Infection by Endosymbiotic “ Male-Killing ” Bacteria in Coleoptera

Wolbachia, Rickettsia, Spiroplasma and Cardinium are endosymbiotic and intracellular bacteria known to cause numerous disorders in host reproduction, reflected in their common name “male-killers”. In this study, 297 beetle species from various taxonomic groups were screened with the use of molecular markers for the presence of infection by any of these endosymbionts. Wolbachia was found to be the most common “male-killer” among beetle hosts as it infected approx. 27% of species. Rickettsia, Spiroplasma and Cardinium were much less prevalent as they infected: 8%, 3% and 2%, respectively, of the studied beetle species. This is the first report of Cardinium presence in beetle hosts. Incidences of co-infection of two bacteria taxa were very rare and only two weevil species were found to be infected by three different bacteria. These findings suggest that endosymbiotic bacteria inhabit their host at various levels of prevalence and that particular taxa usually infect different hosts, suggesting some competition among “male-killers”. This is the first study that simultaneously verifies infection status by all major endosymbiotic bacteria in hundreds of species (based on coleopterans).

Evolution of mutualistic symbioses between unicellular and multicellular organisms was key to starting a new era for many life forms.Not only did it accelerate the evolution of eukaryotic organisms, but most herbivores would not be able to digest without their symbionts (MARGULIS & FESTER 1991).Insects are hosts to a wide range of microorganisms called endosymbionts that live within the host body or cells (MARGULIS & CHAPMAN 2009).The relationship between host and endosymbiont vary from mutualistic (the host derives a fitness benefit) to parasitic (the host suffers a decline in fitness), whereas the majority of bacteria are probably commensals (BUCHNER 1965;ITURBE-ORMAETXE & O'NEILL 2007).The specific group of endosymbionts are so called "male-killing" bacteria.The best known are two genera of á-proteobacteria: Wolbachia (Anaplasmataceae) and Rickettsia (Rickettsiaceae), others like Spiroplasma (Tenericutes) and Cardinium (Bacteroidetes) are much less studied (BOVÉ 1997;CASPI-FLUGER et al. 2011;KAJTOCH & KOTÁSKOVÁ 2018;ZCHORI-FEIN & PERLMAN 2004).Wolbachia has been reported from arthropods and filarial nematodes around the world (TAYLOR & HOERAUF 1999;WERREN & WINDSOR 2000), also Rickettsia seemed to be a common bacterial symbiont of arthropods (PERLMAN et al. 2006).In arthropods, they can manipulate host reproduction through "male-killing" (JIGGINS et al. 2001;LAWSON et al. 2001), cytoplasmic incompatibility (POINSOT et al. 2003), parthenogenesis induction (HAGIMORI et al. 2006;STOUTHAMER et al. 1999) and feminization of genetic males (HIROKI et al. 2002).These effects on host reproduction and development could result in diversification of populations and consequently lead to speciation.On the other hand, many species naturally infected by "male-killing" bacteria (e.g.Drosophila melanogaster; HOFFMANN et al. 1998) do not manifest disruption in sex ratio, while the endosymbiont influences other host traits such as: increasing insulin signaling levels (IKEYA et al. 2009), female fecundity (FRY et al. 2004;FRY & RAND 2002) or inducing host resistance to viral infections (TEIXEIRA et al. 2008) and confers protection against wasp parasitism (XIE et al. 2010).Moreover Wolbachia may be an obligatory bacteriocyte-associated nutritional mutualist (HOSOKAWA et al. 2010), highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.
Beetles (Coleoptera) are the most species-rich and diversified order of insects in the world, including approximately 386,000 known species (OELIPIÑSKI et al. 2011).Knowledge about endosymbiont prevalence within this group is poorly established.Only a few studies so far have found Wolbachia with Rickettsia and/or Spiroplasma together in beetle hosts (BILI et al. 2016;DUDEK et al. 2017;MAJERUS et al. 2000;PEROTTI et al. 2016;SCHEBECK et al. 2018;TOJU & FUKATSU 2011;WEINERT et al. 2007;WHITE et al. 2015).Wolbachia is the best studied organism in this case, with approx.38% of infection among beetles (KAJTOCH & KOTÁSKOVÁ 2018), next Rickettsia being mostly screened among ladybird beetles (WEINERT et al. 2009) and on single species: the buprestid leaf-mining beetle Brachys tessellates (LAWSON et al. 2001) and bruchid beetle Kytorhinus sharpianus (FUKATSU & SHIMADA 1999).Spiroplasma has been studied mostly on single beetle species, e.g. the ladybird beetle Anisosticta novemdecimpunctata (TINSLEY & MAJERUS 2006), leaf beetles Leptinotarsa decemlineata (CLARK 1982) and Diabrotica undecimpunctata, lampyrid beetle Ellychnia corrusca, cantharid beetles Cantharis bilineatus and Cantharis carolinus (CLARK et al. 1987), bark beetle Pityogenes chalcographus (SCHEBECK et al. 2018).Surprisingly it seems that Cardinium has never been found within beetles (SCHEBECK et al. 2018;ZCHORI-FEIN & PERLMAN 2004).TOJU & FUKATSU (2011) showed that pairs of some endosymbionts (e.g.Wolbachia-Rickettsia and Rickettsia-Spiroplasma) infected the same host more frequently than others.Although interesting, their study focused only on Japanese populations of chestnut weevil Curculio sikkimensis (Curculionidae).On the other hand GOTO et al. (2006) suggested that there is some balance in the number of these bacteria, probably caused by competition within host cells.The complexity of endosymbiont interactions and their effects on their hosts may play a pivotal role for diversification of host populations and consequently on speciation (e.g., by the selective sweep of mtDNA or the whole genome of the infected host with the genome of bacteria; KELLER et al. 2004;MAZUR et al. 2016).Consequently, it was proved that presence of endosymbiotic "male-killers" could bias species identification via barcoding (SMITH et al. 2012).
This study, by verifying infection status of Wolbachia, Rickettsia, Spiroplasma and Cardinium across 297 beetle taxa, aimed to answer the question of prevalence of endosymbionts within their beetle hosts.Moreover, co-occurrence of these bacteria in particular beetle species was analyzed in respect to their potential mutually exclusive distribution within the hosts.

