Endosymbionts in Arthropods, Their Role and Possible Implications for Biological Control Success

Biological control 204

Hidden arsenal: endosymbionts in arthropods, their role and possible implications for biological control success

M.R. McNeill1, N.K. Richards1, J.A. White2 and A. Laugraud1

1AgResearch, Lincoln Science Centre, Private Bag 4749, Canterbury 8140, New Zealand 2University of Kentucky, Department of Entomology, S-225 Agric. Science Center, North Lexington, KY 40546-0091, USA Corresponding author: [email protected]

Abstract Bacterial endosymbionts are common among arthropods including many important pest and beneficial insect species. These symbionts provide either an obligate function, performing essential reproductive or nutritive roles, or are facultative, influencing the ecology and evolution of their hosts in ways that are likely to impact biological control. This includes resistance against parasitoids and modification to parasitoid fecundity. Recent research has shown that endosymbionts are associated with exotic weevil pests found in New Zealand pasture, including the clover root weevil, Sitona obsoletus (=S. lepidus). The role of endosymbionts in insect biology and impacts on biological control is reviewed. For New Zealand plant protection scientists, endosymbiont research capability will provide important insights into interactions among insect pests, plant hosts and biological control agents, which may provide management opportunities for existing and future pest incursions in New Zealand.

Keywords symbiosis, arthropod symbiont diversity, arthropod pests, Wolbachia, symbiont- based control strategies, metagenomics, parasitism.

INTRODUCTION Two exotic Sitona species have been accidently However, a major negative impact was its wide introduced and established in New Zealand. non-target host range. Moroccan M. aethiopoides Sitona discoideus Gyllenhal (Coleoptera: was able to parasitise and develop in a range of Curculionidae) was first found in the 1970s native and introduced beneficial weed biocontrol where it proved to be a significant pest of Curculionidae species (Barratt et al. 1997b), as lucerne (Medicago sativa) stands (Goldson et well as the exotic hosts Listronotus maculicollis al. 1985). The introduction and release of a (Dietz) (McNeill et al. 1999) and S. hispidulus Moroccan strain of Microctonus aethiopoides Fabricius (Sundaralingam et al. 2001). Loan (Hymenoptera: Braconidae) (Stufkens et al. Sitona obsoletus (Gmelin) (= S. lepidus) (Löbl 1987), an endoparasitoid of the adult weevil, led and Smetana 2013) was first discovered in New to a significant reduction in weevil populations Zealand in 1996 (Barratt et al. 1996). Of European and M. sativa yield losses (Kean & Barlow 2000). origin, this pasture pest weevil species significantly

