous virions (Clark Supporting Informaiton). Parallel amplification of the honey bee http://femsle.oxfordjournals.org/ 1978). Arsenophonus spp. have been detected in the gut micro- Lys-1 gene (Harpur and Zayed 2013) was used to verify the DNA biota (Babendreier et al. 2007; Cornman et al. 2012)andinthe quality. Negative and positive controls were included in the anal- hemolymph (Gauthier et al. 2015)ofA. mellifera workers and yses. PCR products were stained using GelRed for 30 min after seems to be abundant in the bees’ body surface (Aizenberg- electrophoresis in 1.2% agarose gel. Bands were visualized un- Gershtein, Izhaki and Halpern 2013). Interestingly, Arsenophonus der UV light. spp. appear to be more abundant in colonies displaying clini- cal symptoms of Colony collapse disorder (CCD; Cornman et al. Queen dissections and screening assays 2012). Arsenophonus spp. have also been recently found in Varroa destructor (Hubert et al. 2015), an ectoparasitic mite, which feeds Laying queens were sampled from A. mellifera colonies, which on the honey bee hemolymph. were found positive for any of the tested bacteria (see above). by Orlando Yanez on July 5, 2016 Since many endosymbionts may be beneficial for their hosts The ovaries and digestive tracts of five A. mellifera queens were (Hansen et al. 2007;Oliveret al. 2010) and may also play a role dissected following standard procedures (Carreck et al. 2013). in honey bee pathology (Evans and Armstrong 2006), it is impor- The remains of the queen’s bodies were preserved for further tant to better understand the role of endosymbionts in honey analyses. The ovaries, spermathecae, digestive tracts, thoraces bees in light of A. mellifera colony losses (Neumann and Car- and heads from additional three A. mellifera queens were also reck 2010; Aebi and Neumann 2011). Indeed, depending on the dissected. DNA was extracted from the dissected queen’s body strain, Wolbachia can protect other hosts against several vec- parts and the whole bodies of surface sterilized A. cerana, A. tored RNA viruses (Teixeira, Ferreira and Ashburner 2008)and dorsata and A. florea queens (Table S2, Supporting Informaiton). can be regarded as part of host immunity (Zindel, Gottlieb and Samples were homogenized using a Mixer Mill MM 300 (RETSCH Aebi 2011). Similarly, Spiroplasma rescues host females from the GmbH, Haan, Germany) machine in TN buffer with 3-mm metal sterilizing effects of nematode parasitism (Jaenike et al. 2010). beads. DNA extraction and PCR reactions were performed as de- Likewise, other endosymbionts may be beneficial for honey bees. tailed before. Positive PCR products were Sanger sequenced to Since