Xyleborus volvulus (Coleoptera Curculionidae) Biology andFungal Associates

Luisa F Cruza Octavio Menocala Julio Mantillaa Luis A Ibarra-Juarezbc Daniel Carrilloa

aTropical Research and Education Center University of Florida Homestead Florida USAbRed de Estudios Moleculares Avanzados Instituto de Ecologiacutea AC Xalapa MexicocCaacutetedras CONACyT Instituto de Ecologiacutea AC Xalapa Mexico

ABSTRACT The ambrosia beetle Xyleborus volvulus Fabricius has been reportedas a potential vector of the plant pathogen Raffaelea lauricola TC Harr Fraedrich ampAghayeva that is affecting avocado orchards in South Florida In this study we examinedits life cycle process of gallery formation gallery structure and fungal associates by rear-ing one generation on avocado sawdust medium under control conditions The adultfoundress excavated a vertical tunnel that constituted the main gallery with a length of25 cm followed by the construction of up to six secondary galleries with a total lengthof 44 cm The time period for one generation (egg to adult) was 28 days Teneral malesemerged 3 days after the emergence of the first females The F1 generation did not sig-nificantly contribute to gallery expansion Four species of Raffaelea and nine yeast spe-cies were recovered from galleries and beetles Raffaelea arxii and Candida berthetii werethe most frequent symbionts recovered from new adults and galleries Candida berthetiidominated during the early stages of the gallery development whereas R arxii wasmost frequent in later stages Other Raffaelea species were inconsistently isolated fromgalleries which suggests a strong association between Xyleborus volvulus and both Rarxii and C berthetii These results suggest that R arxii is the primary nutritional symbi-ont of X volvulus and that yeast species may be pioneer colonizers that assist with thegrowth of fungal symbionts

IMPORTANCE Ambrosia beetles cultivate fungi in tunnels bored into weakened hosttrees This obligate interaction is required for their survival as beetles feed on thesesymbiotic fungi and the fungi benefit from transportation by the beetles Xyleborusvolvulus carries many nonpathogenic symbionts however recently the acquisition ofRaffaelea lauricola (the causal agent of a lethal vascular disease of lauraceous trees)by this beetle has altered its status from wood degrader to potential pest in avo-cado We conducted a study to understand the relationship of this beetle and itsfungal associates Our results show that X volvulus has a multipartite flexible associ-ation with different Raffaelea species The lack of fidelity in the mutualistic associa-tion may explain the acquisition of R lauricola Knowing the beetle biology and itsmutualistic interactions furthers an understanding of the beetlersquos role as a potentialvector and in disease transmission

KEYWORDS artificial medium artificial rearing Candida spp fungal associatesfungus-beetle symbiosis Persea americana Raffaelea Scolytinae Xyleborini

Ambrosia beetles (Coleoptera Curculionidae Scolytinae) comprise a polyphyleticgroup of beetles that practice fungiculture (1) Usually ambrosia beetles are

attracted to stressed decaying or dead trees in which they construct galleries andcultivate their symbionts (2) In the fungus-beetle mutualistic association symbionts areused as nutritional sources by the beetles and their offspring and provide them with

Citation Cruz LF Menocal O Mantilla J Ibarra-Juarez LA Carrillo D 2019 Xyleborus volvulus(Coleoptera Curculionidae) biology and fungalassociates Appl Environ Microbiol 85e01190-19 httpsdoiorg101128AEM01190-19

Editor Karyn N Johnson University ofQueensland

Copyright copy 2019 American Society forMicrobiology All Rights Reserved

Address correspondence to Luisa F Cruzluisafcruzufledu

Received 27 May 2019Accepted 23 July 2019

Accepted manuscript posted online 2August 2019Published

INVERTEBRATE MICROBIOLOGY

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vitamins amino acids and sterols (3) while the fungi benefit from dispersal by thebeetles to new suitable host trees (4)

In ambrosia beetles sexual division of labor gregarious development and a highlevel of sociality evidence their adaptation for fungal farming and the obligate mutu-alistic associations (5) Fungal propagules are carried in specialized organs (eg my-cangia) harbored only by females which acquire disperse and cultivate the symbionts(6 7) However males which are flightless remain in galleries exclusively to mate withtheir siblings (8) Scolytinae species are characterized by arrhenotokous reproduction inwhich females are diploid and males are haploid (9 10) This system allows the establish-ment of colonies where successive overlapping generations cooperate in brood care andfungal garden maintenance (11)

Microorganisms transmitted in concert to constitute ambrosial gardens include notonly known nutritional fungal symbionts but also yeasts and bacteria with unknownecological functions (12) Mutualistic fungi are often described in the anamorph genusRaffaelea Arx amp Hennebert emend LR Batra and Ambrosiella Brader emend LR Batra(13) Most of these symbioses are multipartite with a beetle associated with two ormore permanent partners (7) It is thought that the mycangia play a critical role inconferring symbiont specificity (14 15) Fungal symbiont transfer occurs vertically fromthe natal galleries to the offspring and thereafter the symbionts are transmitted by thenew generation of females when they colonize other trees (7)