New Zealand Plant Protection 67: 204-212 (2014) www.nzpps.org © 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.html Biological control 205 impacts on the production and persistence Two broad categories of insect endosymbionts of white clover (Trifolium repens) (Gerard et have been identified, obligate (primary) or al. 2007). Based on its success in controlling facultative (secondary), and both can significantly S. discoideus and wide host range, the suitability of influence the ecology, biology and evolution of the Moroccan M. aethiopoides was evaluated against host (Table 1). Obligate symbionts are maternally S. obsoletus in the laboratory. Sitona obsoletus inherited and generally have a nutritional basis, proved to be an unsuitable host for Moroccan supplying essential nutrients that are lacking in M. aethiopoides with emergence rates from host diets (e.g. Douglas 1998; Baumann 2005) exposed S. obsoletus of 1-6% (Barratt et al 1997a). with symbiosis going back millions of years A further study showed that although up to 50% of (Moran et al. 1993; Wernegreen & Moran 2001). S. obsoletus exposed to Moroccan M. aethiopoides Approximately 10% of all insect species carry were subject to ovipositional attempts, almost nutritional symbionts (Buchner 1965). Primary all (up to 98%) parasitoid larvae removed from symbionts typically occupy specialized host dissected weevils were non-viable (McNeill et organs called bacteriomes (Baumann 2005) al. 2000). While there are genetic differences and, because the symbiosis is obligate, neither between M. aethiopoides strains (Phillips et al. the bacteria nor the insect is viable without the 2002), the result was intriguing given the ability other. Two well known obligate endosymbionts of this parasitoid to successfully develop in a include Buchnera aphidicola, which infects the pea wide range of alternative hosts (Barratt et al. aphid Acyrthosiphon pisum and provides its host 1997b; McNeill et al. 1999; Sundaralingam et with essential amino acids (Douglas 1998), and al. 2001). However, a possible causal agent was Wigglesworthia glossinidia, which is a symbiont identified when preliminary screening in 2000 of the tsetse fly Glossina morsitans morsitans and found that S. obsoletus was infected with either aids its host in vitamin synthesis (Nogge 1981). Rickettsia or Wolbachia bacterial endosymbionts Weevils are ancestrally infected with the primary (N.K. Richards, AgResearch, unpublished data). endosymbiont Nardonella. In the West Indian Wolbachia has previously been implicated in sweet potato weevil, Euscepes postfasciatus, removal defending a weevil host species against parasitism of this endosymbiont resulted in significantly (Hsiao 1996). Control of S. obsoletus was lighter body weight and lower growth rate than eventually achieved with the release of an Irish the control insects, an effect that persisted in the strain of M. aethiopoides (Gerard et al. 2006), offspring (Kuriwada et al. 2010). but the reason behind resistance to parasitism In comparison, facultative or secondary by the Moroccan strain remained of interest due endosymbionts are not essential for host to its implications for future biological control survival and their presence can be neutral, programmes. In a recent genetic diversity profile beneficial or detrimental to the host (Oliver study, S. obsoletus was found to be infected by one et al. 2010; Vorburger & Gouskov 2011). strain of Wolbachia and two strains of Rickettsia, Facultative endosymbionts exhibit a more none of which was found in S. discoideus (J.A. recently developed association, are sometimes White, University of Kentucky, unpublished data). horizontally transferred between hosts and live Although the function of these endosymbionts in the haemolymph of the insects (not specialised in relation to the biological control of these two bacteriocytes). Common examples include Sitona species has yet to be examined in detail, the Wolbachia, Rickettsia, Cardinium, Spiroplasma, role of endosymbionts in insect biology and the Hamiltonella, Regiella and Serratia, and can be implications for biological control in general are found in the host as single species (e.g. Lachowska reviewed below. et al. 2010) or as multiple species infections (e.g. Toju & Fukatsu 2011; Ishii et al. 2013). What are endosymbionts? An endosymbiont can be described as any Role of facultative endosymbionts in defence organism that lives within the body or cells of Facultative endosymbionts have been documented another organism, i.e. forming an endosymbiosis. to significantly impact on the success of biological

© 2014 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.html Biological control 206 control (e.g. Hedges et al. 2008; Oliver et al. (A. pisum) hosts to survive and reproduce. Strains 2010; Xie et al. 2010; Table 1). For example, the of H. defensa have been found to vary in the degree extensively studied aphid endosymbionts have of protection conferred against A. ervi (Degnan & been shown to provide defence against parasitoids Moran 2008). Thus strains that have lost the phage and fungi, improve heat tolerance and increase exhibit decreased protection against the parasitoid fecundity (Oliver et al. 2010). Rickettsiella has (Oliver et al. 2009), while conversely, the presence been shown to have an important role in aphid of the endosymbiont can influence parasitoid colouration (Tsuchida et al. 2010), which in turn behaviour. Oliver et al. (2012) showed that A. ervi potentially influences attractiveness to natural discriminates between symbiont-infected and enemies (Tsuchida et al. 2014). Hamiltonella uninfected aphids and changes its oviposition defensa and Serratia symbiotica improve defensive behaviour accordingly, depositing a single egg properties in the pea aphid, Acyrthosiphon pisum in uninfected aphids, but two or more eggs in (Oliver et al. 2003), as does Regiella insecticola H. defensa-infected aphids. The suggestion that in the peach-potato aphid, Myzus persicae oviposition choices may be largely determined (Vorburger et al. 2010). It is the toxins encoded by infection status led to the comment that that by a bacteriophage of H. defensa (APSE) that an evolutionary arms race between A. pisum and kill larvae of the solitary endoparasitoid wasp the parasitoid may be mediated by a bacterial Aphidius ervi, thereby allowing the pea aphid symbiosis (Oliver et al. 2012).

