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Fungal Ecology 35 (2018) 116e126

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Fungal Ecology

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Developmental biology of Xyleborus bispinatus (Coleoptera:Curculionidae) reared on an artificial medium and fungal cultivationof symbiotic fungi in the beetle's galleries

L.F. Cruz a, *, S.A. Rocio a, b, L.G. Duran a, b, O. Menocal a, C.D.J. Garcia-Avila c, D. Carrillo a

a Tropical Research and Education Center, University of Florida, 18905 SW 280th St, Homestead, 33031, FL, USAb Universidad Aut�onoma Chapingo, Km 38.5 Carretera M�exico - Texcoco, Chapingo, M�ex, 56230, Mexicoc Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, Unidad Integral de Diagn�ostico, Servicios y Constataci�on, Tec�amac, 55740, Estado deM�exico, Mexico

a r t i c l e i n f o

Article history:Received 10 January 2018Received in revised form10 July 2018Accepted 12 July 2018Available online 23 August 2018

Corresponding Editor: Peter Biedermann

Keywords:Ambrosia beetleAmbrosia fungiArtificial rearingAvocadoFungal farmingLaurel wiltRaffaelea speciesScolytinaeSymbiosis

* Corresponding author.E-mail address: luisafcruz@ufl.edu (L.F. Cruz).

https://doi.org/10.1016/j.funeco.2018.07.0071754-5048/© 2018 Elsevier Ltd and British Mycologic

a b s t r a c t

Survival of ambrosia beetles relies on obligate nutritional relationships with fungal symbionts that arecultivated in tunnels excavated in the sapwood of their host trees. The dynamics of fungal associates,along with the developmental biology, and gallery construction of the ambrosia beetle Xyleborus bispi-natus were elaborated. One generation of this ambrosia beetle was reared in an artificial medium con-taining avocado sawdust. The developmental time from egg to adult ranged from 22 to 24 d. The meantotal gallery length (14.4 cm and 13 tunnels) positively correlated with the number of adults. The mostprevalent fungal associates were Raffaelea arxii in the foundress mycangia and new galleries, and Raf-faelea subfusca in the mycangia of the F1 adults and the final stages of the galleries. Raffaelea sp. PL1001,Raffaelea subalba and seven yeast species were also recovered. These results indicate flexibility in theassociation between X. bispinatus and its symbiont species, and that this beetle may use more than oneRaffaelea species as a food source. These results are important, as X. bispinatus has been associated withthe transmission of Raffaelea lauricola, the causal agent of laurel wilt, a lethal disease affecting avocadotrees.

© 2018 Elsevier Ltd and British Mycological Society. All rights reserved.

1. Introduction

Through coevolution, a symbiotic nutritional relationship hasarisen between ambrosia beetles (Coleoptera: Curculionidae: Sco-lytinae) and ambrosia fungi (Farrell et al., 2001; Mueller et al.,2005). In this mutualistic interaction, ambrosia beetles proliferatein the low nutrient environment within the sapwood of their hosttrees (Bleiker et al., 2009), while concurrently ambrosia fungiachieve their permanence and dissemination by being acquired andtransported by beetles (Six, 2012). The establishment of the fungalgarden is critically important to the reproductive success of each ofthe newly founded colonies (Biedermann et al., 2009, 2013). Theseambrosia gardens, however, contain a variety of bacteria, yeasts,

al Society. All rights reserved.

and filamentous fungi in addition to ambrosia fungi (Haanstad andNorris, 1985).

Ambrosia beetles can have one or multiple ambrosia fungi(Bateman et al., 2015; Kostovcik et al., 2015). Most fungal symbiontsof ambrosia beetles belong to the phylum Ascomycota (Ascomy-cota: Sordariomycetes), in the orders Ophiostomatales and Micro-ascales, and the species are classified in the anamorph generaAmbrosiella (Microascales: Ceratocystidaceae) and Raffaelea(Ophiostomatales: Ophiostomataceae) (Dreaden et al., 2014a;Dreaden et al., 2014b; Mayers et al., 2015). Generally, these fungalsymbionts are saprobes of stressed, dying, and dead trees (Beaver,1989; Kirkendall et al., 1997). Exotic, invasive ambrosia beetlespecies may carry plant pathogens among their symbionts, whichmay be very damaging pests of healthy trees (Hulcr et al., 2011).

Ambrosia symbiont spores are carried by female beetles in aspecialized cuticular pocket, termed a mycangium, the anatomicallocation of which varies among beetle species, for example, in the

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Xyleborini, it may be mandibular, mesonotal or elytral (Six, 2003;Hulcr and Stelinski, 2017), and in Xyleborus bispinatus it ismandibular. The spores are inoculated during tunnel excavation(Six, 2012). The immature stages develop feeding on fungi and thenew generation of adults fill their mycangia via feeding in the natalgalleries before dispersing (Li et al., 2018). These transmissionprocesses strengthen the link and fidelity between the fungalsymbionts and the beetles (Six, 2012). Although the mycangiaensure reliable fungus transmission, the interaction of sympatricspecies co-occurring in the same host tree allows horizontalacquisition of new symbionts (Carrillo et al., 2014).

Horizontal movement of symbionts plays an important role inthe epidemiology of laurel wilt in avocado trees (Persea americana),a lethal disease of the Lauraceae, caused by the fungal pathogenRaffaelea lauricola (Ophiostomatales: Ophiostomataceae), andmainly transmitted by the Asian redbay ambrosia beetle, Xyleborusglabratus (Curculionidae: Scolytinae) (Rabaglia et al., 2006;Fraedrich et al., 2008). However, X. glabratus has not been detectedbreeding in avocado trees (Carrillo et al., 2012). Instead, nine otherspecies of ambrosia beetles that breed in avocado, including X.bispinatus, carry the pathogen and perhaps act as an alternativevector (Carrillo et al., 2014; Ploetz et al., 2017).

Despite the knowledge gained to date, regarding the interactionof X. bispinatus with R. lauricola (Carrillo et al., 2014; Saucedo et al.,2017; Menocal et al., 2018). There is a dearth of information on thebasic biology of this beetle species and the mutualistic fungal as-sociations during its life cycle. Moreover, due to their cryptic lifestyle, the study of ambrosia beetles in their native environment isextremely difficult. Nevertheless, the development of artificialsubstrates has facilitated the study of this taxonomic group underlaboratory conditions (Mizuno et al., 1997; Mizuno and Kajimura,2002, 2009; Biedermann et al., 2009; Castrillo et al., 2012; Maneret al., 2013; Cooperband et al., 2016; Menocal et al., 2017). Theprimary goal of the current study was to gain insight into thebiology of X. bispinatus and its fungal associates. The specific ob-jectives were to: (1) elucidate the beetle's life cycle and its devel-opmental stages, (2) determine the process of gallery construction,(3) survey the fungal repertoire associated with the galleries andwith different developmental stages of X. bispinatus reared in anartificial medium, and (4) explore the possible existence of antag-onistic interactions of the X. bispinatus symbionts againstR. lauricola. The findings of this study will establish whether theassociation of X. bispinatus with its fungal symbionts is species-specific or multipartite and flexible throughout its life cycle.

