Symbiosis DOI 10.1007/s13199-017-0514-3

Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the pathogen of , 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 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, , 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 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 , 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 (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, a range of 15% to 100% in these systems. The vertical transmission of these fungi to of the individuals that were assayed), compared to 60% of the new natal galleries and the obligate dependence of the beetles assayed X. glabratus (Ploetz et al. 2017c). on the nutritional symbionts would be expected to promote Ambrosia beetles depend upon their fungal symbionts for fidelity in these systems and, thus, a prevalence of nutritional development, sexual maturation and reproduction (Norris Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the laurel wilt pathogen of avocado,...

the exception of R. arxii, β-tubulin regions. In addition, the identity of R. lauricola was confirmed with taxon-specific microsatellite markers described by Dreaden et al. 2014b. Voucher specimens of the symbionts that were used in this study have been deposited at the Westerdijk Fungal Biodiversity Institute (formerly Centralbureau von Schimmelcultures, CBS) (Utrecht, The Netherlands).

2.2 Artificial media and inoculation with fungal symbionts

A modified artificial medium, described by Menocal et al. (2017), consisted of 240 g of avocado sawdust, 36 g agar, 6gsucrose,6gstarch,6gyeast,12gcasein,1.5g Wesson’s salt mixture, 0.421 g tetracycline, 3 mL germ oil, 3 mL peanut oil, 6 mL ethanol and 1600 mL distilled water. Sterile medium (20 mL in 50 mL plastic centrifuge tubes CentriStar™, Corning) was inoculated with 500 μlof 8×106 conidia/mL of a given symbiont. Media were colo-

nized for 10 days prior to use, and sterile deionized H2Owas Fig. 1 Life cycle of Xyleborus bispinatus. In clockwise order, an egg (top used as a control treatment. Each treatment was replicated image) is followed by a larva, pupa, adult male and adult female. Scales with 24 single tube experimental units. associated with the different life stages are 1 mm (illustration: Randy Fernandez) 2.3 Feeding of unfertilized females and colony initiation 1972, 1979; Kingsolver and Norris 1977). However, little is known about the role phytopathogenic symbionts play in the life Female pupae with non-developed mycangia (Six and Paine cycles of these insects. Although some of those that predominate 1998) were obtained from laboratory stock cultures of in a given beetle (vector) must play a primary, nutritional role, X. bispinatus maintained for 11 generations. Pupae were others might increase the fitness of a given beetle by increasing rinsed in sterile water for 4–5 s to remove phoretic contami- the suitability of a susceptible host tree by reducing host defenses nants, and were placed (fed) on 2-week-old potato dextrose (inducing disease development), thereby facilitating colonization agar (PDA) cultures of a given symbiont for 2 days. Control by, and brood development of, the beetle partner. As suggested pupae were exposed solely to PDA and transferred to water- above, additional, nonpathogenic and non-nutritional reasons for treated medium. After pupae become sclerotized adults, fe- the prevalence of a given associate are also possible. males were surface-disinfested with 70% ethanol for 10 s, In the present study, no-choice assays were conducted to as- dried with sterile filter paper, and individuals were introduced sess the response of X. bispinatus to different ambrosia fungi, to 24 pre-colonized tubes of each of the above symbionts or including R. lauricola. Artificial diets were used to determine the noncolonized, control tubes (one female per tube). Four entry in vitro interaction between these fungi and foundress females holes, 1 cm deep, were made on the medium surface to en- and her progeny. courage gallery formation. Tubes were capped with lids fitted with a 1-cm metallic screen opening for airflow (Fig. 2), and maintained in darkness under laboratory conditions at 25 °C 2 Materials and methods and 75% relative humidity. Observations of behavior and col- ony development were recorded biweekly until colonies were 2.1 Symbiont recovery and identification dissected.

Fungal symbionts were isolated from beetles reared from laurel 2.4 Data collection wilt-affected logs of avocado harvested in Miami-Dade County, FL USA. Raffaelea lauricola, R. arxii and Since females that were used to establish these colonies were R. subalba were obtained from pre-oral mycangia of unfertilized, all progeny were males. Each tube (experimental Xyleborus bispinatus,andAmbrosiella roeperi from mesotho- unit) was evaluated for brood production, survival of racic mycangia of Xylosandrus crassiusculus. Symbionts were foundress females and male progeny, female fecundity, fungal identified with partial sequences of the large sub unit (LSU) content of mycangia and galleries, and the number of entry and small sub unit (SSU) of ribosomal DNA, and, with holes and gallery length. Saucedo J.R. et al.

