J Chem Ecol (2013) 39:989–1002 DOI 10.1007/s10886-013-0318-8

REVIEW ARTICLE

The Bark Beetle Holobiont: Why Microbes Matter

Diana L. Six

Received: 13 May 2013 /Revised: 19 June 2013 /Accepted: 27 June 2013 /Published online: 12 July 2013 # Springer Science+Business Media New York 2013

Abstract All higher organisms are involved in symbioses Introduction with microbes. The importance of these partnerships has led to the concept of the holobiont, defined as the animal or plant In their recent paper on symbiosis, Gilbert et al. (2012) state with all its associated microbes. Indeed, the interactions “for animals, as well as plants, there have never been in- between insects and symbionts form much of the basis for dividuals.” Undeniably, for higher organisms, symbiosis is a the success and diversity of this group of arthropods. Insects hard and fast rule. Every species is intricately involved with a rely on microbes to perform basic life functions and to myriad of associates—some obligate, some facultative—that exploit resources and habitats. By “partnering” with mi- profoundly influence their evolution, physiology, and life crobes, insects access new genomic variation instantaneous- history. The study of symbiosis has undergone a revolution ly allowing the exploitation of new adaptive zones, influenc- in recent years. Once considered the study of curiosities, it ing not only outcomes in ecological time, but the degree of has taken front center stage as the major discipline describing innovation and change that occurs over evolutionary time. In the basis of life on Earth. Groundbreaking work in insect this review, I present a brief overview of the importance of systems has been a major part of this revolution. insect-microbe holobionts to illustrate how critical an under- In this review, I make the argument that to study an insect is standing of the holobiont is to understanding the insect host to study the holobiont. Defined as the animal or plant with all its and it interactions with its environment. I then review what is associated microbes (Margulis and Fester 1991), the holobiont known about the most influential insect holobionts in many is a concept that has been with us for some time, but one that has forest ecosystems—bark beetles and their microbes—and only recently gained support as a primary unit of study in how new approaches and technologies are allowing us to ecological and evolutionary systems (Gilbert et al. 2012; illuminate how these symbioses function. Finally, I discuss Zilber-Rosenberg and Rosenberg 2008). I begin with a brief why it will be critical to study bark beetles as a holobiont to overview of the ubiquity, complexity, and remarkably understand the ramifications and extent of anthropogenic intertwined nature of insect-microbial symbioses. I then turn to change in forest ecosystems. describing the state of knowledge of the bark beetle holobiont. Bark beetles are among the most ecologically and economically Keywords Symbiosis . Mutualism . Endosymbiont . important insects in forests worldwide. Their enormous influ- Ectosymbiont . Fungi . Yeast . Bacteria . Dendroctonus . ence in these ecosystems is made possible only through micro- Ips . Ophiostoma . Grosmannia . Ogataea . Rhanella bial alliances that support their exploitation of trees. Thus, symbiosis in bark beetle systems is not only interesting to study, but critically important to understanding the ecology, diversity, and functional roles of these insects. Finally, I discuss why a holobiont approach is not a choice, but a requirement, for D. L. Six (*) understanding how anthropogenic change, particularly climate Department of Ecosystem and Conservation Sciences, change and the movement of bark beetle holobionts around the The University of Montana, Missoula, MT 59812, USA globe, will affect these important symbioses, and subsequently, e-mail: [email protected] the ecosystems within which they exist. 990 J Chem Ecol (2013) 39:989–1002

A Brief Introduction to Symbioses Among Insects The ramifications of such highly interwoven systems are and Microbes huge. Strong interdependence and vertical transmission has led to co-cladogenesis in several hemipteran-bacterial systems Symbioses with microbes form much of the basis for the (Gruwell et al. 2007; Jousselin et al. 2009). A strong reliance amazing success and diversity of insects. Insects rely on on the host also has led to extreme genome reduction in the microbes to perform basic life functions and to exploit re- bacteria (McCutcheon and Moran 2012). The genomes of sources and habitats. By “partnering” with microbes, insects insect-associated endosymbiotic bacteria are five to ten times can access new genomic variation instantaneously allowing smaller than those of free-living bacteria (McCutcheon and them to exploit new adaptive zones more easily and rapidly Moran 2012). The mechanisms behind reduction are not well (Janson et al. 2008). Although hosts and symbionts possess understood, but likely they involve a combination of small separate genomes, once they associate, the holobiont be- effective population sizes, deletional bias for pseudogenes, comes the unit upon which selection acts (Gilbert et al. losses of large stretches of genes whose functions are support- 2012; Zilber-Rosenberg and Rosenberg 2008). The extent ed by the host, and overall relaxed selection in a simplified to which the association is obligate or facultative, and the environment. Loses of genes encoding for traits supplied by degree of partner fidelity or flexibility, influences not only the partner has been termed compensated trait loss and occurs outcomes in ecological time, but the degree of innovation in a number of insect-bacterial systems (Ellers et al. 2012). and change that occurs over evolutionary time. Genome reduction in symbionts can be extreme. The smallest known genome is that of Tremblaya—one of the mealybug symbionts (McCutcheon and Moran 2012). At An exemplar: Sap-sucking Hemiptera 138,927 bp, it is far smaller than what has been estimated in the past to constitute the minimum size of a genome. The extent A perfect illustration of where the pairing of insects and of genome streamlining in Tremblaya and some other endo- microbes has allowed the exploitation of an immense but symbiotic bacteria raises the question of whether these symbi- otherwise refractory environmental niche are the intricate onts can still be considered distinct organisms or should now be mutualisms that occur between sap-sucking Hemiptera and considered organelles (McCutcheon and Moran 2012). their primary and secondary endosymbionts. The primary Hemiptera also possess secondary symbionts. These bac- symbionts are obligate and transmitted vertically from mother teria are facultative, do not live in specific host-derived to offspring and provision the host with essential amino acids structures, can be transmitted horizontally as well as verti- (Douglas 1998; Moran et al. 2008). In turn, the symbionts live cally, and occur only in a portion of the host population protected within specialized structures in the host’sbodyand (Oliver et al. 2010). While the insects do not require these receive nutrients from the host (Moran et al. 2008). There is symbionts, the symbionts replicate only within the host. much more to many of these partnerships, however, than a Some confer protection against parasitoid wasps and simple exchange of benefits. In some, such as aphids, the entomopathogens. For example, the pea aphid has little exchange is relatively straight forward. The insect partners ability to encapsulate parasitoids and relies on a facultative with Buchnera, a bacterium that possesses the complete set of symbiont, Hamiltonella defensa, for protection (Oliver et al. pathways needed to provide amino acids the host is not able to 2005). However, there is a twist. The symbiotic bacterium acquire from sap (Shigenobu et al. 2000). Others are more alone cannot do this. It must be infected with the bacterio- complex. For example, the sharpshooter, Homalodisca phage, APSE which has two biotypes, APSE-2 and APSE-3 coagulata, possess multiple primary symbionts that divide (Oliver et al. 2009). Hamiltonella defensa with APSE 2 up the labor—in this case, the bacteria Sulcia muelleri and confers little resistance to parasitoids, while the symbiont Baumannia cicadellinicola are complementary, providing dif- containing APSE 3 confers near complete resistance. ferent amino acids, but together deliver the complete set (Wu Interestingly, when aphids are infected with H. defensa lack- et al. 2006). The story gets more complicated in the mealybug, ing phage, titers of H. defensa reach very high levels and host Planococcus citri (McCutcheon and von Dohlen 2011). These fitness drops dramatically (Weldon et al. 2013). This sug- insects have two bacterial symbionts, but instead of the bac- gests the phage plays a role in regulating symbiont abun- teria living individually within the host, one, Moranella dance and consequently in alleviating symbiont competition endobia, lives within the other, Tremblaya princeps. These with the host for nutrients. While either biotype of phage can bacteria also divide the workload, but instead of each having regulate the symbiont, only APSE-3 provides protection complete pathways for different amino acids, they possess against parasitism. In the field, aphids infected with APSE- only partial pathways indicating an amazing degree of coor- 3 rapidly spread to near fixation under parasitism pressure. In dination of intermediates that must be shuttled between the contrast, aphids lacking H. defensa proliferate in the absence symbionts as well as to and from the insect host (McCutcheon of parasitoids (Oliver et al. 2008). In this system, there and von Dohlen 2011). appears to be a cost to harboring the symbiont that is offset J Chem Ecol (2013) 39:989–1002 991 by the benefit of being protected from parasitism. Similarly, termites diverged from cockroaches, all except one lineage for the red gum lerp psyllid, the prevalence of a secondary lost Blattabacterium and acquired a new set of complementa- symbiont also is related to parasitism pressure (Hansen et al. ry gut symbionts more appropriate for the exploitation of 2007). In each case, the ability to have flexible associations wood (Sabree et al. 2012). Blattabacterium is present only with facultative symbionts appears to be a considerable in the basal termite, Mastotermes darwiniensis (Bandi et al. advantage to the host. 1995), where it has a highly degraded genome and no longer Some secondary symbionts of hemipterans may confer mo- possesses the genes needed to produce the amino acids and re than one type of benefit. The