Sampling
Beetle species were collected during several field trips organized across central, eastern, and southeastern Europe from 2014 to 2017.Beetle species examined in this study were mostly collected in Poland, Slovakia, Romania, and Bulgaria; some taxa were also taken from Czechia, Germany, Austria, Hungary, Croatia, Greece, Ukraine, and Belarus (192 sites in total, Suppl.Table 1).Beetles were caught in various habitats using numerous entomological techniques (using sweep-net, sieve, light-capturing, traps for scavengers, searching in dead wood, mushrooms, etc.) by experienced entomologists -specialists in various groups of beetles.After collection specimens were immediately preserved in 96% ethanol and afterwards deposited in laboratories in -20ºC.Next, taxonomists assigned the collected beetles to species level.The nomenclature adopted in this study follows that of the Catalogue of Palaearctic Coleoptera (LÖBL & SMETANA 2003-2013).Finally 297 species were selected for molecular examination.The majority of these species were analyzed using two to five or more specimens; only some (mainly rare taxa) could be examined using a single representative.These beetles belonged to 37 families and 204 genera.

Laboratory methods
Before DNA extraction all specimens were cleaned using ethanol and distilled water in order to reduce the risk of external contamination.DNA was extracted from the whole insect body (for beetles up to ap-proximately 10 mm length; only from abdomen for larger specimens) using the Nucleospin Tissue kit (Macherey-Nagel), following the manufacturer's instructions.Prior to bacteria screening, the quality of DNA isolates was assessed by amplification cytochrome oxidase, subunit I of mtDNA (with use of primers specific for beetles from FOLMER et al. 1994;HEBERT et al. 2003) and for almost all isolates amplification of this gene was successful (see Suppl.Table 1).The  (2004).However, these primers were found to amplify not only targeted bacterial taxa, but also some others (e.g.primers for Spiroplasma resulted in sequences identified as Streptococcus) or the obtained sequences were of very poor quality, that is chromatograms from these samples contained multiple double peaks or high background, prevents their unambiguous use in further analyses (this concern mainly trials for Cardinium).Consequently, new primer sets were designed based on 16S sequences downloaded from GenBank separately for Spiroplasma and Cardinium (Suppl.Table 2) and used under PCR conditions as follows: 4 min at 95ºC, then 35 cycles of 35 sec at 95ºC, 1 min at 55ºC (both for Spir and Car primers) and 2 min at 72ºC, followed by 10 min at 72ºC.After DNA purification (Exo-BAP Kit; EURx, Poland), the PCR fragments were sequenced using a BigDye Terminator v.3.1.Cycle Sequencing Kit (Applied Biosystems) and resolved on an ABI 3100 Automated Capillary DNA Sequencer.All newly generated sequences (both from beetles and bacteria) were submitted to GenBank (see Suppl.Table 1) for accession numbers.