Fig. 2 Schematic illustration of the artificial rearing system used in this study. a Adult females and eggs of Xyleborus bispinatus surrounded with a fungal layer in a secondary (2°) gallery. b Tube in which primary (1°), secondary (2°) and tertiary (3°) galleries have been produced in the artificial medium. Note female foundress and eggs in the indicated secondary gallery, and the screen opening on the tube cap. c Frass/refuse generated by foundress female (illustration: Randy Fernandez)

Forty-five days after the introduction of foundress females, of the tubes were of the desired treatment. For example, all colonies were dissected and eggs, larvae, pupae, dead and of the 24 original tubes only 17 R. lauricola,16R. arxii, live males, total brood, and female survival were recorded 13 R. subalba and 18 A. roeperi diets were colonized (Fig. 1). Fungi growing on top of the media were removed with the desired fungus and not contaminated; only the and the numbers of entry holes, other than the initial artificial latter tubes were considered in these analyses (Table 1). holes, were counted. From the primary gallery, the length of Several Raffaelea units, from which no symbiont was all galleries along and within the artificial media plug was recovered from both the galleries and foundress measured with a plastic flexible ruler (Fig. 2). The location mycangia, were added as units of the no symbiont treat- of the brood chamber, and whether it was located in single or ment. Unexpectedly, R. subfusca,whichwasalsorecov- multiple galleries, was recorded (Fig. 2). ered in several of the contaminated tubes mentioned Serial dilutions (1:1, 1:10, 1:100) of macerated mycangia and 2-mm2-samples of the gallery surface were used to calculate colony-forming units (CFUs) of the dif- Table 1 Timeline for developmental stages of Xyleborus bispinatus reared on different Raffaelea spp. The week during which a life stage ferent symbionts. To recover Raffaelea spp., dilutions were was first observed and the length of time it was produced is indicated plated on malt-extract agar plus cycloheximide and s- treptomycin (CSMA) (Harrington 1981),amendedwithri- R. lauricola Eggs Eggs Eggs Eggs fampicin and ampicillin and for A. roeperi PDA amended (17) Larvae Larvae Larvae Larvae Larvae with streptomycin and ampicillin. Non-inoculated fungi Pupae Pupae Pupae recovered from each experimental unit were counted and Males Males identified and only tubes with the inoculated symbiont R. arxii (16) Eggs Eggs Eggs Eggs were included in a given symbiont treatment. Larvae Larvae Larvae Larvae Pupae Pupae Pupae 2.5 Statistical analyses Males Males R. subalba Eggs Eggs Eggs Eggs A non-parametric statistical analysis was performed with (13) Larvae Larvae Larvae Larvae the statistical software R v. 3.3.2 (R Development Core Pupae Pupae Pupae Team 2016) to determine whether the fungal symbionts Males Males affected the reproductive behavior of X. bispinatus. R. subfusca Eggs Eggs Eggs Tubes in which the inoculated symbiont was recovered (15) Larvae Larvae Larvae from mycangia and/or galleries were considered as single Pupae Pupae experimental units of a given treatment. Despite the pre- Males cautions that were taken to ensure that foundress females Week 1 Week 2 Week 3 Week 4 Week 5 were exposed to only one fungus or water, only some Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the laurel wilt pathogen of avocado,... above, was the predominant symbiont in 15 tubes of the arxii and R. subalba diets lasted until week 4, and on R. subfusca water control treatment; it was considered as an additional diet lasted from weeks 2 to 4. Pupae were produced from weeks treatment below. Raffaelea subfusca had been recovered 3to5onR. lauricola, R. arxii, R. subalba diets, and from weeks previously from X. bispinatus (Saucedo, unpublished da- 4to5ontheR. subfusca diet. Similarly, male adults were ob- ta), and we presume it was introduced via phoretic con- served from weeks 4 to 5 on R. lauricola, R. arxii and R. subalba tamination of foundress females. diets, but only during week 5 on R. subfusca diet (Table 1). None Mean egg, larva and pupa production, the number of dead of the assayed individuals on A. roeperi and no-symbiont treat- and live males, total brood, foundress survival, female fecun- ments were able to initiate a colony. dity, gallery length and the number of entry holes were com- pared with the Kruskal-Wallis rank sum test. This test can be 3.3 Dissection of artificial diets applied to one-way data with non-normal distribution with more than two groups, and the p value (0.05) is divided by Although there were no significant differences among the the number of comparisons made (15); thus, values <0.003 mean total broods produced by X. bispinatus females on were significantly different. The Kruskal-Wallis rank sum test the different Raffaelea diets (Table 2), differences be- was also used to assess CFU differences for fungal symbionts tween these treatments and the A. roeperi and no- in mycangia and galleries, as well as to compare the frequen- symbiont diets (on which no progeny were produced) cies of brood chamber locations. Results were adjusted with were significant (Kruskal-Wallis test = 57.243, df =5, the Benjamin-Hochberg method and a post-hoc Dunn test p = 4.507e-11). Similarly, egg (p = 0.002, df =5),larva (Dunn 1964) was performed to classify which diets signifi- (p = 6.914e-09, df = 5) and pupa (p = 1.909e-06, df =5) cantly impacted the reproductive behavior and survival of the production were significantly different on the A. roeperi beetles. Simple linear regressions between the total brood and andno-symbiontdietscomparedtotheRaffaelea diets, gallery lengths produced by X. bispinatus females on the dif- but did not differ among the Raffaelea diets. ferent diets were performed with the statistical software R v. The mean survival rate for female foundresses was signif- 3.3.2. icantly higher on the Raffaelea diets than on the A. roeperi and no-symbiont diets (p =5.068e-09,df = 5). Female survival rates were 100% on the R. arxii diet, 94% on the R. lauricola 3Results diet, 93% on the R. subfusca diet, and 77% on the R. subalba diet, whereas only 39% of the females survived on the A. 3.1 Symbiont identification roeperi diet, and 13% on the no-symbiont diet. Male survival was significantly different on Raffaelea diets compared with LSU, SSU, and β-tubulin sequences for isolates of A. roeperi A. roeperi and no-symbiont diets (p =1.432e-08,df = 5), but (accession CBS142880 in the Westerdijk Fungal Biodiversity no significant differences were observed among the Raffaelea Institute), R. lauricola (CBS142879), R. subalba (CBS142877) diets (Table 2). and R. subfusca (CBS142881) in this study were 99–100% Mean female fecundity was significantly higher on matches for the consensus sequences of these species in Raffaelea diets than on A. roeperi and no-symbiont diets GenBank; LSU and SSU sequences for the isolate of R. arxii (p =3.043e-13,df = 5), and fecundity rates mirrored those were also 99–100% matches (GenBank accession numbers for for foundress survival, as 100% of the females on R. arxii diet A. roeperi: LSU-MF138153, SSU-MF138158, β-tubulin- produced progeny, vs 94% of those on R. lauricola,93%of MF138161; R. lauricola: LSU-MF138152, SSU-MF138157, those on R. subalba and 67% of those on R. subfusca diets. β-tubulin-MF138160; R. subalba: LSU-MF138150, SSU- There were no significant differences in fecundity among the MF138155, β-tubulin-MF138163; R. subfusca:LSU- different Raffaelea diets. MF138154, SSU-MF138159, β-tubulin-MF138162; and R. Although mean gallery length was longer on the Raffaelea arxii: LSU-MF138151, SSU-MF138156). We were unable to diets than on the A. roeperi (5.3 cm) and no-symbiont diets amplify the β-tubulin region for R. arxii, which may be typical (3.8 cm), only those on the R. lauricola (7.54 cm) and R. arxii for this species (Dreaden et al. 2014a). In addition, the taxon- (8.44 cm) diets were significantly greater (p =0.003,df =5) specific microsatellite markers of Dreaden et al. (2014b)were (Table 2). With the exception of the significant difference amplified for the isolate of R. lauricola. between the A. roeperi (0.67) and R. subfusca diets (0.07), the average of number entry holes per tube did not differ 3.2 Colony initiation among the other treatments (p =0.02,df =5)(Table2). The simple linear regression model did not correlate gallery length Egg production was observed until week 4 on all diets, except and total broods that were produced with different diets with that based on R. subalba (week 3) (Table 1). Larvae were ob- the exception of that based on R. arxii (R2 = 0.36576, served from weeks 1 to 5 on R. lauricola diet, whereas on R. P =0.013)(Fig.3). Saucedo J.R. et al.