secondary symbiont, Rickettsia vitamins required by cockroaches (Sabree et al. 2012). Within insecticola, protects pea aphids against the entomopathogenic the ‘higher’ termites, the Termitidae, there is evidence of sweep- fungus, Pandora neoaphidis (Ferrari et al. 2012), while also ing losses of genes associated with their wood-degrading gut enhancing the performance of some genotypes of the aphid on symbiont communities as well as of subsequent gene invasion some plant species (Leonardo and Muiru 2003). Some second- events (Zhang and Leadbetter 2012) indicating that symbiont ary symbionts also confer protection from heat. In the pea communities can be dynamic and adaptive over time. aphid, heat decreases the number of bacteriocytes and numbers Honeydew-feeding ants, whose diets are high in sugar but of cells of the primary symbiont, Buchnera aphidicola, leading lacking in nitrogen, are consistently associated with nitrogen- to poor overall condition, lower fecundity, and higher mortality fixing bacteria in the Rhizobiales. However, incongruent ant and (Wernegreen 2012). However, when aphids are infected with symbiont phylogenies indicate that these associations are dy- the secondary symbiont, Serratia symbiotica, they retain sig- namic and that ants have acquired symbionts from this bacterial nificantly more bacteriocytes and primary symbiont cells after group independently several times (Russell et al. 2009). heat treatment suggesting that S. symbiotica safeguards the Some of the most striking nutritional ectosymbioses involve primary symbiont from heat damage (Montllor et al. 2002). insects and fungi. These include the leaf-cutter ants (Attini). While these ants are considered the major herbivores in new Additional Examples of Insect-Microbe Symbioses world tropical ecosystems, they do not feed directly on plant material. Rather, they compost leaves in fungal gardens (Mueller Not just Hemiptera, but all insects likely meet challenges et al. 2005). The cultivated fungi are the sole source of food for inherent in their lifestyles by exploiting the genes of microbes. the larvae of the ants, providing carbohydrates, amino acids, and Partnering with microbes to meet nutritional challenges is a sterols as well as producing the enzymes required to degrade pervasive strategy (Douglas 2013). Few foods are high in plant defensive compounds, carbohydrates and proteins (De Fine nutrients, and even fewer provide a balanced diet. Highly Licht et al. 2013). The gardens contain not only the mutualist mobile insects such as migratory locusts can alter foraging fungus, Leucocoprinus, but also the mycoparasitic fungus behavior to make up for diet imbalances (Raubenheimer and Escovopsis, and several bacterial species. An actinobacterium, Simpson 2010). However, most insects are relatively seden- Pseudonocardia, can be found on the integuments of the ants tary or limited to a single host plant species or genus, or other and was once thought to be a highly co-evolved associate similarly narrow ranges of foods. These insects typically deal important in suppressing Escovopsis, which unchecked, can with nutritional challenges through the exploitation of mi- destroy the mutualist fungus garden leading to the collapse of crobes. For example, tsetse flies feed on blood which is high the colony (Reynolds and Currie 2004). More recently, it has in protein but lacking in B vitamins. The flies rely on an been found there are many Pseudonocardia strains associated endosymbiotic bacterium, Wigglesworthia, to provide thia- with attine ants, and many are similar to free-living strains mine and likely others (Snyder et al. 2010). Similarly, cock- indicating they may be frequently acquired from the environ- roaches rely on the endosymbiont, Blattabacterium, for essen- ment (Kost et al. 2007). The emerging view is that some aid in tial amino acid and vitamin production (Sabree et al. 2009). protecting ants against ant diseases, while other Pseudonocardia As with hemipteran symbionts, the primary symbionts asso- and members of other genera, including Burkholderia and ciated with tsetse flies and cockroaches are obligate, possess Streptomyces, provide defense for the fungal gardens (Barke reduced genomes, and cannot live independent of their host et al. 2010;Haederetal.2009; Mueller 2012; Seipke et al. (Toh et al. 2006). However, even in cases of extreme 2011). The association of the mutualist fungus with the ants is interdependence between host and symbiont, it appears that characterized by a complex pattern of repeated de novo acquisi- such associations are not always locked in stone. While sym- tion of fungi and horizontal transmission of fungal cultivars bionts that undergo extreme changes due to a symbiotic life- between species (Mikheyev et al. 2010). In this ectosymbiois, style are not likely to be able to revert to a free-living state or the host is dependent upon a fungus from this group for survival, to shift hosts, a number of studies indicate insects can some- while the fungal symbionts are capable of a free-living existence times replace their symbionts as they move into new adaptive (Vo et al. 2009). Interestingly, in this symbiosis, it is the host ants zones or undergo environmental perturbations (Sabree et al. that exhibit compensated trait loss, losing genes related to nutri- 2012; Zhang and Leadbetter 2012). For example, when ent acquisition (Suen et al. 2011). It is not known if the fungus 992 J Chem Ecol (2013) 39:989–1002 also exhibits functional trait loss and genome reduction. Given living trees, some infest living trees without killing them, and its ability to live independent of the host, this may be less likely. others attack dead or dying trees. Regardless of which strategy One group of insect symbionts that are severely understudied is used, all bark beetles face the challenge of feeding on woody is the gut microbes. In the past, only culturable species could be tissues low in particular nutrients. They also must contend with characterized, which may have biased surveys toward less fas- toxic plant defensive compounds. These challenges are met tidious, transient environmental microbes, while potentially through partnering with an array of microbial partners, partic- missing many species likely to be important to the insect. ularly fungi and bacteria. Culture-independent molecular methods have improved our The typical bark beetle life cycle consists of male–female ability to survey, not only the diversity and functional groups pairs entering a tree and constructing galleries under the bark. present, but also to characterize how gut communities respond As the female moves along, she lays eggs along the walls of to shifts in environmental conditions, food quality and quantity, the gallery. Once the eggs hatch, the larvae begin to tunnel and age of the host, and challenges by pathogens and environmental feed in the phloem (a few species later move into the outer toxins such as host plant defensive compounds and pesticides bark) (rev. in Six and Wingfield 2011). Larvae of tree-killing (Kikichi et al. 2012;Kochetal.2012; Priya et al. 2012). and secondary beetles construct individual feeding galleries, In some cases, gut communities are fairly specific and while parasitic species (those that feed in living trees) tend to highly resistant to perturbation. For example, in bumble form cavities where they feed as a group, likely as a strategy bees, the gut community exhibits low diversity and remark- that circumvents the resin defenses of the living host (Wood able homeostasis even when food availability is altered and 1982). The beetles also feed on microbes they bring with them the insect is challenged with immune system-priming treat- into the tree. Feeding on these microbes is unavoidable given ments of heat-killed bacteria (Koch et al. 2012). In other their ubiquity once galleries are established. Some, such as cases, the bacterial community appears to be more diverse, or yeasts and bacteria, colonize gallery walls, while the filamen- dynamic, or both. In the European firebug, Pyrrhocoris tous fungi ramify throughout the phloem and sapwood apterus, the fore- and hind-gut of the insect are dominated (Adams and Six 2008). by transient food-derived bacteria that can vary by geograph- ic region, while the mid-gut region is dominated by a stable The Fungal Symbionts microbiota (Sudakaran et al. 2012). The insect feeds on seeds of Malvaceae that are avoided by other insects due to heavy Bark beetles are associated with an array of filamentous concentrations of plant defensive compounds. The mid-gut fungi and yeasts. Some are incidentals or commensals, but bacteria of these bugs likely play a role, not only in others provide nutritional benefits and may aid in detoxifi- degrading complex dietary compounds, and but also in de- cation of tree defensive compounds and pheromone produc- toxification (Sudakaran et al. 2012). tion (Ayres et al. 2000; Bleiker and Six 2007; Brand et al. Many insect-microbe symbioses have been described. I 1977; Davis et al. 2011;SixandPaine1998). Best studied are have only touched upon a few here. My goal is not to review the filamentous fungi, particularly Ascomycetes often collec- all insect-microbe symbioses, but to demonstrate that no tively termed ‘ophiostomatoid fungi.’ These include four sex- insect operates as an individual, but rather that each exploits, ual genera, Ophiostoma, Ceratocystiopsis, Grosmannia,and and is often dependent upon, the expanded genetic repertoire Ceratocystis (Kirisits 2004;Zipfelet al. 2006). Ophiostoma, of its associated microbial community. Recent studies, sup- Grosmannia,andCeratocystiopsis form a monophyletic ported by advances in technology, have begun to reveal group in the Ophiostomatales distinct from Ceratocystis, amazing degrees of innovation and adaptation due to sym- which is in the Microascales (Zipfel et al. 2006). The two biosis, which past studies were only able to hint at. Much major fungal groups differ in their host plant affiliations. work is needed, particularly on systems that have strong Most Ophiostomatales are found with conifers, while most influences in Earth’s ecosystems and that are increasingly Ceratocystis are associated with angiosperms (Harrington being affected by anthropogenic change. One such system is 1993). Bark beetles that colonize angiosperms also commonly the bark beetle holobiont. carry non-ophiostomatoid Ascomycetes, including Geosmithia (Kolarik et al. 2008), whose effects on beetles are for the most part unknown (Kolarik et al. 2008, 2011). Three bark beetle The Bark Beetle Holobiont species are associated with Basidiomycetes in the genus Entomocorticium (Hsiau and Harrington 2003; Paine and The Beetle Host Birch 1983; Whitney et al. 1987). The phloem diet of bark beetles is low in nitrogen and usable Bark beetles, like many insects, exploit a highly specific niche. sterols. Some beetles appear to deal with these deficiencies by For most, the niche is the phloem layer of trees. These beetles consuming large amounts of phloem (phloeophagous), while use a wide range of strategies to access phloem. Some kill others consume less phloem and supplement their diet with J Chem Ecol (2013) 39:989–1002 993 fungi (mycophloeophagous) (Ayres et al. 2000; Bentz and Six Some, such as D. rufipennis and Dendroctonus murrayane 2006; Bleiker and Six 2007). The latter group typically possess (lodgepole pine beetle), consistently carry specific fungal mycangia, specialized integumental structures for carrying fun- partners suggesting these may be nutritional mutualisms gi, and exhibit specific associations with two or three fungal (Six and Bentz 2003; Six et al. 2011a). Others, such as Ips partners that provide nutritional benefits (Six 2012). Non- typographus (European spruce beetle), do not have consis- mycangial beetles may or may not have specific fungal sym- tent partners and are not likely to rely on fungi for nutrition bionts (Kirisits 2004; Six and Bentz 2003; Six et al. 2011a); (Giordano et al. 2013; Kirisits 2004; Six and Wingfield whether any are mycophloeophagous remains to be shown. 2011). Still others may have facultative associations with For at least three mycophloeophagous species, Dendroctonus fungi. For example, Ips pini (pine engraver) commonly ponderosae (mountain pine beetle) D. frontalis (southern pine carries Ophiostoma ips. This beetle has greater brood pro- beetle), and D. brevicomis (western pine beetle), nitrogen sup- duction in the presence the fungus, but may not require it for plementation by specific mutualistic partners is critical for de- survival (Kopper et al. 2004). Dependence on fungi also velopment and survival (Ayres et al. 2000; Bleiker and Six 2007; appears to be related to whether beetles colonize conifers Six and Paine 1998). While the beetles are restricted to living and or angiosperms. Obligate mutualisms with fungi have been feeding only in the phloem and/or the outer bark, the fungi grow found only with bark beetles infesting conifers. Bark beetles in the phloem and the sapwood. All woody tree tissues are low in colonizing angiosperms are not known to rely on fungi for nitrogen; however, the massive volume of sapwood relative to nutrition although many are vectors (Kolarik et al. 2008; the very small amount of phloem present in a tree indicates that Webber 1990). This difference may be an artifact of the lack the sapwood may constitute a substantial reservoir in terms of the of studies investigating mycophagy in angiosperm systems, total nitrogen available. The fungi tap into sapwood nitrogen and or may indicate that angiosperms and conifers are inherently transport it to the phloem and bark where beetle larvae feed, different adaptive zones and that the beetles that exploit them increasing the nitrogen content by up to 40 % (Bleiker and Six do so in very different ways. 2008). Mycophloeophagous beetles that colonize conifers com- Not all bark beetle-associated fungi are equal when it comes monly have two, and sometimes more, specific filamentous to concentrating nitrogen. Cook et al. (2010) found that one fungal associates. The fungi contribute to the nutrition of the associate of D. ponderosae, Grosmannia clavigera,concentrates beetle although the degree of benefit conferred varies. nitrogen better than the other associate, Ophiostoma montium. Typically, one is better than the other(s) supporting greater This difference may account for why beetles developing with G. brood production, size, and survival (Ayres et al. 2000; clavigera are larger and have greater survival than beetles de- Bleiker and Six 2007; Six and Paine 1998). veloping with O. montium (Bleiker and Six 2007; Goodsman The relative prevalence of fungal associates with a beetle et al. 2012; Six and Paine 1998). The poor performance of bark appears to be primarily determined by temperature (Addison beetles in the presence of some commensal ophiostmatoid fungi et al. 2013; Six and Bentz 2007; Hofstetter et al. 2006a). may be due to their lack of ability to concentrate nitrogen. For Each fungus possesses a different optimal temperature for example, D. frontalis larvae that feed in phloem colonized by O. growth as well as different upper and lower limits, although minus often tunnel for long periods without growing, and then considerable overlap can occur at intermediate temperatures die, apparently due to starvation (Barras 1970). (Hofstetter et al. 2006a; Rice et al. 2008; Six and Paine Another nutritional challenge for insects using plants is ac- 1997). Fluctuations in temperature within and among years quiring sterols. Insects require sterols in their diet that are used to results in fluctuations of the relative prevalence of the fungi produce hormones for molting and reproduction (Clayton 1964). over time (Hofstetter et al. 2006a; Six and Bentz 2007). Plants produce sterols but mostly in forms unusable by insects. While overall warmer sites may be dominated by warm- Fungi, however, produce ergosterol, which is usable by most loving associates and cooler sites by cool-loving ones, fluc- insects and is known to be an important nutrient supplied by tuations in temperature, as well as the migration of beetles fungi in other insect-fungus symbioses (Maurer et al. 2005; between sites with different thermal profiles, may reduce the Norris et al. 1969). Ophiostomatoid fungi associated with potential that any one fungus excludes the other(s) over time D. ponderosae and D. rufipennis (spruce beetle) contain very (Addison et al. 2013). Because the fungi differentially affect high concentrations of ergosterol (Bentz and Six 2006) beetle fitness, their relative prevalence in a population is suggesting the fungi may meet the sterol needs of their host expected to influence host beetle dynamics. Beetle popula- beetles. Newly eclosed D. ponderosae adults that do not feed on tions associated mainly with the ‘best’ fungus should have spores do not reproduce (Six and Paine 1998), a common effect higher productivity and survival, and thus be more respon- of a lack of usable sterols in an insect’s diet (Clayton 1964). sive to changes in environmental favorability. For mycangial bark beetle species, the use of fungi as a Filamentous fungi are not the only fungal partners of bark nutritional supplement appears to be obligate. For non- beetles. Yeasts are extremely common and are found with all mycangial species, the role of fungal partners is unclear. developmental stages. They can be found on the outer surface 994 J Chem Ecol (2013) 39:989–1002 of beetles, in their guts and oral secretions, within mycangia, have potentially conflicting effects on host beetle fitness. and on gallery walls (Adams et al. 2008; Bridges et al. 1984; Volatiles associated with yeasts and filamentous fungi are used Davis et al. 2011; Lewinsohn et al. 1994;Luetal.1957; for host location by some natural enemies of bark beetles Rivera et al. 2009; Shifrine and Phaff 1956; Whitney and (Adams and Six 2008;Booneetal.2008). Volatiles released Farris 1970). Those found in guts do not appear to specialize by microbes can elicit responses by the host plant, the host in any particular region (Rivera et al. 2009). Some gut yeasts insect, natural enemies, and co-occurring microbial symbionts, can convert host tree defensive chemicals to beetle phero- alike (Davis et al. 2013; Groenhagen et al. 2013,) and the results mones, however, the insect is not dependent upon them for of several recent studies indicate the need for more study on their this function (Hunt and Borden 1990). roles in mediating host behavior and symbioses. Yeasts provide a variety of benefits in several insect Other yeasts have been found to affect the growth of fila- systems (Ganter 2006; Rohlfs and Kurschner 2010; Vega mentous fungal associates. Adams et al. (2008)testedthe and Dowd 2005). However, the roles yeasts play in bark effects of several yeasts isolated from galleries on growth of beetle systems remain unclear. Most are highly variable in the two symbiotic fungi of D. ponderosae. They found that a their occurrence indicating that they are not required by the Basidiomycete yeast and a Candida sp. greatly enhanced the insect (Rivera et al. 2009). A few species are found with growth of O. montium, while suppressing the growth of G. several bark beetle species and may be bark beetle specialists clavigera. In contrast, the yeast, Pichia scolyti, had no effect on (Rivera et al. 2009). Some appear limited in their distribution the growth of either fungus. If and how such effects observed to beetles colonizing particular conifer genera suggesting in vitro translate to actual effects on fungal partners in vivo and they may be sensitive to host tree defensive chemistry subsequently to effects on host fitness is not known. Because (Rivera et al. 2009). most yeasts are not specific and do not penetrate the phloem but Only one yeast species has been found to be a relatively remain confined to gallery walls, effects are likely to be highly consistent associate of a beetle host. Ogataea pini is present in localized and variable over time. Like many aspects of the mycangia of approximately 60 % of D. brevicomis (western beetle holobiont, the yeasts will require more attention before pine beetle) and also can be isolated from the exoskeleton (Davis any generalizations can be made. et al. 2011;ShifrineandPhaff1956;). While not present in high enough frequencies to indicate it is required, the yeast is prev- The Bacterial Symbionts alent enough that it may exert substantive, although inconsistent, effects either directly or indirectly on the beetle host. Davis et al. The bacterial communities associated with bark beetles are (2011) found that volatiles produced by O. pini in vitro signif- just beginning to be studied in earnest, in part because of icantly enhanced the growth of one of the mutualistic fungi of D. advances in technology, but also due to an increasing recog- brevicomis, while inhibiting the growth of an entomopathogen, nition of the importance of bacterial symbioses in insect Beauveria bassiana. This same yeast also may act to alter the ecology and evolution (Clark et al. 2010; Duron and Hurst chemical environment within the tree during the early stages of 2013; Oliver et al. 2010). Bacteria are ubiquitous with bark beetle development when the concentration of defensive beetles. They can be found on exoskeletons, in oral secre- chemicals