2. Materials and methods

2.1. Collection of beetles

X. bispinatus is a neotropical species, distributed throughoutSouth America, Central America, and along southeastern NorthAmerican coast (Fraedrich et al., 2008). Previously, X. bispinatuswasundistinguished from Xyleborus ferrugineus; however, these twospecies were later separated based on differences in morphologicalcharacters (Atkinson et al., 2013). For the current study, fullysclerotized X. bispinatus females were collected from avocado logsshowing signs of active infestation (i.e., excavated materialextruded from the beetle galleries). Logs were collected in January2017 from an avocado grove in Homestead, Florida, USA (25� 490

4500 N, 80� 480 0900 W) and kept in emergence chambers, asdescribed in Carrillo et al. (2012), i.e., Rubbermaid 2643-60 BRUTE166 L containers made of opaque plastic each with two 8.3 cmdiameter holes cut into opposite sides one near to the top and theother near to the bottom of the container. Into each of the holes a0.9146 L clear Mason jar without a lid was fitted with the bottom of

the jar oriented outward from the main chamber. This was done bygluing the threadedmetal band into the hole and then screwing thetop of the jar into the metal band. A moistened paper towel wasplace into the jar. Infested avocado log segments were placed intoeach container and its top was closed tightly with an opaque lid.After 1 week, dark brown females (fully sclerotized) emerged fromthe logs, and they were collected daily from the clear jars intowhich they had been attracted by the light. These females wereidentified under a stereoscopewithout immobilization according toRabaglia et al. (2006), and the X. bispinatus specimens wereretained for use in the experiments.

2.2. Artificial medium and rearing conditions

Sawdust was produced following the methodology described byCastrillo et al. (2011). Briefly, healthy avocado logs (without anysign of beetle infestation) from an avocado orchard in Miami-DadeCounty (25� 290 3800 N, 80� 28’ 5300 W) were debarked and dried at75 �C for 4 days. Sawdust was obtained from the xylem sapwoodlayer using a sander. Large particles of sawdust were eliminatedusing a 12mm sieve. Medium preparation was carried out ac-cording to Castrillo et al. (2011). The medium consisted of 75 g ofavocado sawdust, 20 g agar, 10 g sucrose, 5 g starch, 5 g casein, 5 gyeast, 1 g Wesson salt mixture, 0.35 g streptomycin, 5mL 95%ethanol, 2.5mL wheat germ oil, and 500mL distilled water. Solidingredients were first homogenized, and then incorporated withthe liquid materials. The medium was autoclaved at 121 �C and 15PSI for 30min; mixed thoroughly to re-suspend settled ingredients,and 15mL was poured into each 50mL polypropylene tube underaseptic conditions. The tubes were loosely capped and the mediumwas allowed to dry and solidify for 1 week under a laminar flowhood (Castrillo et al., 2011).

Prior to infesting the medium in the tubes, the beetles weresurface sterilized by immersing for 5 sec in 75% ethanol to eliminatesurface contaminants. The medium was perforated with a sterileneedle to facilitate initial boring. One female was introduced intoeach of sixty tubes. Tubes with infested medium were incubatedhorizontally at 25± 1 �C under a LD 16:8 h photocycle for 38 d.

2.3. Medium dissection and fungal isolation from galleries

Fifty-five tubes containing successful colonies were processed inthis study. Dissection of the medium was performed twice a weekfor 5 weeks. Five tubes were randomly selected each time (i.e., 10tubes per week) and dissected under the stereoscope. The mediumplugs were carefully drawn out of the tube and dissected along thegallery tunnels. Gallery length was measured, quantitative de-scriptions of the structures of the main and secondary gallerieswere recorded, and beetle developmental stages were located andcounted. For fungal isolation, samples from those gallery wallsoccupied by immature beetle stages, the gallery entrance, and theextruded frass were acquired once aweek with a sterile needle, andstreaked on plates of cycloheximide-streptomycin-malt agar(CSMA) a semi-selective medium for ophiostomatoid fungi(Harrington et al., 2010), and potato dextrose agar amended with0.2 g/L streptomycin (PDAþ). These plates were incubated 7e10 d atroom temperature.

2.4. Symbiont isolation from beetles in different developmentalstages

Six adult females from the initial foundress group, five larvae,five tenerals, and five fully sclerotized females F1 progeny, werecollected when first observed while dissecting the colonized me-dium. Each individual obtained from a different colony was surface

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sterilized with 75% ethanol for one minute and subsequentlywashed three times with sterile water. Heads and bodies of adultfemales, both foundresses and F1 offspring, were aseptically excisedand separately processed to isolate the symbionts associated witheither the mycangium or the gut. Heads and bodies were individ-ually macerated in 400 mL deionized sterile water; 100 mL sub-sample were plated on CSMA, and an additional 100 mL subsamplewas plated on PDAþ. Plates were incubated at room temperature for7e10 d, until fungal colonies were visible and distinguishable.

2.5. Identification of isolates

Isolates were initially selected for subculturing based on colonymorphology. A representative of each morphotype was transferredto a fresh Petri dish containing either PDAþ or CSMA. Afterobtaining pure fungal cultures from the different morphotypes d

each from a single spore d DNA was isolated from mycelia and/orspores using a modified cetyl trimethyl-ammonium bromide(CTAB) protocol (Doyle and Doyle, 1987). The fungal isolates weretyped by sequencing a section of the nuclear large subunit 28S ri-bosomal DNA (rDNA) using primers LR0R/LR5 (Vilgalys and Hester,1990) and the small subunit rDNA with primers NS1/NS4 (Whiteet al., 1990). PCR products were purified using ExoSAP-IT (Affime-trix, CA, USA) following the manufacturer's protocols and Sangersequencing was performed by Eurofins genomics (Louisville, KY).The NCBI Basic Local Alignment Search Tool (BLAST) was used toidentify the nucleotide sequences.