3.4 Mycangia and gallery content and location of brood chamber

Entry holes Mean CFUs of the inoculated (presented) fungi within the mycangia of females on Raffaelea diets were significantly different than those on A. roeperi and no-symbiont diets (p =2.718e-10,df =5),asA. roeperi was neither recovered from mycangia nor galleries of that treatment (although it did 7.54±0.65 a 0.53±0.14 ab Gallery length (cm) 5.3±0.63b 0.67±0.14a 3.8±0.67b 0.19±0.14ab colonize the medium surface). Mean CFUs recovered from mycangia of females on R. lauricola diet, 1228, were higher than on other Raffaelea diets, but with no significant differ- ences compared to the other Raffaelea diets (mean CFUs rang- ing from 579 to 649). The frequency with which a given Foundress fecundity (%) 0b 0b species was recovered from mycangia (frequency of recovery) also did not differ among the Raffaelea species, but it was significantly different compared to the A. roeperi and no- symbiont diets (p =1.359e-11,df = 5). Although the inoculat- ed symbiont was recovered from 100% of the galleries in the survival (%) 39b 13b different Raffaelea diets, only R. lauricola was recovered from 100% of the assayed mycangia; R. arxii was recovered in 75% of these assays, R. subalba 77%, and R. subfusca 67% (Table 3). Although there were significant differences in CFU numbers in the galleries of Raffaelea diets compared to the A. 0b 0b roeperi and no-symbiont diets (p = 8.32e-14, df = 5), there =0.003)