is the highest, and potentially, the most damaging to tions and guts, in frass, and on the walls of galleries (Adams both the beetle and its mutualistic filamentous associates. Davis et al. 2008; Cardoza et al. 2006; Morales-Jimenez et al. and Hofstetter (2011) observed that the concentration of defen- 2012). Most work to date has concentrated on describing sive monoterpenes not only affected the growth of O. pini, but the bacteria found in the guts of conifer-colonizing bark was in turn also affected by the presence of the yeast. Most beetles. A commonality that quickly emerges from these monoterpenes inhibited the growth of the yeast; however, its studies is that the communities are strikingly species-poor growth was significantly enhanced in the presence of myrcene relative to those of many other insect groups, particularly and terpinolene (Davis and Hofstetter 2011). The presence of O. other wood-feeding insects (Adams et al. 2010; Delalibera pini also altered the rate of loss of several monoterpenes in et al. 2005, 2007; Morales-Jimenez et al. 2009; Sevim et al. phloem over time in phloem. In the presence of O. pini, Delta- 2012). This low diversity may reflect the dietary require- 3-carene declined at a greater rate than in controls, while con- ments of the insect and/or the toxic defensive chemicals centrations of terpinolene and alpha-pinene increased substan- encountered in conifer phloem. tially (Davis and Hofstetter 2011). While in some systems, Only a few studies have focused on the potential for gut yeasts have been shown to reduce the toxicity of plant defenses bacteria to contribute to bark beetle nutrition. Three species (Starmer and Aberdeen 1990), in the D. brevicomis system, it of bacteria isolated from D. rhizophagus exhibited cellulytic appears that O. pini may actually extend the period of time after activity in vitro, but none was consistently associated with tree colonization that beetle larvae and symbiotic fungi are the host (Morales-Jimenez et al. 2012). In contrast, Rahnella exposed to some toxic compounds while reducing their expo- aquatilis, a bacterium capable of nitrogen fixation, was con- sure to others. This is not the only instance where fungal partners sistently isolated from all stages of the beetle. This bacterium J Chem Ecol (2013) 39:989–1002 995 has been found in the guts of many bark beetles, and may different colonization strategies may be reflected in the gut increase the availability of nitrogen, particularly for species bacterial community. Only one study has investigated this such as D. rhizophagus that do not appear to have fungal possibility. It found bacteria in the gut of D. valens, aparasite, mutualists that play this role. Given that nitrogen fixation are more tolerant of monoterpenes than those in the gut of D. only occurs under anaerobic conditions, future research on ponderosae, a tree-killer (Adams et al. 2011). this bacterium should focus on areas of the gut that can In all cases, bark beetles must somehow degrade or detoxify support this function. tree defensive chemicals to survive. Recent studies indicate this Carbohydrates may not be limiting in bark beetle diets, function may be provided by bacteria. The gut bacterial com- which may explain why cellulose-degrading bacteria are not munity of D. ponderosae is highly enriched with genes in- major components of their gut communities. Delalibera et al. volved in terpene degradation (Adams et al. 2013). The com- (2005) compared cellulose degradation by microbial commu- munity is dominated by four genera, Pseudomonas, Serratia, nities in guts of a wood-boring cerambycid beetle, Saperda Rahnella,andBurkholderia, all of which contain these genes. vestita, and two bark beetles, I. pini and D. frontalis. Cellulytic In addition, Serratia, Rahnella, and Brevundimonas from the bacteria were consistently present in the wood borer gut but gut of D. ponderosae significantly reduce levels of monoter- not in the guts of the bark beetles. This difference in bacterial penes in vitro (Boone et al. 2013). Although some overlap functional groups may reflect a difference in carbohydrate occurs in the ability of the bacteria to degrade various terpenes, availability in the diets of the insects. Bark beetles and wood for the most part, degradation byeachbacteriumiscomple- borers both feed as early larval instars in phloem, which is mentary rather than redundant (Boone et al. 2013). Bacteria in primarily cellulose but also contains relatively high concen- these genera are commonly found in dead wood, indicating that trations of simple sugars. However, wood borers soon move the beetles may exploit a broad array of environmental mi- into the sapwood, which is primarily cellulose. Ips pini com- crobes to meet their complete needs for degradation of host pletes development in the phloem, while D. frontalis moves defensive chemistry. It has also been suggested that a mycangial into the outer bark, a substrate even more nutrient deficient fungus of D. ponderosae, G. clavigera, may play a role in than sapwood (Wood 1982). For the wood borer, cellulytic monoterpene degradation because it can use monoterpenes as bacteria are critical to obtain carbohydrates (Delalibera et al. a carbon source and possesses a number of genes that detoxify 2005). In contrast, I. pini may obtain sufficient carbohydrates defensive chemicals and regulate their levels within fungal cells from sugars in phloem, while D. frontalis may use phloem (DiGuistinietal.2011;Wangetal.2013). However, the fungus sugars until it moves into bark, after which it derives all its does not reduce concentrations of monoterpenes in vitro, and its nutrition from its symbiotic fungi. growth is, as well as that of the other symbiont, O. montium,is Bark beetles must also contend with a toxic environment. strongly inhibited in the presence of these compounds (Boone Conifers are heavily defended by phenolics and oleoresin et al. 2013; Paine and Hanlon 1994;). These results, taken terpenoids (Keeling and Bohlmann 2006) that negatively af- together, indicate that it is most likely the bacteria that fulfill fect both beetles and their symbiotic fungi (Kopper et al. 2004; this critical function for the beetle. Any detoxification capabil- Raffa and Smalley 1995). All bark beetles encounter these ities possessed by the fungus likely function to protect it rather defenses, although the degree and length of time of exposure than the beetle. varies by ecological strategy. It has been assumed that tree- Only a couple of studies have investigated the structure and killers experience the highest levels of chemical defenses composition of bark beetle gut bacterial communities. These when they initially colonize a tree, but that after mass attack indicate that communities may be either variable or conserved. overwhelms the tree, defenses begin to decline in a more or The gut community of D. valens, a parasite, varies both in less linear fashion. However, recent work in lodgepole pine composition and richness by geographic region (Adams indicates that levels of monoterpene defenses increase sub- et al. 2010), while those of D. ponderosae, a tree killer, are stantially in the period after attack and are maintained at high broadly similar across environments (Adams et al. 2013). levels for at least a month (Clark et al. 2012). Thus, beetle More comparative work is needed to understand how specific progeny and fungal associates in their initial stages of devel- or labile gut communities are, and how their structure and opment must contend with an even more toxic environment composition relates to functions required by the beetle host. than did their parents at the time of colonization. Secondary In recent years, there has been considerable interest in the beetles colonizing dead and dying trees experience lower use of bacteria by insects for protection (Kaltenpoth 2009). levels of tree defenses than do tree-killers. However, when This topic has received the most attention in the leaf-cutter secondary beetles colonize trees killed by tree-killing beetles, ant mutualism described previously (Currie et al. 1999;Mueller a common behavior, they may also encounter high levels of 2012). Another remarkable system is the beewolves, where defensive chemicals. Parasitic beetles contend with high wasps use bacteria contained in pockets in their antennae to levels of tree defenses for their entire developmental period. protect their young from harmful soil microbes (Kaltenpoth Differences in the chemical environments of beetles with et al. 2012; Koehler and Kaltenpoth 2013). The first suggestion 996 J Chem Ecol (2013) 39:989–1002 that bark beetles may make use of bacteria for protection was Regardless of whether or not bark beetles make use of made by Cardoza et al. (2006) who observed adults of the spruce bacteria for protection from antagonistic fungi, bacteria, for beetle, D. rufipennis, spreading oral secretions across portions of better or for worse, interact with the mutualistic fungal associ- their galleries. They posited this behavior was used to distribute ates. Adams et al. (2008) studied the effects of two bacteria, one beneficial bacteria for protection from antagonistic fungi, such isolated from walls of galleries of D. ponderosae (Micrococcus as Aspergillus and Trichoderma, who reduce beetle oviposition sp.) and the other a putative endophyte (Bacillus pumilus) rates and survival. Bacteria in the secretions were found to isolated from uninfested pine sapwood, on the growth of the inhibit the antagonistic fungi; however, they also inhibited beetle’s two mycangial fungi in vitro. They found that, overall, Leptographium abietinum (Cardoza et al. 2006), the dominant O. montium grew better in the presence of Micrococcus sp. than fungal symbiont of the beetle (Aukema et al. 2005;Sixand it did by itself, while the converse was true for G. clavigera. Bentz 2003). They speculated that L. abietinum may be a However, B. subtilis inhibited both fungi. Adams et al. (2009) competitor with the beetle, using the beetle as a dispersal agent, tested for effects of volatiles producedbybacteriaisolatedfrom and then competing with it for food once in the tree. A follow up galleries and the host tree defensive compound, alpha-pinene, experiment was conducted to test for effects of L. abietinum on on the growth of fungi associated with D. ponderosae, D. the beetle (Cardoza et al. 2008). Adult D. rufipennis were placed valens, and I. pini. They also found variable effects with some into phloem with and without the fungus to assess weight bacteria enhancing and others inhibiting fungal growth. The change, gallery construction, and egg-laying. No effect on addition of