2.6. Antagonistic assays of the X. bispinatus fungal symbiontsversus R. lauricola

In the present study, the pathogen R. lauricola was not detectedin galleries and the different developmental stages of the beetle. Todetermine if the absence of R. lauricolawas related to the ability ofany of the X. bispinatus fungal associates to inhibit the growth of thepathogen, a dual culture technique previously described byCampanile et al. (2007) was deployed to test antagonistic activity.An isolate of R. lauricola was recovered from an infected avocadotree and identified with the primer sets previously reported byDreaden et al. (2014a). Agar disks (5mm diam) obtained from 2week-old pure fungal cultures of the symbionts and from thepathogen were transferred onto Petri dishes each containing eitherCSMA or the beetle rearing medium. Each disk was placed 3 cmfrom a R. lauricola disk. These dishes were incubated at roomtemperature for 2 weeks. The experiment was repeated three times

Table 1Fungal isolates: identity and frequency of isolation from gallery entrances, tunnels and p

Fungal species Closest relative GeneBank accession no

LSU (% Similarity) SSU (

Raffaelea subfusca KR018422 (100) KJ90Raffaelea arxii KR018419 (99) EU17Raffaelea subalba KX267102 (100) KJ90Raffaelea sp. PL1001 KJ909293 (99) KJ90Candida nemodendra NG055146 (99) EU01Candida berthetii KY106320 (98) AB05Candida sp. NRRL Y-27127 EF550293 (98) EF55Saccharomycopsis synnaedendra EU057559 (99) EU05Ambrosiozyma monospora EU011590 (99) JQ69Ambrosiozyma ambrosiae EU011593 (99) EU01Alloascoidea spa JQ689066 (93) JQ69Bionectria ochroleuca AY686634 (99) GU11Stilbocrea macrostomab GQ506004 (99) AY48

a Closest match Alloascoidea africana.b Only recovered from foundresses gut.

and three dishes were used for each isolate each time. The exper-iment was performed for 13 isolates recovered from galleries andthe beetle's developmental stages (Table 1). The isolate's suppres-sive effect was determined by calculating the percentage of inhi-bition in radial growth according to Gaigole et al. (2011).

3. Results

3.1. Life cycle and developmental stages of X. bispinatus

In the artificial medium, eggs were first observed at 4e7 d aftercolony initiation (DACI), with a maximum number at 11 DACI. Eggswere frequently found as clusters, with a maximum number of 24eggs located mostly at the distal ends of the secondary galleries(Figs. 1 and 2D). Larvae (all instars) were first observed 8e10 DACI;the maximum number was observed 18 DACI. They appeared ingroups of up to 15 individuals mostly in the secondary galleries(Figs. 1 and 2D). Pupae were first observed 15e17 DACI with a peaknumber at 25 DACI. They were distributed irregularly in main andsecondary galleries (Figs. 1 and 2D). Female tenerals were firstobserved 18e21 DACI, with a maximum number at day 28, whilemales were observed 22e24 DACI with a maximum number at 33days. Tenerals were distributed somewhat irregularly in main andsecondary galleries (Figs. 1 and 2D). Fully sclerotized F1 generationfemale and male adults were observed at days 22e24 and 29e31respectively, with a maximum number at 35 days for both (Fig. 1).The developmental time from egg to adult was 22e24 d (Fig. 1).Eggs were white, translucent, and oval shaped. Larvae were cur-culioniform, legless, cream colored, and with a white head capsule.Pupae were white and motionless. The first mature fertilized fe-male was recorded emerging through the surface of the mediumduring 24e25 DACI. By the end of the experiment (38 DACI), themale:female ratio was 1:6 and one of the five colonies did notcontain any males. The mean number of individuals per develop-mental stage ±SD at 38 d was as follows: 2.2± 2.16 larvae; 0.6± 1.3pupae; 2± 2.1 teneral males; 6.2± 4.5 teneral females; 0.4± 0.54adult males; and 8± 7 adult females. No eggs were observed duringthe dissection of the medium at 38 d.

3.2. Construction of the gallery system

During the first week, each foundress female excavated themain gallery in parallel to the walls of the tube, and, it reached anaverage length of 1.48 cm after 1 week (Fig. 2A and B). Extension ofthe length of the main gallery continued for 4 weeks to reach an

rotruding material (frass) during the entire study (n¼ 55).

. Frequency of isolation per location (n¼ 25) (%)

% Similarity) Frass Entrance Tunnel

9306 (99) 72 60 720279 (99) 68 76 529304 (99) 0 16 329294 (99) 4 24 121709 (99) 32 32 284883 (99) 36 56 480431 (99) 28 32 367527 (98) 8 8 48881 (99) 40 32 321673 (99) 28 24 648925 (92) 64 56 602755 (99) 36 32 329693 (99)

Fig. 1. Developmental stages of X. bispinatus reared in avocado sawdust based artificialmedium at 25 �C. Mean number of individuals in the different developmental stagesrecorded twice per week (n¼ 5).

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 119

average length of 3.2 cm. After 1 week, the female foundresses hadstarted construction of secondary galleries near the middle or theend of the main tunnel. The aggregate length of the secondarygalleries increased gradually with brood size (Fig. 2A). Ovipositionstarted with the construction of the first secondary gallery, andpeak oviposition was observed after 1e2 weeks when the averagenumber of secondary galleries was three with a mean length of2.3 cm. During the larval and pupal periods, i.e., 3e4 weeks, thefoundresses continued to extend existing galleries and excavatenew ones, which measured 6.2e7.48 cm in length. The fastestgrowth of the gallery system coincided with the appearance of the(F1) adults after 4e5 weeks when the galleries reached their finalsizes (Fig. 2B). A positive correlation (R2¼ 0.9792) was found be-tween the number of adults and the cumulative length of second-ary galleries (Fig. 2C). By the end of the experiment at 38 DACI, thegallery system, in the artificial medium, consisted of a main tunnel

Fig. 2. Xyleborus bispinatus gallery system construction in avocado sawdust based artificiaGallery construction process related to brood development. (C) Mean number of adults plotand location of beetle developmental stages: *egg clusters, : larvae groups, A pupae,

excavated perpendicular to the surface of the medium, with anaverage length of 3.2 cm and up to 13 secondary galleries with acumulative average length of 14.44 cm (Fig. 2D). In a few cases,tertiary galleries extending from secondary galleries were observed(Fig. 2D).

3.3. Frequencies of fungal species in extruded frass, at tunnelentrances and on tunnel walls

Nucleotide sequencing of fungal isolates recovered from gallerywalls, gallery entrances, and extruded frass (Table 1) revealed thepresence of four Raffaelea species (Raffaelea subfusca, Raffaelea arxii,Raffaelea subalba, Raffaelea sp. PL1001), seven yeast-like species(Candida nemodendra, Candida berthetii, Candida sp. NRRL Y-27127,Saccharomycopsis synnaedendra, Ambrosiozyma monospora,Ambrosiozyma ambrosiae, Alloascoidea sp.) and two bionectraceousfungi (Stilbocrea macrostoma and Bionectria ochroleuca) (Table 1).R. subfuscawas the most abundant species recovered in the gallerytunnel in 72% of the tubes (n¼ 25), followed by R. arxii found in 52%of the colonies. Among the yeast species, A. ambrosiae (64%) andC. berthetii (48%) were themost frequently isolated ones. All isolateswere recovered from all three locations, with exception ofR. subalba, which was found at the entrances and within the gal-leries, but not in the extruded frass material (Table 1).

