Symbiosis DOI 10.1007/s13199-017-0514-3 Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the laurel wilt pathogen of avocado, Raffaelea lauricola J. R. Saucedo1 & R. C. Ploetz1 & J. L. Konkol1 & M. Ángel2 & J. Mantilla1 & O. Menocal1 & D. Carrillo1 Received: 18 May 2017 /Accepted: 22 September 2017 # Springer Science+Business Media B.V. 2017 Abstract Ambrosia beetles subsist on fungal symbionts that the beetle were positively impacted. However, no fungus was they carry to, and cultivate in, their natal galleries. These sym- recovered from, and reproduction did not occur on, the bionts are usually saprobes, but some are phytopathogens. A. roeperi and no symbiont diets. These results highlight the Very few ambrosial symbioses have been studied closely, flexible nature of the ambrosial symbiosis, which for X. and little is known about roles that phytopathogenic symbi- bispinatus includes a fungus with which it has no evolutionary onts play in the life cycles of these beetles. One of the latter history. Although the Bprimary^ symbiont of the neotropical symbionts, Raffaelea lauricola, causes laurel wilt of avocado, X. bispinatus is unclear, it is not the Asian R. lauricola. Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this Keywords Nutritional symbiont . Laurel wilt . Avocado . pathosystem. We examined the response of a putative, alter- Raffaelea lauricola . Xyleborus bispinatus native vector of R. lauricola, Xyleborus bispinatus,toartificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice 1 Introduction assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, Xyleborine ambrosia beetles (Curculionidae: Scolytinae) have R. arxii, R. subalba and R. subfusca, all of which had been symbiotic nutritional relationships with fungi (Farrell et al. previously recovered from field-collected females of 2001; Mueller et al. 2005). Foundress females disseminate X. bispinatus. However, no development was observed in their fungal partners to, and propagate them in, natal galleries the absence of a symbiont or on another symbiont, that they bore in host tree xylem. Gardens of these fungi serve Ambrosiella roeperi, recovered from another ambrosia beetle, as their and their progeny’s primary food, and enable them to Xylosandrus crassiusculus. In the no-choice assays, mycangia utilize a protected, but nutrient-poor environment for repro- of foundress females of X. bispinatus harbored significant duction, the xylem (Bleiker et al. 2009). colony-forming units of, and natal galleries that they produced Xyleborine ambrosia beetles have a haplodiploid reproduc- were colonized with, the respective Raffaelea symbionts; with tion system, also known as arrhenotoky, in which diploid fe- each of these fungi, reproduction, fecundity and survival of males develop from fertilized and haploid males from unfer- tilized eggs (Cognato et al. 2011; Farrell et al. 2001;Norris 1979;Normark2003). Males are flightless, and are generally * R. C. Ploetz restricted to their natal gallery in which they mate with their [email protected] mother and sisters. In contrast, mature females disperse from the natal colony and are responsible for establishing broods in new sites, which depend upon the ambrosial gardens that they 1 Tropical Research and Education Center, University of Florida, 18905 SW 280th Street, Homestead, FL 33031-3314, USA establish and manage. Regardless of the female’s mating status, her reproductive 2 Facultad de Agrobiología Presidente Juárez-Universidad Michoacana de San Nicolas de Hidalgo, 60170 Uruapan, Michoacan, success and the numbers of offspring that she produces de- Mexico pends on the availability of nutritional symbionts for the Saucedo J.R. et al. developing colony. Females carry their nutritional fungi (the associates, as was presumed by many early workers (Batra so-called Bambrosia^) in specialized structures in their bodies 1966 ; Baker and Norris 1968). However, common associates called mycangia or mycetangia (Six 2003). Mycangia are ab- could be adapted to survive or multiply in or on the insect or in sent in male xyleborines, and females of a given species pos- their natal galleries; it should not be assumed that associates sess a single type. Pre-oral mycangia, which are small sacs in serve nutritional roles. For example, Freeman et al. (2016) the mandibles, are found in some taxa, whereas elytral reported that of the three species that predominated in the mycangia, small cavities at the base of pronotum adjacent to Euwallacea nr. fornicatus system they studied, larvae com- the scutellum, and mesothoracic mycangia, large invagina- pleted their development when fed on only two of the fungi, tions between the