alpha-pinene either amplified, reduced, or reversed weight gain or adult survival was seen, but gallery length and these effects. It should not be surprising that different bacteria thenumberofeggslaidwerereducedinthepresenceofthe colonizing gallery walls affect fungal associates in different fungus, although number of eggs per cm was higher in the ways. Like the yeasts that share this niche, their effects are likely presence of the fungus. The type of relationship the beetle has to be localized and sporadic depending on their prevalence and with this fungus remains unclear. However, in nature, the fungus consistency with the beetle. Because they are confined to gallery is found with 80-100 % of adult beetles (Aukema et al. 2005; walls, indirect effects on the host through effects on the fungal Six and Bentz 2003) and colonizes phloem around nearly all associates may ultimately be weak and highly variable. beetles during development. This indicates that the bacteria are To date, most work on bark beetles and microbes have not highly effective in excluding L. abietinum in vivo, although involved surveys of microbial associates or investigations of it is inhibited in vitro. A closer look is needed to determine if the tree defensive responses to symbiotic fungi. Only a few oral bacteria indeed function in protecting the beetle from this systems have been investigated in depth to determine the fungus or the antagonistic saprophytic fungi. If L. abietinum is outcomes of interactions among hosts and symbionts. Most indeed a competitor with the beetle, the bacteria may provide of these studies have focused on a relatively few North some advantage. However, if it is a mutualist, the outcome of the American tree-killing beetles. To develop a less biased and interaction may be quite the opposite. Studies are needed to more comprehensive understanding of these symbioses, an investigate how L. abietinum influences larval nutrition, devel- increase in the number of studies focusing on interactions opment and survival to better clarify its relationship with the between secondary and parasitic beetles and microbes from beetle. Likewise, studies investigating in vivo effects on other regions of the world are needed. Aspergillis and Trichoderma will help clarify the roles of the oral bacteria in protectionofdevelopingbrood. The next study to propose that bark beetles use bacteria for A Holobiont Approach to Understand the Effects protection was by Scott et al. (2008) who found that mycangia of Anthropogenic Change of D. frontalis can contain an actinobacterium, Streptomyces sp., that in vitro, inhibits the antagonistic fungus, O. minus,more Anthropogenic effects, particularly climate change and the than it inhibits one of the mycangial fungi, Entomocorticium sp. movement of exotics, are altering the roles of many bark A. They suggested that the bacterium provides a mechanism of beetles and their microbes in forest ecosystems. In many selectivity for the mycangium that acquires the beetle’stwo cases, these changes will irrevocably transform the symbioses, mycangial fungi but does not acquire the antagonist. It remains themselves (Addison et al. 2013;Six2009; Six et al. 2011b). unclear, however, whether the bacterium is required for this task. Many effects of climate change on bark beetle systems are Streptomyces are commonly isolated from many locations on already apparent. Several outbreaks of bark beetles of unprec- bark beetles, but to date, no consistent association of these edented size in North America in recent years highlight the bacteria with bark beetles has been found (Hulcr et al. 2011). rapid response these insects can have to changes in tempera- Follow up studies are needed to determine how prevalent ture and host tree susceptibility (Raffa et al. 2008). Current Streptomyces sp. is with D. frontalis, its actual efficacy in vivo, outbreaks differ from those in the past in several important and to explain how the second mycangial fungus of the beetle ways. They are larger and more severe, and for at least one avoids being excluded from the mycangium. species, D. ponderosae, they have involved an expansion of J Chem Ecol (2013) 39:989–1002 997 the beetle’s geographic, elevational, and host tree range (Bentz as leaving the beetle to develop primarily with the less beneficial et al. 2010; Logan et al. 2010; Safranyik et al. 2010), as well as associate, O. montium. Over time, this should result in negative the expansion of the range of its symbionts (Lee et al. 2007). effects on beetle population dynamics and potentially a contrac- Outbreaks of bark beetles in the past were correlated with tion of the geographic range where the beetle can survive. abnormally hot dry periods and ended when there was a return Dendroctonus ponderosae is not the only bark beetle likely to to ‘normal’ cooler wetter conditions (Bentz et al. 2009). be affected in such a manner. Dendroctonus frontalis and D. However, with anthropogenic warming, a return to normal brevicomis are also in symbiosis with multiple mutualistic fungi, conditions is not expected. Rather, warming is expected to each with different temperature tolerances. Likewise, these bee- increase, and in many areas higher temperatures will be accom- tle holobionts are prone to decoupling as warming increases panied by decreasing precipitation. This combination is pre- (Hofstetter et al. 2006a). dicted to enhance the productivity and survival of some beetles Not only mutualistic fungi will be affected by a warming coincident with increasingly more weakened trees across vast climate, but also commensals and antagonists. While decreases areas leading to outbreaks of increasing severity and duration. or increases in the prevalence of some fungi may have no effect While warming is currently enhancing the success of on host beetles, some may have significant impacts. One fungus, some beetle species, over time increasingly warmer condi- O. minus, a strong antagonist of the bark beetle, D. frontalis, tions are expected to begin to have negative effects. These may play a major role influencing the dynamics of this beetle in effects are likely to occur directly through reductions in the future. Ophiostoma minus is not so much an associate of the forest habitat and impacts on beetle development including beetle (although it can be vectored loosely on the exoskeleton) larval desiccation and seasonal asynchronicity, as well as as one of phoretic mites that use the beetle to disperse from tree indirectly through effects on symbionts. to tree (Hofstetter et al. 2006b). The mites benefit from feeding Fungi, like insects, are highly sensitive to temperature. on the fungus; however, when the beetle feeds in portions of the Increasing temperatures are predicted to lead to the margin- tree colonized by this fungus, they die (Barras 1970). Relatively alization or loss of fungal partners restricted to growing warm periods support high mite populations and consequently under cool conditions (Addison et al. 2013). In symbioses high levels of the fungus within D. frontalis-colonized trees where partners are redundant in their benefits to the beetle, (Hofstetter et al. 2006b). The amount of tree colonized by the the beetle host may remain in symbiosis with ‘back up’ fungus has been shown to be inversely related with beetle partners, although ultimately they may become more vulner- population growth; the more O. minus in the phloem, the greater able to complete partner loss or range contractions if the the decline in the beetle population the following year remaining symbiont(s) cannot grow under all conditions (Hofstetter et al. 2006b). Additionally, the greater the degree of experienced by the insect at particular sites (Six and Bentz colonization of O. minus inatree,thelessareathatiscaptured 2007). When fungal partners are complementary, the loss of by the mutualistic fungi. If warming trends support greater levels any one symbiont could be devastating. of mites, and thus greater prevalence of O. minus,thenD. While cool-loving fungi are predicted to be lost over time, frontalis and its symbiotic fungi may be at risk in the future warm-tolerant fungi should be favored at least initially, al- with cascading effects on southern US forests with the dampen- though as their upper limits of temperature tolerance are ing of a natural disturbance agent. reached, they too will be marginalized or lost. Host population Temperature-driven shifts in fungi also may affect beetles dynamics and population viability will either be positively or indirectly through effects on natural enemies. The symbiotic negatively affected depending on whether or not the symbiont fungi release volatiles that are used by some parasitoids to favored by warming is the most beneficial or the least. locate their bark beetle hosts (Adams and Six 2008; Boone Evidence supports a strong role of temperature in determining et al. 2008). Losses or shifts in the prevalence of these the relative prevalence of fungal partners with hosts beetles as associates may ultimately reduce the ability of natural ene- well as their geographic range. Dendroctonus ponderosae is mies to locate hosts and regulate beetle populations. associated with G. clavigera and O. montium across its entire How a changing climate will affect the symbiotic yeasts range, but the cool-tolerant G. clavigera tends to dominate in and bacteria associated with bark beetles is unknown. Given populations in the far north. Likewise, a third cool-loving the strong potential that these microbes, particularly the gut mycangial symbiont, L. longiclavatum is found in the northern bacteria, may be important associates affecting beetle fitness, part of the beetle’s range, but only rarely further south (Lee et al. this is clearly an area in need of investigation. 