3.4. Symbiont species composition of fungal gardens during beetledevelopment

After 7 d, when the foundresses started laying eggs, R. arxii wasthe most frequent associate in the freshly excavated galleries, beingpresent in four of five (4/5) colonies tested at this time, whileR. subfuscawas recovered at the low frequencies of 1/5. In contrast,neither R. subalba nor R. sp. PL1001 were detected (Fig. 3). Yeast-like organisms found in the ambrosia gardens of 7 d includedA. ambrosiae (5/5) Alloascoidea sp. (4/5), C. berthetii (3/5), andC. nemodendra (2/5) (Fig. 3).

After 14 d, when larvae were the predominant developmental

l medium. (A) Mean (n ¼ 5) length of primary and secondary galleries over time. (B)ted against gallery length. (D) Schematic representation of gallery construction weeklytenerals, Cmature female.

Fig. 3. Frequency of isolations of the fungal associates recovered from the gallery tunnels once per week (n¼ 5) during five weeks.

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126120

stage in the galleries, R. arxii continued to be the most frequentfungal associate, found in five of five galleries. R. subfuscawas foundat a higher frequency (4/5) than during the previous week. Incontrast, R. subalba and R. sp. PL1001 were not detected at 14 d(Fig. 3). In addition to the yeast species recovered at 7 d, threespecies: Candida sp. NRRL Y-27127 (2/5), S. synnaedendra (1/5) andA. monospora (2/5) were now also observed growing in the tunnels.C. berthetii and Alloascoidea sp. were the most frequent yeast spe-cies (5/5) (Fig. 3).

After 3 weeks (21 DACI), when pupae started to appear, R. arxiiwas not detected in the galleries. However, R. subfuscawas found inall galleries (5/5) and R. subalba was found for the first time, beingpresent in four of five galleries. However, Raffaelea sp. PL1001 wasstill not detected (Fig. 3). Also, B. ochroleuca was recovered for thefirst time from the galleries (2/5). With exception of C. berthetii andS. synnaedendra, all the yeast species that were found at 14 d were

also recovered at 21 d. Among them, A. ambrosiae was the pre-dominant species being found in four of five galleries.

After 4 weeks (28 DACI), when the number of tenerals peaked e

although larvae and pupae were still present e R. subfuscacontinued to show the highest frequency (5/5), while R. arxii, whichhad not been found in the third week, was found in 3/5 galleries,and R. subalba was found only in one of five galleries, while Raf-faelea sp. PL1001 was not detected (Fig. 3). The composition of theyeast community was similar to that at 21 d, althoughC. nemodendrawas not recovered from the colonies at 28 d (Fig. 3).

In the last week of colony dissection (35 DACI), the populationconsisted mainly of adults and the number of larvae and pupae haddecrease. Now Raffaelea sp. PL1001 was recovered for the first timefrom the galleries, while R. arxii was no longer detected. Similarisolation frequencies were obtained for R. subfusca (3/5), R. subalba(3/5), and Raffaelea sp. PL1001 (3/5). The yeast species isolated at

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this time included Candida sp. NRRLY-27127 (4/5), A. monospora (3/5), C. berthetii (1/5), Alloascoidea sp. (1/5) and B. ochroleuca. (Fig. 3).

3.5. Fungal associates of X. bispinatus developmental stages

R. arxiiwas the most frequent (6/6 beetles; Fig. 4) and abundant(average± SE/female head¼ 578.3± 167.4 CFU) fungal associate inthe mycangia of mature foundresses. Other Raffaelea species,including R. subfusca (3/6, 109.3± 49.8 CFU) and R. subalba (4/6,25.5± 10.5 CFU), were isolated from the beetle mycangia and gut,respectively. Four species of yeast were also recovered: C. berthetii(6/6; 176.3± 45.91 CFU) isolated from the mycangia, andS. synnaedendra (1/6; 8 CFU) and A. ambrosiae (1/6; 48 CFU) isolatedfrom the gut. A. monospora was isolated from both mycangia (1/6,172 CFU) and the gut (1/6, 8 CFU). Another fungal species identifiedas S. macrostoma (4/6, 36.5± 13.26 CFU) was also found in thebeetle gut (Figs. 4 and 5).

Fig. 4. Frequency of fungal species isolated from

In agreement with the fungal composition of the galleries dur-ing weeks two to three, isolates obtained from the larvae corre-sponded with three Raffaelea species: R. arxii with the highestfrequency (4/5, 35.5± 9.1 CFU), R. subfusca (3/5, 43.3± 19.9) andR. subalba (2/5, 155± 5 CFU). In addition, four yeast species;A. monospora (4/5, 64± 33.1 CFU), S. synnaedendra (2/5,34± 22 CFU), Candida sp. NRRL Y-27127 (1/5, 556 CFU), andAlloascoidea sp. (1/5, 80 CFU) were also identified (Figs. 4 and 5respectively).

Three Raffaelea species were isolated from teneral females atsimilar frequencies, i.e., R. subfusca (2/5, 458± 132 CFU), R. arxii (1/5, 8 CFU), R. subalba (2/5, 28± 24 CFU). There was no predominantspecies among the yeasts, Candida sp. NRRL Y-27127 (2/5,271± 101 CFU), A. ambrosiae (2/5, 8 CFU), A. monospora (1/5,335.5 CFU) and Alloascoidea sp. (1/5, 10) (Figs. 4 and 5).

Like the fungal gardens in the fourth week, R. subfusca was themost frequent (5/5) and abundant fungal associate

the different beetle developmental stages.

Fig. 5. Quantification of fungal associates recovered from the different developmental stages of Xyleborus bispinatus.

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126122

(645.4± 184.5 CFU) in the mycangia of the F1 generation females,while R. arxii was only detected in one of five F1 females with158 CFU. In addition, four species of yeast were identified amongthe mycangial isolates at high frequencies, i.e., C. berthetii (5/5,149.6± 36.4 CFU), Candida sp. NRRL Y-27127 (5/5,260.8± 137.4 CFU), Alloascoidea sp. (5/5, 78± 11.71 CFU), andA. ambrosiae (2/5, 14± 10 CFU). In addition, C. berthetii (5/5,224± 143.54 CFU) and Candida sp. NRRL Y-27127 (5/5,812± 534.1 CFU) were also recovered from the beetle gut (Figs. 4and 5).

3.6. Antagonistic assays of X. bispinatus fungal associates versusR. lauricola

The in vitro antagonism assays on plates either of CSMA orartificial rearing medium showed that for most of the isolatesrecovered from the beetle developmental stages and the galleries,there was no antagonism against R. lauricola, i.e., each X. bispinatus

fungal associate grew until its mycelia merged with R. lauricolamycelia. Only Raffaelea sp. PL1001 showed an antagonistic inter-actionwith R. lauricola on CSMA, as demonstrated by the inhibitionof R. lauricola hyphal growth (Fig. 6). On the artificial rearing me-dium, the interaction between R. lauricola and Raffaelea sp. PL1001showed inconsistent results, i.e., only one out of nine platesexhibited inhibition of R. lauricola growth. The mean (n¼ 9) per-centage of inhibition in radial growth± SE on CSMA was41.25± 0.13%.