α were no significant differences among Raffaelea diets. There was a tendency for females on all Raffaelea diets to create most of their brood chambers in the second gallery, 0b 0b followed by the primary gallery (Table 4, Fig. 2). Although the number of brood chambers in primary galleries was sig- nificantly greater on Raffaelea than on A. roeperi and no-

ales Live males Total brood Foundress symbiont diets (on which no progeny were produced), there were no significant differences among the Raffaelea diets. 0b 0b Likewise, there were significant differences on Raffaelea diets compared with A. roeperi and no-symbiont diets for use of the secondary gallery as a brood chamber (p =1.987e-05,df =5), reared on different ambrosial symbionts but no significant differences among the Raffaelea diets. Very few females established broods in the tertiary gallery, 0b 0b and there were no significant differences among the different diets (p =0.3768,df = 5). Interestingly, three females on the R. lauricola diet laid eggs in both secondary and tertiary gal- leries; females did so on no other diet (p =0.01,df =5). Xyleborus bispinatus 0b 0b

4Discussion

A primary symbiont for X. bispinatus has not been report- 1.65±0.52 a 3.12±0.39 a 1.35±0.22 a 0.65±0.13 a 2.12±0.35 a 8.76±0.96 a 94±0.08 a 100 a 0.88±0.43 a0.23±0.17 a 2.06±0.41 a2.13±0.99 a 1.46±0.45 a0b 1±0.23 a 1.4±0.42 a 1.15±0.25 a 0.73±0.24 0.38±0.13 a ab 0.3±0.15 ab 0.07±0.14 1.94±0.36ab a 1.38±0.39 a 1.1±0.37 a 6.31±0.99 a 4.54±1.1 a 100 5.4±1.02 aed. a 77±0.09 a However, 93±0.09 a 77±0.07 a 94±0.06 a 67±0.07 asince 7.04±0.75 ab 8.44±0.67 aRaffaelea 6.24±0.7 ab 0.23±0.16 ab 0.44±0.14 ab 0.07±0.15 b species were recognized pre- viously as the predominant symbionts of Xyleborus species (Harrington and Fraedrich 2010; Campbell et al. 2016;

Colony statistics (Mean±SE) for Ploetz et al. 2017c), the reproductive success of X. bispinatus on four different species of Raffaelea was not unexpected. Nevertheless, nutritional flexibility lauricola , Standard Error . Table 2 Fungal symbiont Eggs Larvae Pupae Dead m R R. arxii R. subalba R. subfusca A. roeperi Nosymbiont 0b SE Data within a column that are followed by different letters are significantly different based on Dunn test ( displayed by X. bispinatus was apparently limited, in that Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the laurel wilt pathogen of avocado,...

Fig. 3 Simple linear regressions of total broods produced by Xyleborus bispinatus on different Raffaelea diets plotted against total gallery length

A. roeperi, the primary symbiont of an ambrosia beetle in number of progeny produced by X. crassiusculus was de- another genus, Xylosandrus crassiusculus, did not support creased on a R. lauricola diet. Nonetheless, the present study X. bispinatus development. appears to be the first to report the nutritional impact of several Little is known about the nutritional impacts fungi have on different symbionts on a given ambrosia beetle species. Better their ambrosia beetle associates. Previously, larvae of understandings are needed for these interactions and why Euwallaceae nr. fornicatus fed on Paracremonium pembeum some of these fungi are used as food sources by these insects. did not complete their life cycle, whereas those reared on Nutrients provided by different fungal diets are key deter- Fusarium euwallaceae and Graphium euwallaceae complet- minants of the fitness and fecundity of ambrosia beetles. X. ed development (Freeman et al. 2016). Similarly, the fungus bispinatus females are synovigenic (produce eggs throughout Paecilomyces variotii had a negative impact on larvae and their life cycle) and, thus, must continuously ingest nutrients adults of Xyleborinus saxesenii Ratzeburg, but the beetle’s to support egg production (Gottlieb et al. 2014). Despite these primary nutritional symbiont, Raffaelea sulfurea, had positive requirements, four species of Raffaelea, including the new effects (Biedermann et al. 2013). In addition, a detrimental encounter phytopathogen R. lauricola, enabled high rates of impact had been shown previously with another nutritional fecundity and significant brood production in X. bispinatus symbiont, as Ott (2007) demonstrated that the fitness of and (Table 2).