2006). At particularly warm sites, the prevalence of G. clavigera The establishment of exotic bark beetles and their symbi- drops to nearly zero, while O. montium dominates almost 100 % onts also alters bark beetle-microbe symbioses and their of the population of beetles (Six and Bentz 2007). Models effects on forest ecosystems. Scolytinae are the single most predict that as climate warms, G. clavigera will be lost from common group of insects intercepted at ports-of-entry, and many populations over time (Addison et al. 2013). The loss of many exotic species are establishedinforestecosystemsaround this fungus will result in a loss of symbiont redundancy, as well the world (Haack 2006). Introduced bark beetles often exploit 998 J Chem Ecol (2013) 39:989–1002 the same niches as native beetles, potentially leading to compet- same ways. This should not be surprising given the great itive exclusion of native beetles and their fungi and a reduction diversity and ecological amplitude in this large subfamily of in native biodiversity (Six et al. 2011b). When native and exotic weevils. We have a long way to go in understanding the bark beetles coexist within the same substrate, there is also a potential beetle holobiont. Work conducted in other symbioses such as for the swapping of microbial associates. If novel beetle vectors the sap-sucking Hemiptera and their endosymbionts and the have different host tree ranges than the original host, this will act other extosymbiotic fungus-farming insects can go a long way to increase the number of tree species exposed to fungal and in informing our approach. Most of all, we need to move other microbial symbionts carried by exotic and native bark forward with an open mind. For bark beetles, most past beetles, alike. Some bark beetle associates are pathogens, and research focused narrowly on the filamentous fungal associ- their movement into naive tree hosts and into new forests is a ates of tree-killing species, virtually ignoring associations serious cause for concern (Wingfield et al. 2010). among other bark beetles and fungi, and with other microbes Little work has been conducted on changes in fungal sym- including yeasts and bacteria. A near complete fidelity to the biont communities of beetles introduced into new environ- paradigm that beetles use pathogenic fungi to overwhelm tree ments. Perhaps the most complete investigation to date has defenses locked research on these systems into a narrow path focused on D. valens (red turpentine beetle). The beetle was for decades (Six and Wingfield 2011). While studies on tree introduced from North America into China in the mid-1980s reactions to fungal inoculations (not reviewed here) have and has since caused massive mortality of Chinese pines (Sun taught us a great deal, the recognition that the fungi play a et al. 2013). This tree-killing behavior is much different from multitude of roles, combined with substantial new information what occurs in the native range where the beetle is a relatively on yeast and bacterial symbionts, is moving us firmly into a benign parasite (Sun et al. 2013;Wood1982). In its native new period of exciting dynamic research. range, the beetle is mainly associated with L. terebrantis, O. A true holobiont approach will also mean moving beyond ips, and L. procerum.However,L. terebrantis has never been the study of reciprocal interactions between beetles and mi- isolated from beetles in China, and isolates of L. procerum from crobes to apply our knowledge of these symbioses to tackle beetles in China exhibit much higher levels of virulence to trees broader ecological problems. For example, currently all than do isolates from North America (Lu et al. 2010). The models that predict range expansions and contractions of bark Chinese strains of L. procerum also increase tree production of beetles in response to a changing climate have been based 3-carene, the most attractive tree host volatile for the beetle, solely on the environmental tolerances of the beetle hosts. which may support greater levels of beetle attack (Lu et al. However, we know that the symbionts often differ from their 2010). The overall community of fungi found with the beetle in hosts in how they respond to environmental factors such as China is dramatically different from that in North America, temperature and moisture. Models that incorporate symbiont indicating the beetle has acquired new associates in its new responses, therefore, will be critical to develop more accurate range, many of whom are likely to have been originally asso- predictions of where host beetles will and will not survive. In ciated with native Chinese Scolytinae (Sun et al. 2013). Such Canada, researchers are already using a combination of geno- shifts in fungal community compositions in new environments mic, genetic, and ecological approaches incorporating the may ultimately alter both native and exotic beetle behavior and fungal symbionts of D. ponderosae to better understand how dynamics with highly unpredictable effects on forests. this beetle-fungus symbiosis will fare in its eastward expan- sion in boreal forests (Diguistini et al. 2011;Riceetal.2007, 2008;RiceandLangor2009; Roe et al. 2011a, b;Tsuietal. Conclusions and Future Directions 2011). Nearly every aspect of our understanding of bark beetle ecology and evolution can be enhanced by including micro- The importance of symbiosis is being increasingly recog- bial symbionts, and for the first time, with powerful new nized as demonstrated by the massive increase in the litera- technologies at our fingertips, it is possible. ture on this topic in just the last five years. This upsurge in interest promises to move our understanding of insect- microbial symbioses forward by massive leaps and bounds. However, as in any rapidly expanding field, it will be impor- tant to retain caution in interpreting findings. Not every References microbe found with an insect is a symbiont, and not every symbiont has a substantive effect on the host. Determining Adams AS, Six DL (2008) Detection of host habitat by parasitoids whether effects observed in vitro translate to actual effects in using cues associated with mycangial fungi of the mountain pine beetle, Dendroctonus ponderosae. Can Entomol 140:124–127 the real world will remain a difficult, but important task. Adams AS, Six DL, Adams S, Holben W (2008) In vitro interactions All bark beetles use microbes to exploit trees; however, not among yeasts, bacteria and the fungal symbionts of the mountain all of them use the same microbes or the same microbes in the pine beetle, Dendroctonus ponderosae. Microb Ecol 56:460–466 J Chem Ecol (2013) 39:989–1002 999

Adams AS, Currie CR, Cardoza Y,Klepzig K, Raffa KF (2009) Effects of frontalis (Coleoptera: Scolytidae) aggregation pheromone by yeast symbiotic bacteria and tree chemistry on the growth and reproduc- metabolites in laboratory bioassays. J Chem Ecol 3:657–666 tion of bark beetle fungal symbionts. Can J For Res 39:1133–1147 Bridges JR, Marler JE, McSparrin BH (1984) A quantitative study of Adams AS, Adams SM, Currie CR, Gillette NE, Raffa KF (2010) the yeast and bacteria associated with laboratory-reared Geographic variation in bacteria commonly associated with the Dendroctonus frontalis Zimm. (Coleopt., Scolytidae). Z Angew red turpentine beetle (Coleoptera: Curculionidae). Environ Entomol 97:261–267 Entomol 39:406–414 Cardoza YJ, Klepzig KD, Raffa KF (2006) Bacteria in oral secretions of Adams AS, Boone CK, Buhlmann J, Raffa KF (2011) Response of bark an endophytic insect inhibit antagonistic fungi. Ecol Entomol beetle associated-bacteria to host monoterpenes and their relation- 31:636–645 ship to insect life histories. Environ Entomol 37:808–817 Cardoza YJ, Moser JC, Klepzig KD, Raffa KF (2008) Multi-partite Adams AS, Aylward FO, Adams SM, Erbilgin N, Aukema BH, Currie symbioses among fungi, mites, nematodes, and the spruce beetle, CR, Suen G, Raffa KF (2013) Mountain pine beetles colonizing Dendroctonus rufipennis. Environ Entomol 37:956–963 historical and naïve host trees are associated with a bacterial Clark EL, Karley AJ, Hubbard SF (2010) Insect endosymbionts: ma- community highly enriched in genes contributing to terpene me- nipulators of insect herbivore trophic interactions. Protoplasma tabolism. Appl Environ Microbiol 79:3468–3475 244:25–51 Addison A, Powell JA, Six DL, Moore M, Bentz BJ (2013) The role of Clark EL, Huber DPW, Carroll AL (2012) The legacy of attack: impli- temperature variability in stabilizing the mountain pine beetle- cations of high phloem resin monoterpene levels in lodgepole pines fungus mutualism. J Theor Biol. doi:10.1016/j.jtbi.2013.06.012 following mass attack by mountain pine beetle, Dendroctonus Aukema BH, Werner RA, Haberkern KE, Illman BL, Clayton MK, ponderosae Hopkins. Environ Entomol 41:392–398 Raffa KF (2005) Quantifying sources of variation and frequency Clayton RB (1964) The utilization of sterols by insects. J Lipid Res 5:3– of fungi associated with spruce beetles: implications for hypothe- 19 sis testing and sampling methodology in bark beetle-symbiont Cook SS, Shirley BM, Zambino P (2010) Nitrogen concentration in relationships. For Ecol Manag 217:187–202 mountain pine beetle larvae reflects nitrogen status of tree host and Ayres MP, Wilkens RT, Ruel JJ (2000) Nitrogen budgets of phloem- two fungal associates. Environ Entomol 39:821–826 feeding bark beetles with and without symbiotic fungi. Ecology Currie CR, Scott TA, Summerbell RC, Malloch D (1999) Fungus- 81:2198–2210 growing ants use antibiotic-producing bacteria to control garden Bandi C, Sironi M, Damiana G, Magrassi L, Nalepa CA, Laudani U, parasites. Nature 398:701–704 Sacchi L (1995) The establishment of intracellular symbiosis in an Davis TS, Hofstetter RW (2011) Reciprocal interactions between the ancestor of cockroaches and termites. Proc Biol Sci 259:293–299 bark beetle-associated yeast Orgataea pini and host plant chemis- Barke J, Seipke RF, Grueschow S, Heavens D, Drou N, Bibb MJ, Goss try. Mycologia 103:1201–1207 RJM, Yu DW, Hutchings MI (2010) A mixed community of Davis TS, Hofstetter RW, Foster JT, Foote NE, Keim P (2011) In- Actinomycetes produce multiple antibiotics for the fungus farming teractions between the yeast Ogataea pini and filamentous fungi ant Acromyrmex octospinosus. BMC Biol 8:109 associated with the western pine beetle. Microb Ecol 61:626–634 Barras SJ (1970) Antagonism between Dendroctonus frontalis and the Davis TS, Crippen TL, Hofstetter RW, Tomberlin JK (2013) Microbial fungus Ceratocystis minor. Ann Entomol Soc Am 63:1187–1190 volatile emissions as insect semiochemicals. J Chem Ecol. doi:10. Bentz BJ, Six DL (2006) Ergosterol content of four fungal symbionts 10076/s10886-013-0306-z associated with Dendroctonus ponderosae and D. rufipennis De Fine Licht HH, Schiott M, Rogowska-Wrzesinska A, Nygaard S, (Coleoptera: Curculionidae, Scolytinae). Ann Entomol Soc Am Roepstorff P, Boomsma J (2013) Laccase detoxification mediates 99:189–194 the nutritional alliance between leaf-cutting ants and fungus- Bentz B, Logan J, Macmahon L, Allen CD, Ayres M, Berg E, Carroll A, garden symbionts. Proc Natl Acad Sci U S A 110:583–587 Hanson M, Hicke J, Joyce L, Macfarlane W, Munson S, Negron J, Delalibera I, Handelsman J, Raffa KF (2005) Contrasts in cellulytic Paine T, Powell J, Raffa K, Regniere J, Reid M, Romme B, activities of gut microorganisms between the wood borer, Saperda Seybold S, Six D, Tomback D, Vandygriff J, Veblen T, White M, vestita, (Coleptera: Cerambycidae), and the bark beetles, Ips pini Witcosky L, Wood D (2009) Bark beetle