Typically ambrosia symbionts are saprophytes and nonpathogenic to their hosttrees however some invasive beetle species are capable of colonizing healthy livingtrees after inoculating them with pathogenic symbionts (16) Examples include thecomplex Fusarium spp transmitted by Euwallacea fornicatus Eichhoff Euwallacea whit-fordiodendrus Schedl and Euwallacea kuroshio Gomez and Hulcr (17ndash20) Ophiostomaulmi and Ophiostoma novo-ulmi transmitted by Scolytus multistriatus Marsham (21) andRaffaelea lauricola transmitted by Xyleborus glabratus Eichhoff (22)

Laurel wilt the disease caused by the fungal pathogen R lauricola has affectednumerous members of the Lauraceae family in the coastal plain of the southeasternUnited States (23) Xyleborus glabratus a species native to Southeast Asia was intro-duced along with its symbiont R lauricola into Georgia (USA) in 2002 (24) Since thenthe beetle-pathogen complex has spread to Virginia Louisiana North and SouthCarolina Mississippi Alabama Arkansas and Texas (httpsouthernforesthealthnetdiseaseslaurel-wiltdistribution-map) This complex was detected in North Florida in2005 and spread throughout the state reaching the avocado (Persea americana Mill)commercial production area in South Florida in 2011 Avocado is perhaps the hostwith the greatest economic impact (25) and the epidemic of laurel wilt of avocado isprobably caused by a pathogen-vector transfer (26 27) Avocado seems to be a poorhost for X glabratus (26) although 13 native and naturalized ambrosia beetle specieshave been reported to breed in avocado 7 of which have been proven to carry Rlauricola and two of them Xyleborus volvulus Fabricius and Xyleborus bispinatus Eich-hoff transmitted the pathogen to healthy avocado under controlled conditions (26)The sympatric distribution of the main vector X glabratus and other ambrosia beetlespecies may facilitate the lateral acquisition of the pathogen by cross-contamination ofadjacent galleries in trees where the beetles coexist (26)

Xyleborus volvulus is found in all tropical and subtropical regions of the New World(28 29) In the United States X volvulus has been reported in Texas North and SouthCarolina Georgia and the Florida East Coast partially overlapping with X glabratus(23) It has a wide host range and is reported in 24 plant families including the Lauraceaewhere it is particularly important due to its link with the transmission of R lauricolaXyleborus volvulus exhibits the characteristic reproductive biology (inbreeding) geneticsystem (arrhenotokous) and behavioral ecology (fungal cultivation) of the Xyleborinitribe (2)

This paper studies the process of gallery formation developmental biology and thefungal associates of X volvulus and aims at gaining a better understanding of thepotential role that this native ambrosia beetle species can play as an alternative vector

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of R lauricola To achieve these objectives we deployed a laboratory rearing systemusing an avocado sawdust medium

RESULTSIdentity of the fungal isolates BLAST search of sequences of the fungal isolates

obtained from the samples collected from frass gallery entrances and from tunnelsindicated the presence of nine yeast species and three Raffaelea species (Table 1)Among them Candida berthetii Candida laemsonensis Candida nemodendra Candidasp strain BCMU and Pichia mexicana were found throughout the galleries at all threeisolation points whereas Zygozyma oligophaga and Ambrosiozyma monospora werefound only at gallery entrances and tunnels respectively Pichia manshurica and Candidacalifornica were only found in frass Candida berthetii and R arxii were the most frequentspecies recovered from the tunnels throughout the experiment Raffaelea sp strainPL1001 and Raffaelea rapaneae were found at low frequencies in tunnels frass or atgallery entrances (Table 1)

Identity of beetle isolates No symbionts or other fungal associates were recoveredfrom surface-disinfected eggs or pupae Raffaelea arxii was the most frequently recov-ered symbiont associated with larvae (5 of 5 larvae) with an average abundance of76 29 CFUlarva followed by A monospora (4 of 5 with 175 93 CFUlarva) andRaffaelea sp (1 of 5 with 10 CFUlarva) (Fig 1) Similar frequencies of the samesymbionts were recovered from teneral adults R arxii (5 of 5 heads with 203 347CFUhead and 3 of 5 bodies with 136 726 CFUbody) A monospora (4 of 5 headswith 5125 3397 CFUhead and 1 of 5 bodies and 5 CFUbody) and Raffaelea sp (2of 5 heads with 285 155 CFUhead and 1 of 5 bodies with 31 CFUbody) (Fig 1)

The most prevalent symbiont recovered from the heads and bodies of fully sclero-tized adult females was C berthetii (5 of 5 heads with 127 378 CFUhead and 2 of 5bodies with 425 225 CFUbody respectively) followed by R arxii (4 of 5 heads with1697 7478 CFUhead and 2 of 4 bodies with 8 4 CFUbody) Raffaelea sp and Amonospora were recovered only from the beetlesrsquo heads (3 of 5 with 141 4839CFUhead) or bodies (1 of 5 with 10 CFUbody) (Fig 1)

Gallery construction brood development and fungal associates in galleries (i)Ten days after foundress introduction (AFI) The foundress excavated the maingallery which reached an average length of 152 023 cm (Fig 2A and B) Construc-tion of secondary galleries started by the end of the first week near the entrance of themain tunnel (Fig 2A) Eggs were first observed at the distal ends of the tunnels laid in

TABLE 1 Isolation of fungal species at each location during the experimenta

Isolate

GenBank accession no( similarity) of the closestmatching sequence toc

Frequency () of isolationby sample type (n 25)