Table 3 Recovery of symbionts from mycangia and natal galleries of Xyleborus bispinatus

Fungal symbiont Mycangia Gallery

Frequency of CFU mean±SE CFU range Frequency of CFU mean±SE CFU range recovery (%) recovery (%)

R. lauricola (n = 17) 100 a 1228±157 a 200–2800 100 108,564±14,492 a 6000–280,000 R. arxii (n = 16) 75 a 621±162 a 0–1600 100 91,243±14,938 a 5500–244,000 R. subalba (n = 13) 77 a 579±179 a 0–1600 100 83,276±16,572 a 200–210,400 R. subfusca (n = 15) 67 a 649±167 a 0–3600 100 151,386±15,428 a 800–320,000 A. roeperi (n =18) 0b 0b 0 0 0b 0 No symbiont (n =16) 0b 0b 0 0 0b 0

Data within columns followed by different letters are significantly different based on Dunn test (α=0.003) SE, Standard Error Saucedo J.R. et al.

Table 4 Number (Mean±SE) of brood chambers in each location Fungal symbiont Primary Secondary Tertiary Secondary (gallery level) and Tertiary

R. lauricola 6 (0.35±0.08) a 7 (0.41±0.09) a 1 3 R. arxii 2 (0.13±0.08) a 11 (0.69±0.1) a 2 0 R. subalba 4 (0.31±0.09) a 5 (0.38±0.11) a 1 0 R. subfusca 2 (0.13±0.08) a 8 (0.53±0.1) a 0 0 A. roeperi 0b 0b 0 0 No symbiont 0 b 0 b 0 0

Data within columns followed by different letters are significantly different based on Dunn test (α=0.003) SE, Standard Error

Recent research suggests that mated females of However, it was not a good predictor of the reproductive be- X. bispinatus increase their reproductive potential in the pres- havior of X. bispinatus. ence of R. lauricola (Menocal et al. 2017). Thus, regardless of Xyleborus species lay eggs in multiple branches of exca- the mating status of X. bispinatus females, R. lauricola can vated galleries, unlike Xyleborinus species that propagate in positively influence their reproduction. Conversely, the repro- large cave-like galleries (Biedermann 2010). Regardless of duction of X. volvulus Fabricius, another ambrosia beetle as- which Raffaelea species they were fed, X. bispinatus females sociated with the transmission of laurel wilt (Carrillo et al. primarily laid eggs in secondary galleries in the present study. 2014), was negatively impacted on R. lauricola diet, as fe- Ambrosia beetles have obligate symbioses with their nutri- males from field-collected bolts, which were colonized with tional partners. Our results indicate that X. bispinatus can pro- other symbionts, had higher total broods on uncolonized diet duce at least one generation on four different species of (11.1) than on R. lauricola diet (3.8) (Menocal et al. 2017). Raffaelea, including the laurel wilt pathogen R. lauricola. In the present study, higher numbers of CFUs of Results from the present study support prior speculation that R. lauricola were recovered from mycangia of X. bispinatus X. bispinatus may be an important alternative vector in the (individual range: 200–2800) than were previously reported in avocado pathosystem (Ploetz et al. 2017c). field-collected females from native Persea spp. (0–53) and Gnotobiotic systems provide invaluable tools for examin- avocado (0–320) that were affected by laurel wilt (Ploetz ing interactions between an insect host and its symbionts et al. 2017c). Although the no-choice conditions that were (Douglas 2011). Ambrosia beetles acquire symbionts in their used in the present study might be expected to increase prop- mycangia and/or on their exoskeletons via feeding in natal agule numbers in mycangia of the assayed females, we note galleries. In the present study, gnotobiotic X. bispinatus were that the numbers of no-choice CFUs of R. lauricola in obtained by surface disinfesting lab-reared pupae and new X. bispinatus resemble those found in X. glabratus (0– adults that were later fed with selected symbionts. Most gno- 11,600 CFUs in Persea spp. and 0–1480 CFUs in avocado) tobiotic X. bispinatus offered sterile media did not produce (Ploetz et al. 2017c). These results highlight the ability of progeny confirming that X. bispinatus, as other ambrosia bee- X. bispinatus to acquire and carry large amounts of tles, has an obligate mutualism with fungal symbionts. R. lauricola, more than enough to kill susceptible avocado However, some surface-disinfested beetles in this study trees (Hughes et al. 2015). established symbioses with R. subfusca, which may have Gallery construction is an important element of ambro- persisted on the pupal cuticle and new adult exoskeleton. sia beetle reproduction. Xyleborus bispinatus females on Alternatively, the fungus may have been carried internally in Raffaelea diets produced tunnels of similar lengths, and a structure other than the mycangia, which are not present in those on R. lauricola and R. arxii diets were significantly the pupal stage (Six and Paine 1998). With the exception of longer than those on A. roeperi andnosymbiontdiets. the R. subfusca experimental units, the majority of new adults Longer galleries of Xyleborus pfeili Ratzeburg resulted in our study selectively interacted with, and established natal in greater brood production of that species (Mizuno and colonies of, the presented fungus. These results may indicate Kajimura 2009). Although our results indicate that longer that the initial feeding period is key in determining which galleries of X. bispinatus were generally more productive, symbiont is used by a developing ambrosia beetle colony. gallery length on only R. arxii diet was significantly re- Moreover, unknown attributes of a symbiont apparently deter- lated to brood production (Fig. 3). mine the food value a fungus provides for a given ambrosia The number of entry holes has been used to estimate pop- beetle partner, as only the Raffaelea symbionts in this study ulation size and describe the phenology of another ambrosia supported reproduction. Research is needed to understand beetle, Euwallacea nr. fornicatus (Cooperband et al. 2016). these temporal and nutritional interactions. Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the laurel wilt pathogen of avocado,...