4. Discussion

4.1. Life cycle and developmental stages of X. bispinatus

The life cycle of one generation of X. bispinatuswas investigatedby infesting an artificial medium based on avocado sawdust and byrearing the F1 progeny from the egg to the adult under laboratoryconditions. The developmental times for larvae, pupae, and adults

Fig. 6. Test of antagonism against the Raffaelea lauricola on CSMA after two weeks ofincubation at room temperature. (A) R. lauricola control plate. (B) Control platemicroscopic view of hyphal growth at the edge of the colony. (C) Inhibition effect ofRaffaelea sp. PL1001 on Raffaelea lauricola. (D) Microscopic view of hyphal growth onthe assay agar plate (C).

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 123

were 8e10, 15e17, 18e21 DACI, respectively, which coincide withthe times reported by Menocal et al. (2018) and Saucedo et al.(2017). The same artificial medium was used in these three sepa-rate investigations, which corroborates the reproducibility of re-sults in this substrate. In the present study, the first fully sclerotizedadult was seen in the various replicates at 22e24 DACI. Similar timeintervals for the development of the adult stage have beenobserved for other artificially reared Scolytinae species, such asXyleborus volvulus (Menocal et al., 2017), X. ferrugineus (Saundersand Knoke, 1967), Xyleborinus saxesenii (Biedermann et al., 2009),Euwallacea fornicatus (Cooperband et al., 2016), Xylosandrus muti-latus (Kajimura and Hijii, 1994), Xyleborus pfeili (Mizuno andKajimura, 2002, 2009), and X. glabratus, in logs (Brar et al., 2013).In our study teneral females eclosed first (21 DACI) followed byteneral males (24 DACI). This female-male sequence has also beendocumented in X. saxesenii (Biedermann, 2010). Although, theopposite order of eclosion of the sexes was found in Xyleborus affinis(Roeper et al., 1980). The earlier development of either the male orthe female has been associated with factors that increase theprobability of mating (Castrillo et al., 2012). Certain differences indevelopment between the sexes are intrinsic to each beetle speciesincluding synchrony of male and female development, male lifespan, and male insemination capacity (Castrillo et al., 2012).Biedermann (2010) hypothesized that the delay of hatching ofmales in X. saxesenii allowed females to reach sexual maturitybefore males. If such a relative delay in the hatch of male eggs werefound to occur also in X. bispinatus, it would explain why femaleswere found to eclose sooner than males in the present study.

By 38 d the average of brood production± SE (n¼ 5) was19± 5.3 individuals, similar to the findings that Menocal et al.(2018) reported in their experiments, i.e., (22.42± 1.72). Broodproduction seems to be species-specific and dependent on thecomposition of the medium, as has been documented in studiesthat tested the effect of medium constituents on offspring numberof different beetle species (Saunders and Knoke, 1967; Mizuno andKajimura, 2002; Biedermann et al., 2009; Castrillo et al., 2012;Maner et al., 2013; Menocal et al., 2017, 2018).

In the tribe Xyleborini, arrhenotokous reproduction, female-biased ratios, and inbreeding polygyny are typical (Kirkendall,1983). The colonies in the present experiment always containedfemales, indicating the fertilized status of the foundresses; and thiswas expected based on their origin as females that emerged fromthe logs, had mated and were ready for dispersion. In contrast,Saucedo et al. (2017) using X. bispinatus virgin females obtainedcolonies only composed of males. We observed only one male-lesscolony at the last dissection time; perhaps no males were foundbecause of the premature death and decomposition of F1 genera-tion males that may have occurred in this colony. In the presentstudy, the male:female ratio was 1:6 at the end of the experiment.This is close to the ratio observed in Xylosandrus germanus in abrood of a similar size (Castrillo et al., 2012). It is likely that the ratiowould increase with the size of the population; however, we wereunable to confirm this, due to the dimensions and characteristics ofthe rearing substrate that limits the amount of food resources andtherefore constrains the size of the population. On the other hand,in wild colonies, the number of males is expected to depend onmale fertility and male lifespan (Biedermann, 2010).

4.2. Gallery construction, expansion, and housekeeping

The development of the brood depends on the growth of sym-biotic fungi in the galleries (Beaver, 1989). The foundress ovipositsonly when the nutritional symbionts have been established in thetunnels (Peer and Taborsky, 2007). In the present study, femalesstarted to oviposit immediately after finishing the construction ofthe main galleries, i.e., by the end of the first week; at this time,fungal growth was evident as a dark layer on the walls of the gal-leries. However, eggs were laid by the foundresses only until thebeginning of the fourth week. The placing of eggs at the distantends of the galleries may be an adaptation to assure the optimal useof both food resources and space. The foundress appears to main-tain the main gallery clear to allow its use as a corridor for themanagement of the ambrosia gardens in the main and secondarygalleries and the care of eggs and larvae.

Once fungal symbionts were established, the colonies started toexpand. In X. mutilatus and X. pfeili there was a direct correlationbetween the number of offspring and the length of the gallerysystem (Kajimura and Hijii, 1994), which suggests that the length ofthe gallery system is a factor onwhich the amount of food availablefor the mother beetle and her brood depends, i.e., space availablefor cultivating fungi (Kajimura and Hijii, 1994; Mizuno andKajimura, 2002). Likewise, in the present study, a positive corre-lation between gallery length and brood size was observed duringthe initial 3 weeks of the colony, and during this period only thefoundress was involved in tunnel excavation. After 3 weeks, asoviposition decreased, the brood size stayed relatively steady. After4 weeks, a positive correlation between the number of new adultsand gallery size was observed, and indeed the greatest growth ofthe gallery system was recorded. The enlargement in gallery sizemay imply an increase in the production of ambrosia fungi, whichwould facilitate the acquisition of fungi for the new generation offemales and the later hatched individuals in the brood. A similarbehavior has been observed in X. pfeili (Mizuno and Kajimura,2002) and X. saxesenii, the latter of which exhibits partial overlapof the parental and F1 generations (Biedermann et al., 2012). The F1females delay their dispersal and cooperate in the care of the am-brosia gardens and the brood, suggesting a high level of sociality(Biedermann et al., 2013). In the present study, eggs were absent atthe last dissection time on 38 DACI even though mature fecundfemales were still present, but which had not oviposited. This couldbe caused by deterioration of the artificial medium.