28S rDNA LSU 18S rDNA SSU FrassGalleryentrance Tunnels

Candida berthetii GU246259 (99) AB054883 (98) 40 55 45Candida laemsonensis AB438205 (99) 25 30 35Pichia mexicana JQ689049 (99) AB013570 (99) 5 15 10Candida nemodendra EU011629 (100) EU011709 (99) 15 5 25Candida sp BCMU BX01 AB285024 (99) AB285024 (99) 5 15 25Pichia manshurica EF550223 (99) KX816338 (99) 5 0 0Candida californica JX188104 (98) LT854931 (99) 5 0 0Zygozyma oligophaga DQ518998 (99) FJ176819 (99) 0 5 0Ambrosiozyma monospora EU011590 (98) JQ698881 (99) 0 0 5Raffaelea arxii EU984298 (100) AY497519 (99) 60 70 60Raffaelea sp PL1001 KJ909293 (99) KJ909294 (99) 15 0 5Raffaelea rapaneae KT182930 (99) 0 5 5Raffaelea spb MG674037 (99) MG673970 (100)aData represent results from five colonies sampled at five time points over 38 daysbRecovered only from beetlescSSU small subunit LSU large subunit

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FIG 1 Developmental stages of X volvulus and frequency of recovery of fungal associates from eachstage

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FIG 2 Xyleborus volvulus colony progression (A) Schematic model of gallery construction and location of beetledevelopmental stages in the galleries (B) Mean length of primary and secondary galleries (n 5) (C) Frequency ofrecovery of the fungal isolates from the X volvulus gallery tunnels at 1-week intervals (n 5) DAFI days after foundressintroduction

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clusters of up to 5 eggs and averaged 10 eggs per colony at day 10 (Fig 2A and 3)Larvae (18 156 larvaecolony) were first observed also at day 10 at the end ofsecondary galleries (Fig 2A and 3) Candida berthetii was recovered more frequently (4of 5 galleries) from the galleries Candida laemsonensis (2 of 5 galleries) R arxii (2 of 5galleries) and R rapaneae (1 of 5 galleries) were also recovered at lower frequenciesThe last was found only at this sampling time and not in later stages of colonydevelopment (Fig 2C)

(ii) Eleven to 17 days AFI From 11 to 17 days AFI the foundress extended the maingallery which reached its final length of 25 021 cm and extended secondarygalleries by approximately 12 cm (Fig 2A and B) The oviposition peak (11 494eggsfoundress) was observed at 14 days AFI (Fig 3) By 17 days AFI the number oflarvae increased to an average of 115 126 per colony and the first pupae wereobserved (day 17 04 04 pupaecolony) near the midpoint of secondary galleries(Fig 2A and 3) Candida berthetii continued to be the most frequent isolate (45galleries) followed by R arxii (25 galleries) C nemodendra (25 galleries) and Claemsonensis (15 galleries) (Fig 2C)

(iii) Eighteen to 24 days AFI From 18 to 24 days AFI the total length of secondarygalleries reached its maximum (44 073 cm) (Fig 2A and B) The number of eggsdecreased (36 095 eggsfoundress) larvae peaked at day 21 (188 356 larvaecolony) and the number of pupae increased to 44 196 pupaecolony (Fig 3) Thefirst teneral adults (13 07 teneral adultscolony) were observed with females at 21days AFI and males at 24 days AFI (Fig 3) Candida berthetii previously the mostfrequently isolated fungus was not recovered at this time (Fig 2C) Raffaelea arxii Claemsonensis and C nemodendra were isolated at similar frequencies (2 of 5 galleries)whereas Candida sp BCMU was isolated from only 1 of 5 galleries (Fig 2C)

(iv) Twenty-five to 31 days AFI The size of the gallery system did not increasesignificantly from 25 to 31 days AFI (Fig 2A and B) The numbers of eggs (11 06eggsfoundress) larvae (61 113 larvaecolony) pupae (18 044 pupaecolony)and teneral adults (08 033 teneral adultscolony) decreased while the first fullysclerotized F1 adults were observed (52 115 F1 adultscolony) (Fig 3) Unlike distri-bution in the other stages the F1 adults were randomly distributed throughout thegalleries (Fig 2A) Raffaelea arxii was the most prevalent species (4 of 5 galleries)followed by Candida sp BCMU (3 of 5 galleries) and C laemsonensis (2 of 5 galleries)(Fig 2C)

(v) Thirty-two to 38 days AFI The size of the gallery system remained stable duringthe last observation period The final gallery system consisted of the main gallery and

FIG 3 Life stages of Xyleborus volvulus during one generation reared on artificial medium Values are themean number (n 5) of developmental stages recorded in biweekly intervals

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up to six secondary tunnels with a cumulative length of 584 073 cm Secondarytunnel length ranged between 03 and 25 cm (Fig 2A) By day 38 F1 adults largelycomprised the gallery population (220 020 mature malescolony and 2080 231mature femalescolony) (Fig 2A and 3) Despite the large adult population no eggswere observed and other developmental stages were found in low numbers (larvae26 098 larvaecolony pupae 32 357 pupaecolony) (Fig 3) Raffaelea arxii (4 of5 galleries) was the most prevalent fungal species Candida nemodendra (2 of 5galleries) Raffaelea sp PL1001 (1 of 5 galleries) and C berthetii (1 of 5 galleries) werealso recovered (Fig 2C)