Acknowledgements We thank James Colee for advice on statistical Douglas AE (2011) Lessons from studying insect symbiosis. Cell Host analyses, and Randy Fernandez for producing Figs. 1 and 2.Thiswork Microbe 10:359–367. https://doi.org/10.1016/j.chom.2011.09.001 was supported, in part, by NIFA grant# 2015-51181-24257 and a schol- Dreaden TJ, Davis JM, de Beer ZW, Ploetz RC, Soltis PS, Wingfield MJ, arship to JRSC from the Mexican government (CONACYT). Smith JA (2014a) Phylogeny of ambrosia beetle symbionts in the genus Raffaelea. Fungal Biol 118:970–978. https://doi.org/10.1016/ j.funbio.2014.09.001 Dreaden TJ, Davis JM, Harmon CL, Ploetz RC, Palmateer AJ, Soltis PS, References Smith JA (2014b) Development of multilocus PCR assays for Raffaelea lauricola, causal agent of laurel wilt disease. Plant Dis 98:379–383. https://doi.org/10.1094/PDIS-07-13-0772-RE Atkinson TH, Carrillo D, Duncan RE, Peña JE (2013) Occurrence of Dunn OJ (1964) Multiple comparisons using rank sums. Technometrics Xyleborus bispinatus (Coleoptera: Curculionidae: Scolytinae) 6:241–252 – Eichhoff in southern Florida. Zootaxa 3669:96 100. https://doi. Farrell BD, Sequeira AS, O’Meara BC, Normark BB, Chung JH, Jordal org/10.11646/zootaxa.3669.1.10 BH (2001) The evolution of agriculture in beetles (Curculionidae: Baker JM (1963) Ambrosia beetles and their fungi with special reference Scolytinae and Platypodinae). Evolution 55:2011–2027. https://doi. to Platypus cylindrus Fabricius. Symposium Society General org/10.1111/j.0014-3820.2001.tb01318.x – Microbiology 13:232 265 Fraedrich SW, Harrington TC, Rabaglia RJ, Ulyshen MD, Ayfieldiii AE, Baker JM, Norris DM (1968) A complex of fungi mutualistically in- Hanula JL, Eickwort JM, Miller DR (2008) A fungal symbiont of volved in the nutrition of the ambrosia beetle Xyleborus ferrugineus. the redbay ambrosia beetle causes a lethal wilt in redbay and other J Invertebr Pathol 11:246–250. https://doi.org/10.1016/0022- Lauraceae in the southeastern United States. Plant Dis 92:215–224. 2011(68)90157-2 https://doi.org/10.1094/PDIS-92-2-0215 Bateman C, Sigut M, Skelton J, Smith KE, Hulcr J (2016) Fungal asso- Francke-Grosmann H (1956) Hautdrusen als Trager der Pilzesymbiose ciates of the Xylosandrus compactus (Coleoptera: Curculionidae, bei Ambrosiakafern. Z Morphol Okol Tiere 45:275–308 Scolytinae) are spatially segregated on the insect body. Environ Francke-Grosmann H (1963) Some new aspects in forest entomology. Entomol 45:883–889. https://doi.org/10.1093/ee/nvw070 Annu Rev Entomol 8:415–438. https://doi.org/10.1146/annurev.en. Batra LR (1963) Ecology of ambrosia fungi and their dissemination 08.010163.002215 by beetles. Trans Kans Acad Sci 66:213–236. https://doi.org/ Francke-Grosmann H (1967) Ectosymbiosis in -inhabiting insects. 10.2307/3626562 Symbiosis 2:141–205 Batra LR (1966) Ambrosia fungi: extent of specificity to ambrosia bee- Freeman S, Sharon M, Dori-Bachash M, Maymon M, Belausov E, Maoz tles. Science 153:193–195 Y, Margalit O, Protasov A, Mendel Z (2016) Symbiotic association Biedermann PH (2010) Observations on sex ratio and behavior of males of three fungal species throughout the life cycle of the ambrosia in Xyleborinus saxesenii Ratzeburg (Scolytinae, Coleoptera). beetle Euwallacea nr. fornicatus. Symbiosis 68:115–128. https:// Zookeys 56:253–267. https://doi.org/10.3897/zookeys.56.30 doi.org/10.1007/s13199-015-0356-9 Biedermann PHW, Klepzig KD, Taborsky M, Six DL (2013) Abundance Gebhardt H, Bergerow D, Oberwinkler F (2004) Identification of the and dynamics of filamentous fungi in the