Discussion
This is the first study which simultaneously examines infection by all major endosymbiotic "male-killing" bacteria (Wolbachia, Rickettsia, Spiroplasma and Cardinium) across numerous taxa of coleopterans.
In nearly 300 beetle species, Wolbachia infection rate was estimated at approximately 27%, which falls within most calculations for different groups of insects (most estimates suggested between 20% and 70% infection rate (HILGENBOECKER et al. 2008;KIKUCHI & FUKATSU 2003;MIURA & TAGAMI 2004) and corresponds well with the average estimate for insects (24% according to ZCHORI-FEIN & PERLMAN 2004 or 23% according to DURON et al. 2008).It is also lower than an estimate based on a systematic review across literature about Wolbachia infection in beetles by KAJTOCH & KOTÁSKOVÁ (2018), suggesting an infection rate of approximately 38%.However, this estimate was based on numerous previous studies, which usually depended on non-random sampling, i.e. these studies were focused on particular taxonomic group of beetles, which were usually selected on the basis of previous knowledge about Wolbachia occurrence.A revision made by KAJTOCH & KOTÁSKOVÁ (2018) indicated that the highest Wolbachia infection rates in beetle species were found for the Curculionidae (81 species), Chrysomelidae (49 species), Hydraenidae (14 species), Buprestidae (13 species), Coccinellidae (12 species) and Dytiscidae (8 species).In all other families only 1-3 species were reported to harbor Wolbachia.Especially weevils seemed to be a group prone to Wolbachia infection according to the literature and the present study.The high prevalence of Wolbachia within Curculionidae had been suggested by previous studies, e.g.approx.40%, according to LACHOWSKA et al. (2010) and 34.8% of Scolytinae according to KAWASAKI et al. (2016).Wide screening in this study showed even higher values of 68% for Curculionidae and 47% for Apionidae species.et al. 2016;TOJU & FUKATSU 2011;WEINERT et al. 2007;WERREN et al. 1994;WHITE et al. 2015).CHEN et al. 1996).

PEROTTI
Previous studies (BILI et al. 2016;HURST et al. 1999a,b;MAJERUS et al. 2000;TINSLEY & MAJERUS 2006;TOJU & FUKATSU 2011;WEINERT et al. 2007) showed that Spiroplasma infects only some species of Staphylinidae, Coccinellidae and Curculionidae.Again, this study substantially extended the number of infected beetles as hosts of Spiroplasma were found in Cantharidae, Chrysomelidae and Scarabaeidae.Infection rate at the level of 3% in beetles could not be compared with any other previous estimate due to a lack of data.Spiroplasma was found to infect less than 7% of insect species (DURON et al. 2008), so infection in beetles is below this average estimate for insects.Surprisingly, although there was no previous evidence of Cardinium infection among beetles (SCHEBECK et al. 2018;ZCHORI-FEIN & PERLMAN 2004), our results for 297 beetle species revealed the presence of this bacteria for the first time in beetle hosts.Six species from three families Chrysomelidae, Curculionidae and Rhynchitidae harbored this bacteria.Data about infection rate of Cardinium in insects are very limited and only ZCHORI- FEIN &PERLMAN (2004) andDURON et al. (2008) suggested that this bacteria infected 6% or 4% of species, respectively.This is quite consistent with the very low infection rate in beetles (2%) found in this study.
Interestingly, in our study co-infection by different endosymbionts occurred rarely and was limited mostly to two bacteria taxa within one host.The only exception was Polydrusus inustus and Tanymecus palliatus, which were host to three endosymbionts: Wolbachia, Rickettsia and Spiroplasma.This may indicate that there is some balance in the number of these bacteria, probably caused by competition within host cells (GOTO et al. 2006).Moreover, specimens from different populations of two species, Paederus caligatus and Paederus limnophilus, were infected by either Wolbachia or Spiroplasma, but never by both of these bacteria.On the other hand our results partly correspondence with the study of TOJU & FUKATSU (2011), which indicated that pairs of some endosymbionts (e.g.Wolbachia-Rickettsia and Rickettsia-Spiroplasma) are more frequent than others.In fact we found eight species that were co-infected by Wolbachia-Rickettsia, four by Wolbachia-Spiroplasma, but we did not found co-infection by Rickettsia-Spiroplasma.There were also only three beetle species which harbored Cardinium and any other "malekilling" bacteria (namely Wolbachia).However, Cardinium as well as Spiroplasma were found in a low number of beetle taxa (2% and 3%, respectively), therefore the rare co-infection of these bacteria with other endosymbionts could be simply caused by missing such rare cases.
Beetles are still an insufficiently examined group in terms of interactions with endosymbiotic bacteria, particularly with those described as "male-killers".Such complex group as beetles (expressed by complex phylogenetic relationships and by extensive ecological connections with each other and numerous other species) makes it a perfect group for studying relationships between endosymbionts and their hosts.This should be investigated in the near future, thanks to the development of new molecular tools, such as Next Generation Sequencing, and extending databases of DNA markers useful for identification of taxa and intraspecific diversity.Progress in knowledge about the relationships of intracellular endosymbionts with their hosts should be focused on identification of bacteria diversity at various levels of organization (e.g. by identification and analyses of co-occurrence of distinct strains) and on examination of effects caused by these bacteria (alone or in conjunction) on infected species, populations and individuals.