The generation time (egg to fully sclerotized adult) of X volvulus was 28 days at25degC 1degC (Fig 3) The first adults leaving the gallery were observed at 30 days AFI Allcolonies had adults of both sexes The sex ratio (femalemale) was 101 The maximumnumber of males per colony was three and the minimum was two There was a positivecorrelation between the brood size and gallery length (Fig 4) but no correlation wasobserved between the size of the colony and the number of F1 adults Ninety percentof the females successfully established colonies

DISCUSSION

Xyleborus volvulus galleries consisted of simple tunnels of about 12 mm in diameterwhich is common for Xyleborus spp (4) Gallery construction by X volvulus involved theexcavation and extension of the main gallery followed by the construction of second-ary galleries where the brood developed A similar construction process was recordedfor Xylosandrus mutilatus (Blandford) (30) and Xyleborus pfeili (Ratzeburg) (31) Gallerylength has been associated with the amount of food required by the brood (30) In thepresent study a positive correlation (R2 05131) between the number of offspringand the total length of the gallery was documented (Fig 4) Kajimura and Hijii (30)suggested that oviposition is resource dependent and that food sources depend inturn on the cultivable area (gallery length) We observed that the foundress excavatesprimary and secondary galleries during the oviposition period and that larval stagesmay contribute to the expansion of secondary galleries (weeks 1 to 3) We could notdetermine whether larvae feed only on fungi (mycetophagous) or on fungi and wood(xylomycetophagous) Biedermann and Taborsky (32) observed cooperation of larvae inthe extension of galleries in Xyleborinus saxesenii Ratzeburg a species that exhibitsxylomycetophagous feeding By the initiation of the pupal period and the emergenceof new adults the gallery length remained nearly constant This was contrary to whatwas recorded by Cruz et al (33) for X bispinatus in which the new adults engaged in

FIG 4 Relationship between the cumulative gallery length and the numbers of offspring

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boring activities that resulted in significant gallery expansion These observations alsocontrast with reports of cooccurrence of all developmental stages in the galleries at 40days AFI which could indicate overlapping generations (34) Boring activity and ovi-position by the new generation of females could be restrained by the dimensions of thesubstrate or more likely be discouraged by the decline of the medium through thepresence of contaminants

The reproductive success of the beetle colony depends on the growth of theirsymbionts in the gallery system (3) Ambrosiella and Raffaelea species are known to bethe primary mutualists of ambrosia beetles During the present study three species ofRaffaelea were recovered from the galleries R arxii R rapaneae and Raffaelea spPL1001 Raffaelea lauricola has been previously reported to be associated with somepopulations of X volvulus (26 27) In this study R lauricola was not recovered from thegalleries or the offspring indicating either that the foundresses were not carrying thepathogen or that if it was present it was undetected due to its scarcity and lowfrequency Raffaelea arxii was the predominant associate in the galleries it was alwayspresent during colony development and was recovered more frequently when theadults predominated Raffaelea arxii was first reported in tunnels excavated by Xylebo-rus torquatus Eichh (a synonym of X volvulus) in Cussonia umbelliferae Sond in SouthAfrica (35) Menocal et al (34) and Saucedo-Carabez et al (36) found that R arxii wasthe most frequent and abundant associate within the mycangia of X volvulus In thecurrent study R arxii was the most frequently isolated fungal associate of larvaeteneral adults and fully sclerotized adults of X volvulus mirroring the content of thegalleries throughout the period of immature development Altogether this suggeststhat X volvulus has a strong association with R arxii which is probably its primarynutritional symbiont The association of X volvulus with other Raffaelea species wasinconsistent and included R rapaneae Raffaelea sp PL1001 and Raffaelea sp The lastof these reported here and by Saucedo-Carabez et al (36) was recovered only from thebeetle developmental stages and not detected in the galleries likely due to its lowabundance Additionally Menocal et al (34) reported variable frequencies of associa-tion with Raffaelea subalba Raffaelea fusca and Raffaelea subfusca It has been hypoth-esized that the presence of various symbionts with functional redundancy facilitatesthe survival of the beetle and its offspring under shifting environmental conditions (37)In some multipartite symbiont systems the preference for a symbiont as a nutritionalsource changes through the developmental cycle of the beetle (38) The mechanism bywhich beetles select their symbionts and prevent the spread of contaminants orunwanted fungi is unknown However the selectivity toward a specific symbiont hasbeen attributed to mycangial glandular secretions (39) and the production of specificvolatiles by the symbiont to attract the beetle (40)

Many beetle systems have been reported to include a complex of yeasts that canexceed the diversity and abundance of mutualistic fungi (34 41) We recovered nineyeast species that were able to grow in CSMA medium (06 gliter cycloheximide 03gliters streptomycin malt extract 15 agar) indicating that they are insensitive tocycloheximide a characteristic reported only for ophiostomatoid fungi (13) The fre-quency of C berthetii in galleries was greater (5 of 5 galleries) than the independentfrequencies of the Raffaelea species during the two initial weeks of the experiment Thismay imply an early establishment of this yeast species prior to the ambrosial symbi-onts Something similar has been reported in the mountain pine beetle Dendroctonusfrontalis Zimm in which larval stages were reported to be mostly associated with avariety of yeast species (42) It remains to be determined whether early yeast coloni-zation has a positive or negative impact on beetle fitness Hypothetically yeasts mayprepare the substrate for growth of mycangial fungi by metabolizing carbohydrates orterpenoids (43) Other potential roles of yeasts in the system include the following (i)providing nutrition (nitrogen and vitamins) for the beetles (ii) detoxification of plantphytochemical defenses and (iii) regulation of fungal growth by producing antagonis-tic metabolites or volatiles that affect the establishment and growth of filamentousfungi including mutualists entomopathogens and opportunistic saprophytes (43ndash45)