complex ambrosia gardens ambrosia fungus of Xyleborus monographus and X. dryographus – of the primitively eusocial beetles Xyleborinus saxesenii Ratzeburg (Curculionidae, Scolytinae). Mycol Prog 3:95 102. https://doi.org/ (Coleoptera: Curculionidae, Scolytinae). FEMS Microbiol Ecol 83: 10.1007/s11557-006-0080-1 711–723. https://doi.org/10.1111/1574-6941.12026 Gottlieb D, Lubin Y, Harari AR (2014) The effect of females mating – Bleiker KP, Potter SE, Lauzon CR, Six DL (2009) Transport of fungal status on male offspring traits. Behav Ecol Sociobiol 68:701 710. symbionts by mountain pine beetles. Can Entomol 141:503–514. https://doi.org/10.1007/s00265-014-1683-1 https://doi.org/10.4039/n09-034 Harrington TC (1981) Cycloheximide sensivity as a taxonomic character in Ceratocystis. Mycologia 73:1123–1129. https:// Campbell AS, Ploetz RC, Dreaden T, Kendra P, Montgomery W (2016) doi.org/10.2307/3759682 Geographic variation in mycangial communities of Xyleborus Harrington, T. C. 2005. Ecology and evolution of mycophagous bark glabratus. Mycologia 108:657–667. https://doi.org/10.3852/15-133 beetles and their fungal partners. pp. 257-291. in: Insect-Fungal Carrillo D, Duncan R, Peña JE (2012) Ambrosia beetles (Curculionidae: Associations. Ecology and Evolution. eds. Vega, F.E. and Scolytinae) that breed in avocado wood in Florida. Fla Entomol 95: Blackwell, M. Oxford Univ Press – 573 579. https://doi.org/10.1653/024.095.0306 Harrington TC, Fraedrich SW (2010) Quantification of propagules of the Carrillo D, Duncan RE, Ploetz JN, Campbell A, Ploetz RC, Peña JE laurel wilt fungus and other mycangial fungi from the redbay am- (2014) Lateral transfer of a phytopathogenic symbiont among native brosia beetle, Xyleborus glabratus. Phytopathology 100:1118–1123. – and exotic ambrosia beetles. Plant Pathol 63:54 62. https://doi.org/ https://doi.org/10.1094/PHYTO-01-10-0032 10.1111/ppa.12073 Hughes MA, Inch SA, Ploetz RC, Er HL, van Bruggen AH, Smith JA Cognato AI, Hulcr J, Dole SA, Jordal BH (2011) Phylogeny of haplo- (2015) Response of swampbay, , and avocado, diploid, fungus growing ambrosia beetles (Curculionidae: Persea americana, to various concentrations of the laurel wilt path- Scolytinae: Xyleborini) inferred from molecular and morphological ogen, Raffaelea lauricola. For Pathol 45:111–119. https://doi.org/ data. Zool Scr 40:174–186. https://doi.org/10.1111/j.1463-6409. 10.1111/efp.12134 2010.00466.x Hulcr J, Stelinski LL (2017) The ambrosia symbiosis: From evolutionary Cooperband MF, Stouthamer R, Carrillo D, Eskalen A, Thibault T, Cossé ecology to practical management. Annu Rev Entomol 62:285–303. AA, Castrillo LA, Vandenberg JD, Rugman JP (2016) Biology of https://doi.org/10.1146/annurev-ento-031616-035105 two members of the species complex Hulcr J, Rountree NR, Diamond SE, Stelinski LL, Fierer N, Dunn (Coleoptera: Curculionidae: Scolytinae), recently invasive in the RR (2012) Mycangia of ambrosia beetles host communities of U.S.A., reared on an ambrosia beetle artificial diet. Agric For bacteria. Microb Ecol 64:784–793. https://doi.org/10.1007/ Entomol 18:223– 237. https://doi.org/10.1111/afe.12155 s00248-012-0055-5 R Development Core Team. 2016. R: A language and environment for Kendra PE, Owens D, Montgomery WS, Narvaez TI, Bauchan GR, statistical computing. Vienna, Austria. The R foundation for statis- Schnell EQ, Tabanca N, Carrillo D (2017) α-Copaene is an attrac- tical computing. ISBN: 3-900051-07-0. https://www.R-project.org/ tant, synergistic with quercivorol, for improved detection of Saucedo J.R. et al.