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126124

4.3. Symbiont dynamics in the fungal gardens and in the beetledevelopmental stages

Ambrosia beetles are known to have an obligate nutritionalrelationship with ambrosia fungi mostly belonging to the poly-phyletic genera Ambrosiella, Raffaelea and Dryadomyces (Dreadenet al., 2014a, 2014b; Mayers et al., 2015). A community of ambro-sia symbiont species within the mycangia and the cultivation ofmore than one nutritional symbiont species is now evident innumerous ambrosia beetle species (Kostovcik et al., 2015). In thisstudy, we were able to recover four species of Raffaelea. Two ofwhich were predominant in the fungal gardens, i.e., R. arxii andR. subalba. R. arxii is the most frequent associate in the mycangia ofX. bispinatus foundresses and subsequently in the fresh fungalgardens during the first weeks of gallery construction. Moreover,R. arxii is the most frequent nutritional resource used by larvae. It isthought that the larva is the stage with the greatest food re-quirements. Therefore, the most dominant fungal symbiont ingalleries during the larval growth period is likely to be crucial forthe development of the beetles (Beaver, 1989). R. arxii seemed todepopulate progressively with the development of the gallery andthe appearance of the adult F1 progeny of X. bispinatus. In contrast,R. subfusca the other prevalent species, whose fraction in the gar-dens increased progressively during the course of the experiment,was the most frequent and abundant associate in the mycangia ofthe mature F1 generation females. R. subalba, the only Raffaelea sp.isolated from the foundress guts, appeared in the galleries in thesecond week and was found at relative low frequencies until theend of the experiment. R. subalba was recovered from larvae andtenerals but not from F1 generation females. Raffaelea sp. PL1001was only found in the galleries at the last dissection time i.e., 38DACI. The distinctively great dominance of R. subfusca, R. arxii, andR. subalba in the gardens and in the different developmental stagesduring this experiment may suggest a preference or a nutritionalrequirement of a particular beetle developmental stage for a spe-cific species of Raffaelea as has been observed in other species.Euwallacea nr. fornicatus harbors three symbionts: Graphiumeuwallaceae, Acremonium pembeum, and Fusarium euwallaceae.Freeman et al. (2016) reported that G. euwallaceae was the pre-dominant symbiont in the initial stages of gallery development andused as a primary food source for larval development, whereasF. euwallaceae was the food source for adult stages (Freeman et al.,2016). Even though, in our experiments the mature stages, foun-dresses and F1 females, did not exhibit the same symbiont speciesdominance, our data may still suggest a certain level of food pref-erence. This might be explored by rearing a second generation inorder to determine whether the foundresses are able to control thegrowth of a given fungal species in the galleries regardless of therelative abundance of various symbiont species in their mycangia.Saucedo et al. (2017) tested the role of R. arxii, R. subfusca, andR. subalba, as nutritional symbionts of X. bispinatus. Their findingsindicate that males of X. bispinatus were able to complete their lifecycle when fed solely on one of these symbionts species, indicatingthe lack of fidelity in this mutualistic interaction, or flexibility in thebeetle's association with its repertoire of symbionts (Saucedo et al.,2017). Interestingly, slightly larger broods were obtained by col-onies fed on R. arxii followed by R. subfusca and R. subalba althoughthese differences were not statistically significant (Saucedo et al.,2017).

Functional redundancy in the symbiont repertoire may result inan advantage by increasing beetle fitness. According to Biedermannet al. (2013), the presence of multiple symbiont species ensures thesurvival of the insects under dissimilar conditions. Thus, if there isany accidental loss of a symbiont species during dispersal, or theconditions are less suitable for one of the symbiont species e.g.,

changes in humidity, in the availability of certain nutrients, or thepresence of an antagonistic organism, it is possible that othersymbionts are able to proliferate and avoid the aposymbiotic stateand failure in the establishment of the new colony. Moreover,multiple symbiont species also may decrease the competition forfood resources among the various beetle developmental stageseven if these beetle stages do not prefer a particular symbiontspecies. In multipartite symbiosis in which what seems to be asingle niche is shared, the optimal conditions for each species mayvary and the performance of a given symbiont member is con-strained by environmental gradients (Six, 2012). The relativedominance of fungal species in this experiment could have beenshaped by the conditions of the medium per se. For instance,Castrillo et al. (2012) found that the type of sawdust in the artificialmedia has an effect on growth rate andmycelial density of differentstrains of Ambrosiella hartigii, a symbiont of X. germanus. Thus, therapid growth in the medium of R. arxii and subsequent decline inthe occurrence and the proliferation of R. subfusca could beconditioned by the medium i.e., depletion of certain nutrients orchange in humidity, and not driven by a developmental stage-specific requirement.

4.4. Antagonistic assays of the X. bispinatus fungal symbiontsversus R. lauricola

R. lauricolawas not recovered from either the beetles at differentdevelopmental stages (n¼ 21) or from the galleries at the differentdissection times (n¼ 25). To determine a possible antagonistic ef-fect of any of the isolates recovered from the experiment, we per-formed an antagonistic assay on CSMA and on the rearing media.The assay demonstrated that only Raffaelea sp. PL1001 was able toinhibit the growth of R. lauricola. However, Raffaelea sp. PL1001 wasonly found in the galleries 38 DAIC. Due to its low frequency, thepresence of Raffaelea sp. PL1001 does not seem to be a determinantfactor to explain the absence of R. lauricola. One possibility is thatR. lauricola was not present in the population used for the experi-ment or that if present, R. lauricola was undetected in the varioussamples taken from galleries and beetles due to its low frequencyand scarcity. Nevertheless, R. lauricola was previously found in agreat percentage of the X. bispinatus tested for the pathogen (Ploetzet al., 2017). Native symbionts should be better adapted to theirbeetle host and consequently carried in larger numbers in theirmycangia allowing them to establish more efficiently in the gal-leries under the experimental conditions used in this study. In theirstudy, Menocal et al. (2018) found low colonization of the mycangiawhen the medium was inoculated with R. lauricola contrary to thefindings of Saucedo et al. (2017). Mature females containing thenative symbionts were used by Menocal et al. (2018), whileSaucedo et al. (2017) performed their study with newly eclosedfemales ensuring the exclusive presence of R. lauricola in theirmycangia. It is worth mentioning that R. lauricola, when present,seems not to alter the community of X. bispinatus fungal symbionts,as observed by Menocal et al. (2018). In their study, media inocu-lated and non-inoculated with R. lauricola exhibit similar fungalsymbiont constituents, which accords with the current study.