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Candida berthetii dominated the mycangia of sclerotized emerging adults but was notisolated from any immature stages (larvae pupae and teneral adults) This suggeststhat yeasts maybe important during the establishment of the new galleries The role ofC berthetii and other yeast as nutritional symbionts requires further investigation

Hulcr and Stelinski (46) stated that only Ambrosiozyma spp are true nutritionalsymbionts of ambrosia beetles and that other yeasts act as opportunistic organismsHowever the yeast species found in this study have been reported consistently inassociation with X volvulus and other ambrosia beetles including (i) C berthetii with Xvolvulus and Platypus externedentatus (httpwwwwesterdijkinstitutenlCollections)(ii) Ambrosiozyma monospora with X volvulus and X bispinatus (33 34) (iii) Candidalaemsonensis with Xyleborus affinis Eichhoff (47) and (iv) Candida nemodendra withXyleborus aemulus Woll (48)

In addition other yeast species were found only at the gallery entrance or on frassexpelled from the galleries by the beetles Zygozyma oligophage was originally isolatedfrom frass of the bark beetle Crossotarsus externedentatus Fairmaire (49) and wasrecently recovered from the mycangia of X volvulus (34) and Pichia mexicana waspreviously isolated from bark beetle species of the genus Dendroctonus (50) To ourknowledge this is the first report of the association of Candida sp strain BCMU BX01C californica and Pichia manshurica with an ambrosia beetle The fungal abundancesand frequencies observed in this study under artificial laboratory conditions may varyfrom the natural conditions due to the differences in the substrate including nutrientcontent and moisture However artificial medium should not affect the diversity offungal species (51) Even though foundresses were not assayed for fungal symbiontsthe fungal community of the assayed offspring did not greatly differ from wild-collected beetles reported by Saucedo-Carabez et al (36)

Similar to other ambrosia beetle members of the tribe Xyleborini X volvulus exhibitsa sib-mating reproductive system and a female-biased sex ratio which is thought to bethe result of a cryptic lifestyle in a habitat with protected food that allows the coexistenceof multiple generations (52) Overall the small number of males per colony in differentspecies of ambrosia beetles may directly reflect a highly efficient mating mechanism thatresults in high fertilization rates (2)

Kirkendall (9) postulated that in Xyleborini with inbreeding polygyny males shouldemerge first to ensure copulation before dispersal In X volvulus males did not emergestrictly before females but were present during the emergence of most females Latedevelopment of males has been observed in X pfeili (31) and X saxesenii (8) A time lagin oviposition of male eggs could be a mechanism to synchronize male and femalereproductive periods if males reach sexual maturity faster than females This time lagwould increase the efficacy of mating (8)

In summary in the present work we studied the developmental biology and fungalassociates of X volvulus Our results indicate that X volvulus has a consistent associationwith R arxii and a less frequent nonobligatory association with other Raffaelea speciesdetermined by their presence in the environment This could be the case for the fungalpathogen R lauricola which has been reported in association with X volvulus (26 36)The exact mechanisms that govern the fidelity of beetle-fungus associations are unknownVertical transmission may support cospeciation in which a symbiont is fixed into amutualistic relationship after selection based on desirable characteristics (7) Howeverhost switches are also common especially between closely related mutualists andcongener beetles (15) which could result in X volvulus acting as a secondary vector ofR lauricola

MATERIALS AND METHODSCollection and rearing of beetles Foundresses were collected from naturally infested logs from

avocado orchards located in Homestead FL (25deg29=38== N 80deg28=53== W) as reported by Cruz et al (33)Fully sclerotized X volvulus females were morphologically identified according to Rabaglia et al (24) andreared in a sterile avocado sawdust medium in 50-ml centrifuge tubes as described in Menocal et al (34)

Fifty-five live beetles were surface sterilized by immersion in 75 ethanol for 5 s to eliminatecontaminants and then individually introduced into the rearing tubes Tubes with beetles were hori-

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zontally stored within plastic containers and incubated at 25degC 1degC and with a darklight 8-h16-hphotoperiod

Colony dissection and fungal isolation from galleries and beetles Ten rearing tubes weredissected every week for 5 weeks The medium was cut along gallery tunnels and all developmentalstages as well as gallery structure and length were recorded Fungi were isolated from the galleryentrance (the oldest part of the gallery which can contain the symbionts initially inoculated by thefoundress) extruded material (frass waste material resulting from gallery maintenance) and tunnels(where developmental stages were enclosed and presumably the nutritional symbionts) using a sterileneedle (one sample per location) The colony samples were streaked on malt extractndash1 agar amendedwith 06 gliter of cycloheximide and 03 gliter streptomycin (CSMA) (13) and on potato dextrose agaramended with 01 gliter of streptomycin (PDA)

Pure fungal isolates were obtained by a single spore culture and identified as described in Cruz et al(33) Briefly DNA was obtained by a modified cetyl trimethyl-ammonium bromide (CTAB) protocol (53)and portions of the nuclear large subunit 28S ribosomal DNA (rDNA) using primers LR0RLR5 (54) and thesmall subunit rDNA with primers NS1NS4 (55) were amplified PCR products were purified usingExoSAP-IT (Affymetrix CA USA) and sequenced in both directions The NCBI Basic Local AlignmentSearch Tool (BLAST) was used to identify the nucleotide sequences