Euwallacea nr. fornicatus (Coleoptera: Curculionidae: Scolytinae). Normark BB (2003) The evolution of alternative genetic systems in in- PLoS ONE 12(6): e0179416 sects. Annu Rev Entomol 48:397–423. https://doi.org/10.1146/ Kingsolver JG, Norris DM (1977) The interaction of Xyleborus annurev.ento.48.091801.112703 ferrugineus (Coleoptera: Scolytidae) behavior and initial reproduc- Norris, D. M. 1972. Dependence of fertility and progeny development of tion in relation to its symbiotic fungi. Ann Entomol Soc Am 70:1–4. Xyleborus ferrugineus upon chemicals from its symbiotes, Pp. 299- https://doi.org/10.1093/aesa/70.1.1 309. In: Insect and mite nutrition. Rodriguez, J. G. (ed). North- Kostovcik M, Bateman C, Kolarik M, Stelisnki LL, Jordal BH, Holland Pub. Co. Amsterdam Hulcr J (2015) The ambrosia symbiosis is specific in some Norris, D. M. 1979. The mutualistic fungi of Xyleborini beetles In: species and promiscuous in others: evidence from pyrose- Insect–Fungus Symbiosis: Nutrition, Mutualism, and quencing. ISME J 9:126–138. https://doi.org/10.1038/ismej. Commensalism. Batra, L. R. (ed). Wiley: New York; 53–63.63 2014.115 Ott, E. P. 2007. Chemical ecology, fungal interactions and forest stand Leach JG, Hodson AC, Chilton SJP, Christensen CM (1940) correlations of the exotic Asian ambrosia beetle, Xylosandrus Observations on two ambrosia beetles and their associated fungi. crassiusculus. M. S. Thesis, Louisiana State University Phytopathology 30:227–236 Ploetz RC, Hulcr J, Wingfield MJ, de Beer ZW (2013) Ambrosia and -associated tree diseases: Black swan events in the pathology? Lynch SC, Twizeyimana M, Mayorquin JS, Wang DH, Na F, Kayim M, – Kasson MT, Thu PQ, Bateman C, Rugman-Jones P, Hulcr J, Plant Dis 95:856 872. https://doi.org/10.1094/PDIS-01-13-0056-FE Stouthamer R, Eskalen A (2016) Identification, pathogenicity and Ploetz RC, Hughes MA, Kendra PE, Fraedrich SW, Carrillo D, Stelinski abundance of Paracremonium pembeum sp. nov. and Graphium LL, Hulcr J, Mayfield AE III, Dreaden TL, Crane JH, Evans EA, — Schaffer BA, Rollins J (2017a) Recovery plan for laurel wilt of euwallaceae sp. nov. two newly discovered mycangial associates – of the polyphagous shot hole borer (Euwallacea sp.) in California. avocado, caused by Raffaelea lauricola. Plant Health Prog 18:51 Mycologia 108:313–329. https://doi.org/10.3852/15-063 77. https://doi.org/10.1094/PHP-12-16-0070-RP Ploetz RC, Kendra PE, Choudhury RA, Rollins JA, Campbell A, Garrett Menocal, O., Cruz, L., Kendra, P., Crane, J., Ploetz, R., Carrillo, D. K, Hughes MA, Dreaden T (2017b) Laurel wilt in native and agri- 2017. Rearing Xyleborus volvulus (Coleoptera: Curculionidae) cultural ecosystems: Understanding the drivers and scales of com- on media containing sawdust from avocado or silkbay, with or plex pathosystems. Forests 8:48. https://doi.org/10.3390/f8020048 without Raffaelea lauricola (: Ploetz RC, Konkol J, Narvaez T, Duncan R, Saucedo JR, Campbell A, ). Environmental Entomology (In press) Mantilla J, Carrillo D, Kendra PE (2017c) Presence and prevalence Mizuno T, Kajimura H (2009) Effects of ingredients and structure of of Raffaelea lauricola, cause of laurel wilt, in different species of semi-artificial diet on the reproduction of an ambrosia beetle, ambrosia beetles in Florida, USA. J Econ Entomol:1–8. https://doi. Xyleborus pfeili (Ratzeburg) (Coleoptera: Curculionidae: – org/10.1093/jee/tow292 Scolytinae). Appl Entomol Zool 44:363 370. https://doi.org/10. Six, D.L. 2003. Bark beetle-fungus symbioses. Pages 97-114 in. Insect 1303/aez.2009.363 Symbiosis. K. Bourtzis, and T. Miller, eds. CRC Press, Boca Raton Mueller UG, Gerardo NM, Aanen DK, Six DL, Schultz TR (2005) Six DL, Paine TD (1998) Effects on mycangial fungi and host tree species The evolution of agriculture in insects. Annu Rev Ecol Evol on progeny survival and emergence of Dendroctonus ponderosae – Syst 36:563 595. https://doi.org/10.1146/annurev.ecolsys.36. (Coleoptera: Scolytidae). Environ Entomol 27:1393–1401. https:// 102003.152626 doi.org/10.1093/ee/27.6.1393