outbreaks in Western and Dendroctonus frontalis (Coleoptera: Curculionidae). Environ North America: Causes and consequences. University of Utah Entomol 34:541–547 Press, Salt Lake City, pp. 1–44 Delalibera I, Vasanthakumar A, Burwitz BJ, Schloss PD, Klepzig KD, Bentz BJ, Regniere J, Fettig CJ, Hansen EM, Hayes JL, Hick JA, Handelsman J, Raffa KF (2007) Composition of the bacterial Kelsey RG, Negron JF, Seybold SJ (2010) Climate change and community in the gut of the pine engraver, Ips pini (Say) bark beetles in the western United States and Canada; direct and (Coleoptera) colonizing red pine. Symbiosis 43:97–104 indirect effects. Bioscience 60:602–613 Diguistini S, Wang Y, Liao N, Taylor G, Tanguay P, Feau N, Henrissat Bleiker K, Six DL (2007) Dietary benefits of fungal associates to an B, Chan S, Hesse-Haridas S, Robertson G, Birol I, Holt R, Marra eruptive herbivore: potential implications of multiple associates on M, Hamelin R, Hirst M, Jones S, Bohlmann J, Breuil C (2011) host population dynamics. Environ Entomol 36:1384–1396 Genome and transcriptome analyses of the mountain pine beetle— Bleiker K, Six DL (2008) Competition and coexistence in a multi- fungal symbiont Grosmannia clavigera, a lodgepole pine patho- partner mutualism: Interactions between two fungal symbionts of gen. Proc Natl Acad Sci U S A 108:2504–2509 the mountain pine beetle in beetle-attacked trees. Microb Ecol Douglas AE (1998) Nutritional interactions in insect-microbial symbi- 57:191–202. oses: aphids and their symbiotic bacteria Buchnera. Annu Rev Boone CK, Six DL, Zheng Y, Raffa KF (2008) Parasitoids and dipteran Entomol 43:17–37 predators exploit volatiles from microbial symbionts to locate bark Douglas AE (2013) Microbial brokers of insect-plant interactions revisited. beetles. Environ Entomol 37:150–161 J Chem Ecol. doi:10.10076/s10886-013-0308-x Boone C, Keefover-Ring K, Mapes AC, Adams AS, Bohlmann J, Raffa Duron O, Hurst GDD (2013) Arthropods and inherited bacteria: from KF (2013) Bacteria associated with a tree-killing insect reduce con- counting the symbionts to understanding how symbionts count. centrations of plant defense compounds. J Chem Ecol. doi:10.10076/ BMC Biol 11:45 s10886-013-0313-0 Ellers J, Kiers ET, Currie CR, McDonald BR, Visser B (2012) Ecolog- Brand JM, Schultz J, Barras SJ, Edson LD, Payne TL, Heddon RL ical interactions drive evolutionary loss of traits. Ecol Lett (1977) Bark beetle pheromones: enhancement of Dendroctonus 15:1071–1082 1000 J Chem Ecol (2013) 39:989–1002

Ferrari J, West JA, Via S, Godfray HCJ (2012) Population genetic antennal symbionts in the rare genus Philanthinus (Hymenoptera, structure and secondary symbionts in host-associated populations Crabronidae). Appl Environ Microbiol 78:822–827 of the pea aphid complex. Evolution 66:375–390 Keeling CI, Bohlmann J (2006) Dipterpene resin acids in conifers. Ganter R F (2006) Yeast and Invertebrate associates. In: Rosa CA and Phytochemistry 67:2415–2423 Gabor P (eds) Biodiversity and ecophysiology of yeasts, Springer Kikichi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K (2012) pp. 303–370 Symbiont-mediated insecticide resistance. Proc Natl Acad Sci U Gilbert SF, Sapp J, Tauber AI (2012) A symbiotic view of life: We have S A 109:8618–8622 never been individuals. Q Rev Biol 87:325–341 Kirisits T (2004) Fungal associates of European bark beetles with Giordano L, Garbelotto M, Nicolotti G, Gonthier P (2013) Character- special emphasis on the ophiostomatoid fungi. In: Lieutier F, ization of fungal communities associated with the bark beetle Ips Day KR, Battisti A, Gregoire J-C, Evans HF (eds) Bark and wood typographus varies depending on detection method, location, and boring insects in living trees in Europe, a synthesis. Kluwer beetle population levels. Mycol Prog 12:127–140 Academic Publishers. Dordrecht, The Netherlands, pp 181–236 Goodsman DW, Erbilgin N, Lieffers VT (2012) The impact of phloem Koch H, Cisarovsky G, Schmid-Hempel P (2012) Ecological effects on nutrients on overwintering mountain pine beetle and their fungal gut bacterial communities in wild bumblebee colonies. J Anim symbionts. Environ Entomol 42:478–486 Ecol 81:1202–1210 Groenhagen U, Baumgartner R, Bailly A, Gardiner MA, Eberl L, Shulz Koehler S, Kaltenpoth M (2013) Maternal and environmental effects on S, Weisskopf L (2013) Production of bioactive volatiles by differ- symbiont-mediated antimicrobial defense. J Chem Ecol. doi:10. ent Burkholderia ambifaria strains. J Chem Ecol. doi:10.1007/ 10076/s10886-013-0304-1 s10886-013-0315-y Kolarik M, Kubatova A, Hulcr J, Pazoutova S (2008) Geosmithia fungi Gruwell ME, Morse GE, Normark BB (2007) Phylogenetic congruence are highly diverse and consistent bark beetle associates: evidence of armored scale insects (Hemiptera: Diaspididae) and their pri- from their community structure in temperate Europe. Microb Ecol mary endosymbionts from the phylum Bacteroidetes. Mol 55:65–80 Phylogenet Evol 44:267–280 Kolarik M, Freeland E, Utely C, Tisserat N (2011) Geosmithia morbida Haack RA (2006) Exotic bark- and wood-boring Coleoptera in the sp. nov., a new phytopathogenic species living in symbiosis with United States: recent establishments and interceptions. Can J For the walnut twig beetle (Pityophthorus juglandis)onJuglans in Res 36:269–288 USA. Mycologia 103:325–332 Haeder S, Wirth R, Herz H, Spiteller D (2009) Candicidin-producing Kopper BJ, Klepzig KD, Raffa KF (2004) Components of antagonism Streptomyces support leaf-cutting ants to protect their fungus and mutualism in Ips pini-fungal interactions: relationship to a life garden against the pathogenic fungus Escovopsis. Proc Natl Acad history of colonizing highly stressed and dead trees. Environ Sci U S A 106:4742–4746 Entomol 33:28–34 Hansen AK, Jeong G, Paine TD, Stouthammer R (2007) Frequency of Kost C, Lakatos C, Bottcher I, Arendholz WR, Redenbach M, Wirth R secondary symbiont infection in an invasive psyllid relates to (2007) Non-specific association between filamentous bacteria and parasitism pressure on a geographic scale in California. Appl fungus-growing ants. Naturwissenschaften 94:821–828 Environ Microbiol 73:7531–7535 Lee S, Kim J-J, Breuil C (2006) Diversity of fungi associated Harrington TC (1993) Diseases of Conifers Caused by Species of with the mountain pine beetle. Dendroctonus ponderosae and Ophiostoma and Leptographium. In: Wingfield MJ, Seifert KA, infested lodgepole pines in British Columbia. Fungal Divers Webber JF (eds) Ceratocystis and Ophiostoma: Taxonomy, Ecol- 22:91–105 ogy and Pathogenicity. American Phytopathological Society, St. Lee S, Hamelin RC, Six DL, Breuil C (2007) Genetic diversity and the Paul, pp 161–172 presence of two distinct groups in Ophiostom clavigerum associ- Hofstetter RW, Klepzig KD, Moser JC, Ayres MP (2006a) Seasonal ated with Dendroctonus ponderosae in BC and the northern Rocky dynamics of mites and fungi and their interaction with southern Mountains. Phytopathology 97:1177–1185 pine beetle. Environ Entomol 35:22–30 Leonardo TE, Muiru GT (2003) Facultative symbionts are associated Hofstetter RW, Cronin JJ, Klepzig KD, Moser JC, Ayres MP (2006b) with host plant specialization in pea aphid populations. Proc R Soc Antagonisms, mutualisms and commensalisms affect outbreak B 270:S209–S212 dynamics of the southern pine beetle. Oecologia 147:679–691 Lewinsohn D, Lewinsohn E, Bertagnolli CL, Partridge AD (1994) Blue Hsiau P-TW, Harrington TC (2003) Phylogenetics and adaptations of stain fungi and their transport structures on the Douglas-fir beetle. basidiomycetous fungi fed upon by bark beetles (Coleoptera: Can J For Res 24:2275–2283 Scolytidae). Symbiosis 34:111–131 Logan JA, Macfarlane WW, Wilcox L (2010) Whitebark pine vulner- Hulcr J, Adams AS, Raffa KF, Hofstetter RW, Klepzig KD, Currie CR ability to climat-driven mountain pine beetle disturbance in the (2011) Presence and diversity of Streptomyces in Dendroctonus Greater Yellowstone Ecosystem. Ecol Appl 20:895–902 and sympatric beetle galleries across North America. Mol Ecol Lu KC, Allen DG, Bollen WB (1957) Association of yeasts with the 61:759–768 Douglas fir beetle. For Sci 3:336–342 Hunt DWA, Borden JH (1990) Conversion of verbenols to verbenone Lu M, Wingfield MJ, Gillette NE, Mori SR, Sun J (2010) Complex by yeasts isolated from Dendroctonus ponderosae (Coleoptera: interactions among host pines and fungi vectored by an invasive Scolytidae). J Chem Ecol 16:1385–1397 bark beetle. PLoS One 2:e1302 Janson EM, Stireman JO, Singer MS, Abbot P (2008) Phytophagous Margulis L, Fester R (1991) Symbiosis as a source of evolutionary insect microbe mutualisms and adaptive evolutionary diversifica- innovation: Speciation and morphogenesis. MIT Press, Boston tion. Evolution 62:997–1012 Maurer P, Debieu D, Leroux P, Malosse C, Riba G (2005) Sterols and Jousselin E, Desdevises Y, Coeur D’Acier A (2009) Fine-scale symbiosis in the leaf-cutter ant Acromyrmex octospinosus (Reich) cospeciation between Brachycaudus and Buchnera aphidicola: (Hymenoptera, Formicidae: Attini). Arch Insect Biochem Physiol bacterial genome helps define species and evolutionary relation- 20:13–21 ships in aphids. Proc R Soc B 276:187–196 McCutcheon JP, Moran NA (2012) Extreme genome reduction in Kaltenpoth M (2009) Actinobacteria as mutualists: general healthcare symbiotic bacteria. Nat Rev Microbiol 10:13–26 for insects? Trends Microbiol 17:529–535 McCutcheon JP, von Dohlen CD (2011) An interdependent metabolic Kaltenpoth M, Yildirim E, Gurbuz MF, Herzner G, Strohm E (2012) patchwork in the nested three-way symbiosis of mealy bugs. Curr Refining the roots of the beewolf-Streptomyces symbiosis: Biol 21:1366–1372 J Chem Ecol (2013) 39:989–1002 1001