A sample of five individuals of each developmental stage (eggs larvae pupae teneral adults andadults) were collected from five additional colony tubes surface disinfected and processed for symbiontisolation and identification Additionally teneral adults and F1 offspring adult females were asepticallyexcised and separately processed to isolate the symbionts associated with either the mycangium (heads)or the gut (abdomen) Identification was carried out using the primers listed above as described in Cruzet al (33)

ACKNOWLEDGMENTSWe thank Joshua Konkol (University of Florida) and Akif Eskalen (University of

California Davis) for suggestions to improve the manuscript We especially thank RandyFernandez for the beetle illustrations We thank Jose Alegriacutea and Rita E Duncan for theexperimental setup

This research was funded by NIFA grant 2015-51181-24257 to Daniel Carrillo

REFERENCES1 Farrell BD Sequeira AS OrsquoMeara BC Normark BB Chung JH Jordal BH

2001 The evolution of agriculture in beetles (Curculionidae Scolytinaeand Platypodinae) Evolution 552011ndash2027 httpsdoiorg101111j0014-38202001tb01318x

2 Kirkendall L Biedermann PH Jordal BH 2015 Evolution and diversity ofbark and ambrosia beetles p 85ndash156 In Vega FE Hofstetter RW (ed)Bark beetles biology and ecology of native and invasive speciesElsevier San Diego CA

3 Beaver RA 1989 Insect-fungus relationships in the bark and ambrosiabeetles p 121ndash143 In Wilding N Collins NM Hammond PM Webber JF (ed)Insect-fungus interaction Academic Press London United Kingdom

4 Maner ML Hanula JL Braman K 2013 Rearing redbay ambrosia beetleXyleborus glabratus (Coleoptera Curculionidae Scolytinae) on semi-artificial media Florida Entomol 961042ndash1051 httpsdoiorg1016530240960343

5 Peer K Taborsky M 2007 Delayed dispersal as a potential route tocooperative breeding in ambrosia beetles Behav Ecol Sociobiol 61729 ndash739 httpsdoiorg101007s00265-006-0303-0

6 Six DL 2003 Bark beetle-fungus symbioses p 97ndash114 In Bourtzis K MillerTA (ed) Insect symbiosis insect symbiosis CRC Press Boca Raton FL

7 Six DL 2012 Ecological and evolutionary determinants of barkbeetle-fungus symbioses Insects 3339 ndash366 httpsdoiorg103390insects3010339

8 Biedermann P 2010 Observations on sex ratio and behavior of males inXyleborinus saxesenii Ratzeburg (Scolytinae Coleoptera) Zookeys 56253ndash267 httpsdoiorg103897zookeys56530

9 Kirkendall LR 1983 The evolution of mating systems in bark and am-brosia beetles (Coleoptera Scolytidae and Platypodidae) Zool J Linn Soc77293ndash352 httpsdoiorg101111j1096-36421983tb00858x

10 Kirkendall LR Kent DS Raffa KF 2010 Interactions among males femalesand offspring in bark and ambrosia beetles the significance of living intunnels for the evolution of social behavior p 181ndash215 In Choe JCCrespi BJ (ed) The evolution of social behavior in insects and arachnidsCambridge University Press Cambridge United Kingdom

11 Biedermann PHW Klepzig KD Taborsky M 2009 Fungus cultivation byambrosia beetles behavior and laboratory breeding success in three

Xyleborine species Environ Entomol 381096 ndash1105 httpsdoiorg1016030220380417

12 Mueller UG Gerardo NM Aanen DK Six DL Schultz TR 2005 Theevolution of agriculture in insects Annu Rev Ecol Evol Syst 36563ndash595httpsdoiorg101146annurevecolsys36102003152626

13 Harrington TC Aghayeva DN Fraedrich SW 2010 New combinations inRaffaelea Ambrosiella and Hyalorhinocladiella and four new speciesfrom the redbay ambrosia beetle Xyleborus glabratus Mycotaxon 111337ndash361 httpsdoiorg105248111337

14 Bracewell RR Six DL 2015 Experimental evidence of bark beetle adap-tation to a fungal symbiont Ecol Evol 55109 ndash5119 httpsdoiorg101002ece31772

15 Skelton J Johnson AJ Jusino MA Bateman CC Li Y Hulcr J 2019 Aselective fungal transport organ (mycangium) maintains coarse phylo-genetic congruence between fungus-farming ambrosia beetles and theirsymbionts Proc Biol Sci 28620182127 httpsdoiorg101098rspb20182127

16 Hulcr J Dunn RR 2011 The sudden emergence of pathogenicity ininsect-fungus symbioses threatens naive forest ecosystems Proc Biol Sci2782866 ndash2873 httpsdoiorg101098rspb20111130

17 Freeman S Sharon M Maymon M Mendel Z Protasov A Aoki T EskalenA OrsquoDonnell K 2013 Fusarium euwallaceae sp novndasha symbiotic fungusof Euwallacea sp an invasive ambrosia beetle in Israel and CaliforniaMycologia 1051595ndash1606 httpsdoiorg10385213-066