Table 1 Endosymbiont functions and taxonomic distributions (not exhaustive). Bacterial taxa Host taxa Described functions References Obligate endosymbionts Numerous taxa Plant sap feeders Host nutrition Baumann 2005, Moran et blood feeders al. 2008 Facultative endosymbionts Wolbachia >30% of arthropod Reproductive manipulations Hsiao 1996, Duron et al. species Host defense, Host fitness 2008, Hedges et al. 2008, Zug & Hammerstein 2012 Cardinium ~4% of arthropod Reproductive manipulations Zchori-Fein & Perlman species, including 2004, Duron et al. 2008 mites, spiders, and parasitic wasps Arsenophonus ~4% of arthropod Reproductive manipulations Duron et al. 2008, species, including Host nutrition Novakova et al. 2009 ticks, whiteflies, triatomine bugs Spiroplasma ~1% of arthropod Reproductive manipulations Duron et al. 2008, species, including Host defense Xie et al. 2010 weevils Rickettsia <1% of arthropod Reproductive manipulations Perlman et al. 2006, species, including Host fitness Duron et al. 2008, ticks and beetles Himler et al. 2011 Hamiltonella Aphids, whiteflies Host defense Oliver et al. 2010 Regiella Aphids Host defense Oliver et al. 2010 Serratia Aphids Host defense Oliver et al. 2010 Protection from environmental stress Rickettsiella Pea aphid Host colouration Tsuchida et al. 2010

The well studied insect endosymbiont mediated transmission of endosymbionts was Wolbachia has been shown to have a range of demonstrated whereby Rickettsia was transferred impacts on its hosts, including cytoplasmic from the whitefly (Bemisia tabaci) to a plant, reproductive incompatibility, thelytoky or moved inside the phloem, and then acquired by parthenogenesis, feminisation of males, male other whiteflies (Caspi-Fluger et al. 2012). The (son) - killing, increase in mating success of consequence for biological control programmes infected males via sperm completion and are that potential failures may occur through complete dependence on bacteria for egg a previously permissive host acquiring production (see Lachowska et al. 2010). Its role ‘resistance’ through the acquisition of defensive in parasitoid defence was first documented in endosymbionts (e.g. Zindel et al. 2011). a study of the western strain of alfalfa weevil, Hypera postica (Gyllenhal) where the presence of Multiple endosymbiont infections Wolbachia caused high mortality of the braconid Hosts are often infected with multiple wasp larvae, M. aethiopoides (Hsiao 1996). endosymbiont species, making it difficult to Wolbachia was absent in Eastern and Egyptian unravel which strain(s) are playing key roles strains of H. postica, which are permissive hosts in the host’s ecology. For example, a survey of for the parasitoid (Hsiao 1996). The presence endosymbionts in natural populations of the of Wolbachia in the Western strain also blocks chestnut weevil Curculio sikkimensis in Japan cross breeding with individuals from the Eastern identified six distinct endosymbiont lineages, and Egyptian strains through cytoplasmic at different infection frequencies and with incompatibility (Hsiao 1996). different geographical distribution patterns (Toju & Fukatsu 2011). Multiple endosymbiont Transfer between species infections were very common, with Endosymbionts can have significant phen- endosymbiont infection frequencies significantly ological and ecological consequence on their correlated with climatic and ecological factors hosts (e.g. Oliver et al. 2010; White 2011), (Toju & Fukatsu 2011). Studies on the virulence ones that can have potential use in biological of leafhoppers for rice plants are a good example control of arthropod pests (Zindel et al. 2011). of the complexity of endosymbiont interactions The survival and persistence of facultative and host response. The Macrosteles leafhoppers, endosymbionts in insects primarily depends on M. striifrons and M. sexnotatus, known vectors vertical maternal transmission, but horizontal of phytopathogenic phytoplasmas, were found transmission also can occur. The transfer of to support two obligate endosymbionts and five facultative endosymbionts between individuals facultative endosymbionts (Ishii et al. 2013).The of the same aphid species and across aphid authors proposed that interactions between the species has been demonstrated in numerous obligate and facultative endosymbionts, and the laboratory studies (see Oliver et al. 2010), as phytopathogenic phytoplasmas within the same well as between parasitoids (Watanabe et al. host insects, may affect vector competence of 2013). Transmission from host to parasitoid the leafhoppers. Endosymbionts may also allow has also been demonstrated in the laboratory leafhoppers to rapidly develop virulence to (Chiel et al. 2009; Gehrer & Vorburger 2012). For resistant rice varieties (Ferrater et al. 2013). instance, the parasitoids Aphidius colemani and In terms of the dynamics of endosymbiont- Lysiphlebus fabarum have been shown to transfer arthropod associations, symbiont-driven H. defensa and R. insecticola by sequentially evolution can be quite rapid (White 2011). stabbing infected and uninfected individuals of Examples include an evolutionary shift in the their host, black bean aphid, Aphis fabae (Gehrer consequence of Wolbachia infection on fruit fly & Vorburger 2012). However, no parasitoids that (Drosophila simulans). Initially, this symbiont emerged from symbiont-infected aphids were reduced fly fecundity, but after only 20 years of infected with symbionts (Gehrer & Vorburger coadaptation with the host, infected flies exhibited 2012). In another study, the potential of plant- a fecundity advantage over uninfected females