4.5. Yeasts and bionectraceous fungi associated with fungal gardensand beetle developmental stages

In addition to the Raffaelea spp., seven species of yeast wererecovered from galleries and different developmental stages of thebeetle. These included C. berthetii, Candida sp. NRRL Y-27127,C. nemodendra, A. ambrosiae, A. monospora, S. synnaedendra and,Alloascoidea sp. With the exception of C. berthetii, which was onlyfound associated with the adult stages (mother and F1 generation

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 125

adult daughters), the yeast species seem not to have a regularpattern for their appearance in the galleries and their presenceseemed to depend on what randomly a certain beetle develop-mental stage was carrying at a given dissection time. According tothe CBS-database from theWesterdijk Fungal Biodiversity Institute,C. berthetii was found associated with the galleries constructed bybark and ambrosia beetles species in various host-plants, includingthe tunnels of X. volvulus in Cussonia umbellifera (Araliaceae),Platypus externedentatus in Macaranga capensis (Euphorbiaceae),and P. externedentatus in Ficus sycomorus (Moraceae) (west-erdijkinstitute.nl/Collections). Similarly, C. nemodendrawas initiallyisolated from tunnels of pin-borer beetles, Xyleborus aemulus, inSouth Africa (westerdijkinstitute.nl/Collections). Candida sp. NRRLY-27127 was identified among the mycangial fungi of X. glabratus(Harrington and Fraedrich, 2010). The genus Ambrosiozyma is well-known to be associated with the galleries and/or different devel-opmental stages of beetles in the groups Platypodidae or Scoly-toidea, as observed in samples obtained in South Africa, USA, andJapan (van der Walt, 1972). S. synnaedendra has been isolated fromthe tunnels of P. externedentatus in South Africa (Van Der Walt andScott, 1971). The genus Alloascoidea was recently distinguishedfrom Ascoidea (Kurtzman and Robnett, 2013); the members ofAscoidea are all known to be associated with decaying wood, barkbeetles, and insect galleries in trees (de Hoog and Smith, 2011). Inspite of their ubiquity and abundance, no studies have been con-ducted to determine their role in the beetle-ambrosia fungimutualistic system of these two genera. Yeast species have beenconsidered to be non-specific, opportunistic, commensals or para-sites, due to their non-specific relationship with different species ofbark or ambrosia beetles (Blackwell, 2017). It is not known if othergenera of yeasts besides Ambrosiozyma, which are considered trueambrosia symbionts, are functionally associated with ambrosiabeetles (Hulcr and Stelinski, 2017). Hypothetical functions havebeen established based on their enzymatic profiles and on theirproduction of volatiles (Davis, 2015), such as mediation ofcompetitive interactions, production of semiochemicals, defensesagainst plant toxins, and nutritional supplements or nutrientcycling (Davis, 2015).

In addition to the Raffaelea and yeast species, two bio-nectraceous fungi (Ascomycota: Hypocreales: Bionectriaceae),were identified. These were B. ochroleuca and S. macrostoma. Thereis little information regarding these two fungi. They seem likely tohave been introduced accidentally in the beetle-gallery system.Both of them are known to occur on living plant material.B. ochroleuca has been identified as a plant endophyte in variousspecies (Paul et al., 2013). This species can also produce metaboliteswith antibacterial and antifungal activity (Samaga et al., 2014) andbe entomopathogenic (Guesmi-Jouini et al., 2014).

To summarize, we reported the developmental biology ofX. bispinatus and documented its fungal symbionts. Based on ourresults, we conclude that the social behavior of X. bispinatus isconditioned by its biology. Asynchrony in the development ofmales and females results in delayed mating and dispersion.During this period, F1 females cooperate in gallery construction,maintenance of the fungal gardens, to assure a sufficient supplyof food for the brood, and brood care of the already establishedcolonies. Our results show a flexible nutritional interaction ofX. bispinatus with its mutualistic symbionts, as evidenced bytheir associations with the different development stages. Theseassociations may be both developed by adaptationdas could bethe case of native symbionts identified in this workdor inci-dentally acquired, as is clearly the case with R. lauricola. Ourresults also indicate that R. lauricola does not seem to be nega-tively affected by other symbionts with the exception of Raffaeleasp. PL1001.

Acknowledgements

Thanks to Waldemar Klassen and Jorge E. Pe~na (University ofFlorida) for suggestions to improve the manuscript. Thanks to JulioMantilla, Jose Alegría, and Rita E. Duncan for experimental set-up.This research was funded by NIFA grant 2015-51181-24257 toDaniel Carrillo.

References

Atkinson, T.H., Carrillo, D., Duncan, R.E., Pe~na, J.E., 2013. Occurrence of Xyleborusbispinatus (Coleoptera: Curculionidae: scolytinae) Eichhoff in southern Florida.Zootaxa 3669, 96e100.

Bateman, C., Kendra, P.E., Rabaglia, R., Hulcr, J., 2015. Fungal symbionts in threeexotic ambrosia beetles, Xylosandrus amputatus, Xyleborinus andrewesi, andDryoxylon onoharaense (Coleoptera: Curculionidae: scolytinae: Xyleborini) inFlorida. Symbiosis 66, 141e148.

Beaver, R.A., 1989. Insect-fungus relationships in the bark and ambrosia beetles. In:Wilding, N., Collins, N.M., Hammind, P.M., Webber, J.F. (Eds.), Insect-fungusInteractions. Academic, London, United Kingdom, pp. 121e143.

Biedermann, P.H.W., 2010. Observations on sex ratio and behavior of males inXyleborinus saxesenii Ratzeburg (Scolytinae, Coleoptera). ZooKeys 56, 253e267.

Biedermann, P.H.W., Klepzig, K.D., Taborsky, M., 2009. Fungus cultivation by am-brosia beetles: behavior and laboratory breeding success in three xyleborinespecies. Environ. Entomol. 38, 1096e1105.

Biedermann, P.H.W., Klepzig, K.D., Taborsky, M., Six, D.L., 2013. Abundance anddynamics of filamentous fungi in the complex ambrosia gardens of the primi-tively eusocial beetle Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculioni-dae, Scolytinae). FEMS Microbiol. Ecol. 83, 711e723.

Biedermann, P.H.W., Peer, K., Taborsky, M., 2012. Female dispersal and reproductionin the ambrosia beetle Xyleborinus saxesenii Ratzeburg (Coleoptera; Scolytinae).Mitt. Dtsch. Ges. Allg. Angew. Entomol 18, 231e235.

Blackwell, M., 2017. Yeasts in insects and other invertebrates. In: Buzzini, P.,Lachance, M.A., Yurkov, A. (Eds.), Yeasts in Natural Ecosystems: Diversity.Springer, Cham, pp. 397e433.

Bleiker, K.P., Potter, S.E., Lauzon, C.R., Six, D., 2009. Transport of fungal symbionts bymountain pine beetles. Can. Entomol. 141, 503e514.

Brar, G.S., Capinera, J.L., Kendra, P.E., McLean, S., Pe~na, J.E., 2013. Life cycle, devel-opment, and culture of Xyleborus glabratus (Coleoptera: Curculionidae: scoly-tinae). Fla. Entomol. 96, 1158e1167.

Campanile, G., Ruscelli, A., Luisi, N., 2007. Antagonistic activity of endophytic fungitowards Diplodia corticola assessed by in vitro and in planta test. Eur. J. PlantPathol. 117, 237e246.

Carrillo, D., Duncan, R.E., Pe~na, J.E., 2012. Ambrosia beetles (Coleoptera: Curculio-nidae: scolytinae) that breed in avocado wood in Florida. Fla. Entomol. 95,573e579.

Carrillo, D., Duncan, R.E., Ploetz, J.N., Campbell, A.F., Ploetz, R.C., Pe~na, J.E., 2014.Lateral transfer of a phytopathogenic symbiont among native and exotic am-brosia beetles. Plant Pathol. 63, 54e62.