18 Carrillo D Cruz L Kendra P Narvaez T Montgomery W Monterroso A DeGrave C Cooperband M 2016 Distribution pest status and fungalassociates of Euwallacea nr fornicatus in Florida avocado groves Insects755 httpsdoiorg103390insects7040055

19 Lynch SC Twizeyimana M Mayorquin JS Wang DH Na F Kayim MKasson MT Thu PQ Bateman C Rugman-Jones P Hulcr J Stouthamer REskalen A 2016 Identification pathogenicity and abundance of Para-cremonium pembeum sp nov and Graphium euwallaceae of thepolyphagous shot hole borer (Euwallacea sp) in California Mycologia108313ndash329 httpsdoiorg10385215-063

20 Gomez DF Skelton J Steininger MS Stouthamer R Rugman-Jones PSittichaya W Rabaglia RJ Hulcr J 2018 Species delineation within theEuwallacea fornicatus (Coleoptera Curculionidae) complex revealed by

Cruz et al Applied and Environmental Microbiology

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morphometric and phylogenetic analyses Insect Syst Divers httpsdoiorg101093isdixy018

21 Brasier CM Kirk SA 2010 Rapid emergence of hybrids between the twosubspecies of Ophiostoma novo-ulmi with a high level of pathogenicfitness Plant Pathol 59186 ndash199 httpsdoiorg101111j1365-3059200902157x

22 Fraedrich SW Harrington TC Rabaglia RJ Ulyshen MD Mayfield AEHanula JL Eickwort JM Miller DR 2008 A fungal symbiont of the redbayambrosia beetle causes a lethal wilt in redbay and other Lauraceae in thesoutheastern United States Plant Dis 92215ndash224 httpsdoiorg101094PDIS-92-2-0215

23 Hughes MA Smith JA Ploetz RC Kendra PE Mayfield AE Hanula JLHulcr J Stelinski LL Cameron S Riggins JJ Carrillo D Rabaglia REickwort J Pernas T 2015 Recovery plan for laurel wilt on redbay andother forest species caused by Raffaelea lauricola and disseminated byXyleborus glabratus Plant Heal Prog 16173ndash210 httpsdoiorg101094PHP-RP-15-0017

24 Rabaglia RJ Dole SA Cognato AI 2006 Review of American Xyleborina(Coleoptera Curculionidae Scolytinae) occurring North of Mexico withan illustrated key Ann Entomol Soc Am 991034 ndash1056 httpsdoiorg1016030013-8746(2006)99[1034ROAXCC]20CO2

25 Evans EA Crane J Hodges A Osborne JL 2010 Potential economicimpact of laurel wilt disease on the Florida avocado industry Horttech-nology 20234 ndash238 httpsdoiorg1021273HORTTECH201234

26 Carrillo D Duncan RE Ploetz JN Campbell AF Ploetz RC Pentildea JE 2014Lateral transfer of a phytopathogenic symbiont among native and exoticambrosia beetles Plant Pathol 6354 ndash 62 httpsdoiorg101111ppa12073

27 Ploetz RC Konkol JL Narvaez T Duncan RE Saucedo RJ Campbell AMantilla J Carrillo D Kendra PE 2017 Presence and prevalence ofRaffaelea lauricola cause of laurel wilt in different species of ambrosiabeetle in Florida USA J Econ Entomol 110347ndash354 httpsdoiorg101093jeetow292

28 Wood SL 1982 The bark and ambrosia beetles of North and CentralAmerica (Coleoptera Scolytidae) a taxonomic monograph Great BasinNaturalist Mem 61ndash1359

29 Gohli J Selvarajah T Kirkendall LR Jordal BH 2016 Globally distributedXyleborus species reveal recurrent intercontinental dispersal in a land-scape of ancient worldwide distributions Evol Biol 1637

30 Kajimura H Hijii N 1994 Reproduction and resource utilization of theambrosia beetle Xylosandrus mutilatus in field and experimental pop-ulations Entomol Exp Appl 71121ndash132 httpsdoiorg101111j1570-74581994tb01778x

31 Mizuno T Kajimura H 2002 Reproduction of the ambrosia beetle Xyleboruspfeili (Ratzeburg) (Col Scolytidae) on semi-artificial diet J Appl Entomol126455ndash462 httpsdoiorg101046j1439-0418200200691x

32 Biedermann PHW Taborsky M 2011 Larval helpers and age polyethismin ambrosia beetles Proc Natl Acad Sci U S A 10817064 ndash17069 httpsdoiorg101073pnas1107758108

33 Cruz LF Rocio SA Duran LG Menocal O Garcia-Avila CDJ Carrillo D2018 Developmental biology of Xyleborus bispinatus (Coleoptera Cur-culionidae) reared on an artificial medium and fungal cultivation ofsymbiotic fungi in the beetlersquos galleries Fungal Ecol 35116 ndash126 httpsdoiorg101016jfuneco201807007

34 Menocal O Cruz LF Kendra PE Crane JH Ploetz RC Carrillo D 2017Rearing Xyleborus volvulus (Coleoptera Curculionidae) on media con-taining sawdust from avocado or silkbay with or without Raffaelealauricola (Ophiostomatales Ophiostomataceae) Environ Entomol 461275ndash1283 httpsdoiorg101093eenvx151

35 Scott DB Du Toit JW 1970 Three new Raffaelea species Trans Br MycolSoc 55181ndash186 httpsdoiorg101016S0007-1536(70)80002-X