(Weeks et al. 2007). In a similar example, the healthy larvae containing multiple parasitoid eggs spread of Rickettsia in field populations of sweet (P.J. Gerard, AgResearch, unpublished data), potato whitefly Bemisia( tabaci) in south-western suggests that an endosymbiont-induced parasitoid USA occurred within 6 years (Himler et al. 2011). defence response may be taking place, similar This was attributed to substantial fitness benefits to that provided by H. defensa in the pea aphid. to infected hosts including higher survival to Furthermore, Northland S. obsoletus samples adulthood and faster development times. were infected with Wolbachia and higher levels of Rickettsia strain 2 compared to weevils sampled in Endosymbionts as a pest management tool Canterbury (J.A. White, University of Kentucky, The use of endosymbionts as a novel tool in the unpublished data). Thus, it is possible that one or biological control of insect pests has also been both of these endosymbionts may play a protective the subject of recent research. In particular, a role against the parasitoid. The elimination of European-funded research programme (EU endosymbionts through heat treatment or selected COST Action FA0701 2012) has investigated the antibiotics (e.g. Hagimori et al. 2006; White et al. potential to target either the symbionts within a 2009), followed by the investigation of subsequent target pest or introduce an endosymbiont into development responses in the target insect a novel host to disrupt the traits contributing populations would be the standard methodologies to the insects pest status (e.g. capacity to vector to apply to such a study. diseases, natural enemy resistance (Douglas Additional studies will include exploring the 2007; Hoffmann et al. 2011)). However, while effect of environmental stresses, such as temperature having great potential in controlling major pests, and age of the host, on endosymbiont titre within developing a robust methodology that permits the insect. This will require designing suitable transfer of an endosymbiont into a novel host, and primers to detect the target endosymbionts and to the rapid multiplication in the target population determine detection thresholds. Recent research while maintaining infection and persistence has shown that certain Wolbachia-infections across generations, are challenges to achieving a are capable of escaping standard PCR screening viable endosymbiont pest management tool. methods by ‘hiding’ as low-titre infections below the detection threshold of standard PCR detection New Zealand endosymbiont research techniques. The use of highly sensitive PCR In contrast to the rapidly evolving research taking technology can enhance the symbiont detection place in the USA and Europe, local research limits substantially (Schneider et al. 2013). In capability on endosymbiont-insect interactions addition, quantitative PCR in conjunction with is only just emerging. This is surprising given real-time detection of the 16S rRNA gene can be the importance of biological control to New used to investigate titres of endosymbiont within Zealand’s broad-acre agriculture and primary the target organism (e.g. Watanabe et al. 2013). plant-based industries. Preliminary investigations It is intended to make use of fluorescencein situ have recently confirmed that both obligate and hybridization (FISH) to identify the location facultative endosymbionts occur in exotic and of endosymbiont bacteria in the host insect as native weevils found in New Zealand pasture demonstrated in Dhami et al. (2012). (J.A. White, University of Kentucky, unpublished A key step in characterising the relationship data). However, research to determine their of identified endosymbionts is through the possible implications in relation to weevil application of phylogenetics. Molecular phenology and impacts on biological control methods that allow the study of both culturable agents is the target for future work. The initial and non-culturable bacteria combined with poor establishment, and the low parasitism the development of cheap high-throughput levels by Irish M. aethiopoides of S. obsoletus in sequencing technologies, known as next-gen Northland, New Zealand (Gerard et al. 2010), sequencing (NGS), have greatly enhanced plus high rates of parasitoid mortality in weevils microbial community diversity research containing only a single parasitoid compared to (Fakruddin & Mannan 2013). Such methods

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