meso- and metanotum, are found in others Fusarium euwallaceae and Graphium euwallaceae;they (Francke-Grosmann 1967; Hulcr and Stelinski 2017). could not discern a role for a third prevalent fungus, Understandings of ambrosial symbioses have evolved as our Paracremonium pembeum (Acremonium pembeum). appreciation of their complexity and the tools with which they Ambrosial fungi are typically saprobes that colonize the inner could be studied have become increasingly sophisticated walls of the natal gallery. In rare cases, the fungal symbionts are (Bateman et al. 2016). These interactions were initially con- pathogens that cause moderate to serious damage on host trees ceived as relationships between a given beetle and a single, (Ploetz et al. 2013). In an extreme case, Raffaelea lauricola,a primary symbiont, the Btrue ambrosial fungus^ (Batra 1963; symbiont of an Asian ambrosia beetle, Xyleborus glabratus,isa Francke-Grosmann 1956, 1963; Leach et al. 1940), even when lethal, vascular pathogen of trees in the southeastern USA, all of multiple species of fungi were consistently associated with a which are in the Lauraceae (Fraedrich et al. 2008). given beetle species (Baker 1963). Batra (1966) later recog- We became interested in the nutritional function of nized that ambrosia beetles Beat one or more auxiliary ambrosia R. lauricola after the fungus began to be isolated from ambro- fungi^. The association with more than one species of fungus sia beetle species other than X. glabratus, soon after the beetle was soon recognized as typical for these insects (Baker and was introduced to the USA (Harrington and Fraedrich 2010). Norris 1968). To date, R. lauricola has been found in nine other ambrosia Subsequent work has sought better information on the beetles, all of which were present in the USA prior to the identity of these fungi. DNA analyses (initially partial se- introduction of X. glabratus (Ploetz et al. 2017a, c). The hor- quences of single genes, but increasingly multi-gene izontal transfer of symbionts among species of ambrosia bee- geneologies) have enabled accurate censuses of the fungi tle had been documented previously (Batra 1966;Harrington (and bacteria) that are associated with these insects 2005;Gebhardtetal.2004), but is poorly understood. Why (Harrington and Fraedrich 2010; Hulcr et al. 2012; and under what circumstances fungal symbionts establish in Kostovcik et al. 2015). A recent meta-analysis provided what new ambrosia beetle species warrants further study, especially is, to date, the most complete pictures of the diverse fungal when it involves the movement of a destructive pathogen to communities that populate the mycangia of these insects. In additional, potential vector species (Ploetz et al. 2017b, c). that study, Kostovcik et al. (2015) inferred with ITS2 pyrose- In recent surveys, X. glabratus was rarely trapped or reared quencing Operational Taxonomic Units (OTUs) of fungi in in laurel wilt-affected trees of avocado (Persea americana the mycangia of Xyleborus affinis Eichhoff, Xyleborus Miller) in Florida (0 of 79,025 ambrosia beetles in Miami- ferrugineus Fabricius and Xylosandrus crassiusculus Dade County and only 11 of 4181 in Brevard County) Motschulsky; each of the communities that were detected (Carrillo et al. 2012;Kendra2017). Rather, other ambrosia was dominated by a core group, that contained eight (48.2% beetle species were associated with these trees. Several of of the OTUs and 78% of the reads), six (42.3% and 64%), and these species harbor R. lauricola (Ploetz et al. 2017c), and 11 (62.4% and 86%) of the OTUs that were observed in the some of them transmitted the pathogen to avocado in no- respective species. In mycangial assays of culturable fungi, choice tests (Carrillo et al. 2014). We describe experiments core suites of symbionts have also been evident. For example, with one of these beetles. three species were the predominate associates of Euwallacea Xyleborus bispinatus (Fig. 1), a close relative of X. nr.fornicatus(Freeman et al. 2016; Lynch et al. 2016), and glabratus (Cognato et al. 2011), is established in South three or fewer fungi were usually present in individuals of Florida (Atkinson et al. 2013), breeds in laurel wilt-affected Xyleborus glabratus Eichhoff (Campbell et al. 2016). avocado trees, and carries R. lauricola frequently (Ploetz et al. Although powerful insight into community structure and 2017c). In prior work with laurel wilt-affected avocado trees, composition has been obtained from such studies, compara- the pathogen was isolated from 35% of 69 individuals of tively few have investigated the function of the detected taxa X. bispinatus (in four experiments,