36 Saucedo-Carabez JR Ploetz RC Konkol JL Carrillo D Gazis R 2018Partnerships between ambrosia beetles and fungi lineage-specific pro-miscuity among vectors of the laurel wilt pathogen Raffaelea lauricolaMicrob Ecol 76925ndash940 httpsdoiorg101007s00248-018-1188-y

37 Six DL Bentz BJ 2007 Temperature determines symbiont abundance in

a multipartite bark beetle-fungus ectosymbiosis Microb Ecol 54112ndash118httpsdoiorg101007s00248-006-9178-x

38 Freeman S Sharon M Dori-Bachash M Maymon M Belausov E Maoz YMargalit O Protasov A Mendel Z 2016 Symbiotic association of threefungal species throughout the life cycle of the ambrosia beetle Euwal-lacea nr fornicatus Symbiosis 68115ndash128 httpsdoiorg101007s13199-015-0356-9

39 Yuceer C Hsu C-Y Erbilgin N Klepzig KD 2011 Ultrastructure of themycangium of the southern pine beetle Dendroctonus frontalis (ColeopteraCurculionidae Scolytinae) complex morphology for complex interactionsActa Zool 92216ndash224 httpsdoiorg101111j1463-6395201100500x

40 Hulcr J Mann R Stelinski LL 2011 The scent of a partner ambrosiabeetles are attracted to volatiles from their fungal symbionts J ChemEcol 371374 ndash1377 httpsdoiorg101007s10886-011-0046-x

41 Endoh R Suzuki M Okada G Takeuchi Y Futai K 2011 Fungus symbi-onts colonizing the galleries of the ambrosia beetle Platypus quercivorusMicrob Ecol 62106 ndash120 httpsdoiorg101007s00248-011-9838-3

42 Bridges JR Marler JE Mcsparrin BH 2009 A quantitative study of theyeasts and bacteria associated with laboratory-reared Dendroctonusfrontalis Zimm (Coleopt Scolytidae) Z Angew Entomol 97261ndash267httpsdoiorg101111j1439-04181984tb03747x

43 Davis TS 2015 The ecology of yeasts in the bark beetle holobiont acentury of research revisited Microb Ecol 69723ndash732 httpsdoiorg101007s00248-014-0479-1

44 Adams AS Six DL Adams SM Holben WE 2008 In vitro interactionsbetween yeasts and bacteria and the fungal symbionts of the mountainpine beetle (Dendroctonus ponderosae) Microb Ecol 56460 ndash 466 httpsdoiorg101007s00248-008-9364-0

45 Davis TS Hofstetter RW Foster JT Foote NE Keim P 2011 Interactionsbetween the yeast Ogataea pini and filamentous fungi associated withthe western pine beetle Microb Ecol 61626 ndash 634 httpsdoiorg101007s00248-010-9773-8

46 Hulcr J Stelinski LL 2017 The ambrosia symbiosis from evolutionary ecol-ogy to practical management Annu Rev Entomol 62285ndash303 httpsdoiorg101146annurev-ento-031616-035105

47 Kostovcik M Bateman CC Kolarik M Stelinski LL Jordal BH Hulcr J 2015The ambrosia symbiosis is specific in some species and promiscuous inothers evidence from community pyrosequencing ISME J 9126 ndash138httpsdoiorg101038ismej2014115

48 van der Walt JP Scott DB van der Klift WC 1971 Five new Torulopsisspecies from South African insect sources Antonie Van Leeuwenhoek37461ndash 471 httpsdoiorg101007BF02218516

49 van der Walt JP von Arx JA Ferreira NP Richards P 1987 Zygozyma gennov a new genus of the Lipomycetaceae Syst Appl Microbiol9115ndash120 httpsdoiorg101016S0723-2020(87)80064-4

50 Rivera FN Gonzalez E Gomez Z Lopez N Hernandez-Rodriguez CBerkov A Zuniga G 2009 Gut-associated yeast in bark beetles of thegenus Dendroctonus erichson (Coleoptera Curculionidae Scolytinae)Biol J Linn Soc 98325ndash342 httpsdoiorg101111j1095-8312200901289x

51 Biedermann PHW Klepzig KD Taborsky M Six DL 2013 Abundance anddynamics of filamentous fungi in the complex ambrosia gardens of theprimitively eusocial beetle Xyleborinus saxesenii Ratzeburg (ColeopteraCurculionidae Scolytinae) FEMS Microbiol Ecol 83711ndash723 httpsdoiorg1011111574-694112026

52 Hamilton WD 1978 Evolution and diversity under bark p 154 ndash175 InMound LA Waloff N (ed) Diversity of insect faunas London UnitedKingdom

53 Doyle JJ Doyle JL 1987 A rapid DNA isolation procedure for smallquantities of fresh leaf tissue Phytochem Bull 1911ndash15

54 Vilgalys R Hester M 1990 Rapid genetic identification and mapping ofenzymatically amplified ribosomal DNA from several Cryptococcus spe-cies J Bacteriol 1724238 ndash 4246

55 White TJ Bruns T Lee S Taylor JW 1990 Amplification and direct sequenc-ing of fungal ribosomal RNA genes for phylogenetics p 315ndash322 In InnisMA Gelfand DH Sninsky JJ White TJ (ed) PCR protocols a guide tomethods and applications Academic Press San Diego CA

Xyleborus volvulus Biology and Fungal Associates Applied and Environmental Microbiology

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