Mikheyev AS, Mueller UG, Abbot P (2010) Comparative dating of Rice AV, Thorman MN, Langor DW (2008) Mountain pine beetle- attine ants and Lepiotaceous cultivar phylogenies reveals coevo- associated blue-stain fungi are differentially adapted to boreal lutionary synchrony and discord. Am Nat 175:E126–E133 temperatures. For Pathol 38:113–123 Montllor CB, Maxmen A, Purcell AH (2002) Facultative endosymbi- Rivera FN, Gonzalez E, Gomex Z, Lopez N, Hernandez-Rodriguez C, onts benefit pea aphid Acythosiphon pisum under heat stress. Ecol Berkov A, Zuniga G (2009) Gut-associated yeast in bark beetles of Entomol 27:189–195 the genus Dendroctonus Erichson (Coleoptera: Curculionidae: Morales-Jimenez J, Zuniga G, Ramirez-Saad HC, Hernandez- Scolytinae). Biol J Linn Soc 98:325–342 Rodriguez C (2012) Gut-associated bacteria throughout the life Roe AD, James PMA, Rice AV, Cooke JEK, Sperling FA (2011a) cycle of the bark beetle Dendroctonus rhizphagous Thomas and Spatial community structure of mountain pine beetle fungal Bright (Curculionidae: Scolytinae) and their cellulytic activities. symbionts across a latitudinal gradient. Microb Ecol 62:347– Microb Ecol 64:268–278 360 Morales-Jimenez J, Zuniga G, Villa-Tanaca L, Hernadez-Rodriguez C (2009) Roe AD, Rice AV, Coltman DW, Cooke JEK, Sperling FAH (2011b) Bacterial community and nitrogen fixation in the red turpentine beetle Comparative phylogeography, genetic differentiation and contrast- (Coleoptera: Curculionidae: Scolytinae). Microb Ecol 58:879–891 ing reproductive modes in three fungal symbionts of a multipartite Moran NA, McCutcheon JP, Nakabachi A (2008) Genomics and evolu- bark beetle symbiosis. Mol Ecol 20:584–600 tion of heritable bacterial symbionts. Annu Rev Genet 42:165–190 Rohlfs M, Kurschner L (2010) Saprophagous insect larvae, Drosophila Mueller UG (2012) Symbiont recruitment versus ant-symbiont co- melanogaster, profit from increased species richness in beneficial evolution in the attine ant-microbe symbiosis. Curr Opin microbes. J Appl Entomol 134:667–671 Microbiol 15:269–277 Russell JA, Moreau C, Goldman-Huertas BB, Fujiwara M, Lohman DJ, Mueller U, Gerardo N, Aanen D, Six DL, Schultz T (2005) The Pierce NE (2009) Bacterial gut symbionts are tightly linked with evolution of agriculture in insects. Annu Rev Ecol Evol Syst the evolution of herbivory in ants. Proc Natl Acad Sci U S A 36:563–595 106:21236–21241 Norris DM, Baker JM, Chu HM (1969) Symbiontic Interrelationships Sabree ZL, Kambhampati S, Moran NA (2009) Nitrogen recycling and between microbes and ambrosia beetles III. Ergosterol as the nutritional provisioning by Blattabacterium, the cockroach endo- source of sterols to the insect. Ann Entomol Soc Am 62:413–414 symbiont. Proc Natl Acad Sci U S A 106:19521–19526 Oliver KM, Moran NA, Hunter MS (2005) Variation in resistance to Sabree ZL, Huang CY, Arakawa G, Tokuda G, Lo N, Watanabe H, parasitism in aphids is due to symbionts not host genotype. Proc Moran NA (2012) Genome shrinkage and loss of nurtrient- Natl Acad Sci U S A 102:12795–12800 providing potential in the obligate symbiont of the primitive ter- Oliver KM, Campos J, Moran NA, Hunter MS (2008) Population mite Mastotermes darwiniensis. Appl Environ Microbiol 78:204– dynamics of defensive symbionts in aphids. Proc R Soc B 210 275:293–299 Safranyik L, Carroll AL, Regniere J, Langor DW, Riel WG, Shore TL, Oliver KM, Degnan PH, Hunter MS, Moran NA (2009) Bactriophages Peter B, Cooke BJ, Nealis VG, Taylor SW (2010) Potential for encode factors required for protection in a symbiotic mutualism. range expansion of mountain pine beetle into the northern boreal Science 325:992–994 forest of North America. Can Entomol 142:415–442 Oliver KM, Degnan PH, Burke GR, Moran NA (2010) Facultative Scott JJ, Dong-Chan O, Yuceer MC, Klepzig KD, Clardy J, Currie CR symbionts in aphids and the horizontal transfer of ecologically (2008) Bacterial protection of beetle-fungus mutualism. Science important traits. Annu Rev Entomol 55:247–266 322:63 Paine TD, Birch MC (1983) Acquisition and maintenance of mycangial Seipke RF, Barke J, Brearley C, Hill L, Yu DW, Goss RJ, Hutchings MI fungi of Dendroctonus brevicomis LeConte (Coleoptera: (2011) A single Streptomyces symbiont makes multiple antifun- Scolytidae). Environ Entomol 12:1384–1386 gals to support the fungus farming ant Acromyrmex octospinosus. Paine TD, Hanlon CC (1994) Influence of oleoresin constituents from PLoS 6:e22028 Pinus ponderosa and P. jeffreyi on growth of the mycangial fungi Sevim A, Gokce C, Erbas Z, Ozkan F (2012) Bacteria from Ips of Dendroctonus ponderosae and D. jeffreyi. J Chem Ecol sexdentatus (Coleoptera: Curculionidae) and their biocontrol po- 20:2551–2563 tential. J Basic Microbiol 52:695–704 Priya N, Ojha A, Kajla MK, Raj A, Rajagopal R (2012) Host plant Shifrine M, Phaff HJ (1956) The association of yeasts with certain bark induced variation in gut bacteria of Helicoverpa armigera. PLoS beetles. Mycologia 48:41–55 One 7:e30768 Shigenobu S, Watanabe H, Hattori M, Sakaki Y, Ishikawa H (2000) Raffa KF, Smalley EB (1995) Interaction of pre-attack and induced Genome sequence of the endocellular bacterial symbiont of aphids monoterpene concentrations in host conifer defense against bark Buchnera sp. APS. Nature 407:81–86 beetle-fungal complexes. Oecologia 102:285–295 Six DL (2009) Climate change and mutualism. Nat Microbiol Rev Raffa KF, Aukema BH, Bentz BJ, Carroll AL, Hicke JA, Turner MG, 7:686 Romme WH (2008) Cross-scale drivers of natural disturbances Six DL (2012) Ecological and Evolutionary determinants of bark prone to anthropogenic amplification: the dynamics of bark beetle beetle-fungus symbioses. Insects 3:339–366 eruptions. Bioscience 58:501–517 Six DL, Bentz BJ (2003) Fungi associated with the North American Raubenheimer D, Simpson SJ (2010) Nutrient balancing in grasshop- spruce beetle, Dendroctonus rufipennis. Can J For Res 33:1815– pers: behavioral and physiological correlates of dietary breadth. J 1820 Exp Biol 216:1669–1681 Six DL, Bentz BJ (2007) Temperature determines the relative abun- Reynolds HT, Currie CR (2004) Pathogenicity of Escovopsis weberi: dance of symbionts in a multipartite bark beetle-fungus symbiosis. the parasite of the attine ant-microbe symbiosis directly consumes Microb Ecol 54:112–118 the ant-cultivar fungus. Mycologia 96:955–959 Six DL, Paine TD (1997) Ophiostoma clavigerum is the mycangial Rice AV, Langor D (2009) Mountain pine beetle-associated blue-stain fungus of the Jeffrey pine beetle, Dendroctonus jeffreyi (Coleop- fungi in lodgepole pine x jack pine hybrids near Grande Prairie, tera: Scolytidae). Mycologia 89:858–866 Alberta (Canada). For Pathol 39:323–334 Six DL, Paine TD (1998) Effects of mycangial fungi and host tree Rice AV, Thormann MN, Langor DW (2007) Virulence of, and interac- species on progeny survival and emergence of Dendroctonus tions among, mountain pine beetle-associated blue-stain fungi on ponderosae (Coleoptera: Scolytidae). Environ Entomol 27:1393– two pine species and their hybrids in Alberta. Can J Bot 85:316–323 1401 1002 J Chem Ecol (2013) 39:989–1002

Six DL, Wingfield MJ (2011) The role of phytopathogenicity in bark and Blackwell M (eds). Oxford University Press, Oxford, pp. 211– beetle-fungus symbioses: a challenge to the classic paradigm. 243 Annu Rev Entomol 56:255–272 Vo T, Meuller UG, Mikheyev AS (2009) Free-living fungal symbionts Six DL, DE Beer ZW, Duong T, Carroll AL, Wingfield MJ (2011a) (Lepiotaceae) of fungus-growing ants (Attini: Formicidae). Fungal associates of the lodgepole pine beetle, Dendroctonus Mycologia 101:206–210 murrayanae. Antonie Van Leeuwenhoek 100:231–244 Wang Y, Lim L, Diguistini S, Robertson G, Bohlmann J, Breuil C Six DL, Poulsen M, Hansen AK, Wingfield MJ, Roux J, Eggleton P, (2013) A specialized ABC efflux transporter GcABC-G1 Slippers B, Paine TD (2011b) Anthropogenic effects on insect- confers monoterpene resistance to Grosmannia clavigera, a microbial symbioses in forest and savanna ecosystems. Symbiosis bark beetle-associated fungal pathogen of pine. New Phytol 53:101–121 197:886–898 Snyder AK, Deberry JW, Runyen-Janecky L, Rio RVM (2010) Nutrient Webber JF (1990) Relative effectiveness of Scolytus scolytus, S. provisioning facilitates homeostasis between tsetse fly (Diptera: multistriatus, and S. kirschii as vectors of Dutch elm disease. Eur Glossinidae) symbionts. Proc R Soc B 277:2389–2397 J For Pathol 20:184–192 Starmer WT, Aberdeen V (1990) The nutritional importance of pure and Weldon SR, Strand MR, Oliver KM (2013) Phage loss and the break- mixed cultures of yeasts in the development of Drosophila mulleri down of defensive symbiosis in aphids. Proc, R. Soc. B. doi:10. larvae in Opuntia tissues and its relation to host plant shifts. In: 1098/rspd.2012.2103, 280 Ecology and Evolutionary genetics of Drosophila. Monographs in Wernegreen JJ (2012) Mutualism meltdown in insects: bacteria con- Evolutionary Biology, Springer, pp. 145–160 strain thermal adaptation. Curr Opin Microb 15:255–262 Sudakaran S, Hassan S, Kost C, Kaltenpoth M (2012) Geographical and Whitney HS, Farris SH (1970) Maxillary mycangium in mountain pine ecological stability of the symbiotic mid-gut microbiota in Euro- beetle. Science 167:54–55 pean firebugs, Pyrrhocoris apterus (Hemiptera: Pyrrhocoridae). Whitney HS, Bandoni RJ, Oberwinkler F (1987) Entomocorticum Mol Ecol 21:6134–6151 dendroctoni gen. et sp. nov. (Basidomycotina), a possible nutri- Suen G, Telling C, Lewyn L, Holt C, Abouheif E, Bornberg-Bauer E, tional symbiote of the mountain pine beetle in British Columbia. Bouffard P, Caldera E, Cash E, Cavanaugh A, Denas O, Elhaik E, Can J Bot 65:95–102 Fave’ M-J, Gadau J, Gibson J, Graur D, Grubbs K, Hagen D, Wingfield MJ, Slippers B, Wingfield BD (2010) Novel associations Harkins T, Helmkampf M, Hu H, Johnson B, Kim J, Marsh S, between pathogens, insects and tree species threaten world forests. Moeller J, Munoz-Torres M, Murphy M et al (2011) The genome N Z J For Sci 40:S95–S103 sequence of the leaf-cutter ant Atta cephalotes reveals insights into Wood SL (1982) The bark and ambrosia beetles of North and Central its obligate symbiotic lifestyle. PLoS Genet 7:e1002007 America (Coleoptera:Scolytidae), a taxonomic monograph. Great Sun J, Min L, Gillette NE, Wingfield MJ (2013) Red turpentine beetle: Basin Nat Mem 6:1–1359 innocuous native becomes tree killer in China. Annu Rev Entomol Wu D, Daugherty SC, van Aken SE, Pai GH, Watkins KL, Kouhri H, 58:293–311 Tallon LJ, Zaborsky J, Dunbar MHE, Tran PL, Moran NA, Eisen Toh H, Weiss BL, Perkin SA, Yamashita A, Oshima K, Hattori M, JA (2006) Metabolic complementarity and genomics of the dual Askoy S (2006) Masive genome erosion and functional adapta- bacterial symbiosis of sharpshooters. PLoS Biol 4:e188 tions provide insights into the symbiotic lifestyle of Sodalis Zhang X, Leadbetter JR (2012) Evidence for cascades of perturbation glossinidius in the tsetse host. Genome Res 16:149–156 and adaptation in the metabolic genes of higher termite gut sym- Tsui CKM, Roe AD, El-Kassaby YA, Rice AV, Alamouti SM, Sperling bionts. MBio 3. doi: 10.1128/mBio.00223-12 FHA, Cooke JEK, Bohlmann J, Hamelin RC (2011) Population Zilber-Rosenberg I, Rosenberg E (2008) Role of microorganisms in the structure and migration pattern of a conifer pathogen, Grosmannia evolution of animals and plants. FEMS Microbiol Rev 32:723– clavigera, as influenced by its symbiont, the mountain pine beetle. 735 Mol Ecol 21:71–86 Zipfel RD, DE Beer ZW, Jacobs K, Wingfield BD, Wingfield MJ Vega FE, Dowd PF (2005) The role of yeasts as insect endosymbionts. (2006) Multigene phylogenies define Ceratocystiopsis and In: Insect fungal associations: Ecology and evolution. In: Vega FE Grosmannia distinct from Ophiostoma. Stud Mycol 55:75–97