Castrillo, L.A., Griggs, M.H., Ranger, C.M., Reding, M.E., Vandenberg, J.D., 2011.Virulence of commercial strains of Beauveria bassiana and Metarhizium brun-neum (Ascomycota: Hypocreales) against adult Xylosandrus germanus (Coleop-tera: Curculionidae) and impact on brood. Biol. Contr. 58, 121e126.

Castrillo, L.A., Griggs, M.H., Vandenberg, J.D., 2012. Brood production by Xylosandrusgermanus (Coleoptera: Curculionidae) and growth of its fungal symbiont onartificial diet based on sawdust of different tree species. Environ. Entomol. 41,822e827.

Cooperband, M.F., Stouthamer, R., Carrillo, D., Eskalen, A., Thibault, T., Coss�e, A.A.,Castrillo, L.A., Vandenberg, J.D., Rugman-Jones, P.F., 2016. Biology of twomembers of the Euwallacea fornicatus species complex (Coleoptera: Curculio-nidae: scolytinae), recently invasive in the USA, reared on an ambrosia beetleartificial diet. Agric. For. Entomol 18, 223e237.

Davis, T.S., 2015. The ecology of yeasts in the bark beetle holobiont: a century ofresearch revisited. Microb. Ecol. 69, 723e732.

de Hoog, G.S., Smith, M., 2011. Ascoidea brefeld & lindau (1891). In: Kurtzman, C.P.,Fell, J.W., Boekhout, T. (Eds.), The Yeasts, a Taxonomic Study, fifth ed. ElsevierScience B.V., Amsterdam, pp. 325e328.

Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities offresh leaf tissue. Phytochemical Bulletin 19, 11e15.

Dreaden, T.J., Davis, J.M., Harmon, C.L., Ploetz, R.C., Palmateer, A.J., Soltis, P.S.,Smith, J.A., 2014a. Development of multilocus PCR assays for Raffaelea lauricola,causal agent of laurel wilt disease. Plant Dis. 98, 379e383.

Dreaden, T., Davis, J., de Beer, Z., Ploetz, R., Soltis, P., Wingfield, M.J., Smith, J.A.,2014b. Phylogeny of ambrosia beetle symbionts in the genus Raffaelea. FungalBiol. 118, 970e978.

Farrell, B.D., Sequeira, A.S., O'Meara, B.C., Normark, B.B., Chung, J.H., Jordal, B.H.,2001. The evolution of agriculture in beetles (Curculionidae: scolytinae andPlatypodinae). Evolution 55, 2011e2027.

Fraedrich, S.W., Harrington, T.C., Rabaglia, R.J., Ulyshen, M.D., Mayfield, A.E.,Hanula, J.L., Eickwort, J.M., Miller, D.R., 2008. A fungal symbiont of the redbayambrosia beetle causes a lethal wilt in redbay and other Lauraceae in thesoutheastern United States. Plant Dis. 92, 215e224.

L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126126

Freeman, S., Sharon, M., Dori-Bachash, M., Maymon, M., Belausov, E., Maoz, Y.,Margalit, O., Protasov, A., Mendel, Z., 2016. Symbiotic association of three fungalspecies throughout the life cycle of the ambrosia beetle Euwallacea nr. for-nicatus. Symbiosis 68, 115e128.

Gaigole, A.H., Wag, G.N., Khadse, A.C., 2011. Antifungal activity of Trichodermaspecies against soil borne pathogen. Asiatic J. Biot. Resour 4, 461e465.

Guesmi-Jouini, J., Garrido-Jurado, I., L�opez-Díaz, C., Ben Halima-Kamel, B., Quesada-Moraga, E., 2014. Establishment of fungal entomopathogens Beauveria bassianaand Bionectria ochroleuca (Ascomycota: Hypocreales) as endophytes on arti-choke Cynara scolymus. J. Invertebr. Pathol. 119, 1e4.

Haanstad, J.O., Norris, D.M., 1985. Microbial symbionts of the ambrosia beetleXyletorinus politus. Microb. Ecol. 11, 267e276.

Harrington, T.C., Fraedrich, S.W., 2010. Quantification of propagules of the laurelwilt fungus and other mycangial fungi from the redbay ambrosia beetle, Xyle-borus glabratus. Phytopathology 100, 1118e1123.

Harrington, T.C., Aghayeva, D.N., Fraedrich, S.W., 2010. New combinations in Raf-faelea, Ambrosiella, and Hyalorhinocladiella, and four new species from theredbay ambrosia beetle, Xyleborus glabratus. Mycotaxon 111, 337e361.

Hulcr, J., Stelinski, L.L., 2017. The ambrosia symbiosis: from evolutionary ecology topractical management. Annu. Rev. Entomol. 62, 285e303.

Hulcr, J., Mann, R., Stelinski, L.L., 2011. The scent of a partner: ambrosia beetles areattracted to volatiles from their fungal symbionts. J. Chem. Ecol. 37, 1374e1377.

Kajimura, H., Hijii, N., 1994. Reproduction and resource utilization of the ambrosiabeetle, Xylosandrus mutilatus, in field and experimental populations. Entomol.Exp. Appl. 71, 121e132.

Kirkendall, L.R., 1983. The evolution of mating systems in bark and ambrosia beetles(Coleoptera: scolytidae and Platypodidae). Zool. J. Linn. Soc. 77, 293e352.

Kirkendall, L.R., Kent, D.S., Raffa, K.F., 1997. Interactions among males, females andoffspring in bark and ambrosia beetles: the significance of living in tunnels forthe evolution of social behavior. In: Choe, J.C., Crespi, B.J. (Eds.), The Evolution ofSocial Behaviour in Insect and Arachnids. Cambridge University Press,pp. 181e215.

Kostovcik, M., Bateman, C., Kolarik, M., Stelinski, L., Jordal, B., Hulcr, J., 2015. Theambrosia symbiosis is specific in some species and promiscuous in others:evidence from community pyrosequencing. ISME J. 9, 126e138.

Kurtzman, C.P., Robnett, C.J., 2013. Alloascoidea hylecoeti gen. nov., comb. nov.,Alloascoidea africana comb. nov., Ascoidea tarda sp. nov., and Nadsonia starkeyi-henricii comb. nov., newmembers of the Saccharomycotina (Ascomycota). FEMSYeast Res. 13, 423e432.

Li, Y., Ruan, Y.Y., Stanley, E.L., Skelton, J., Hulcr, J., 2018. Plasticity of mycangia inXylosandrus ambrosia beetles. Insect Sci. https://doi.org/10.1111/1744e7917.12590.

Maner, M.L., Hanula, J.L., Braman, K., 2013. Rearing redbay ambrosia beetle, Xyle-borus glabratus (Coleoptera: Curculionidae: scolytinae), on semi-artificial me-dia. Fla. Entomol. 96, 1042e1051.

Mayers, C.G., McNew, D.L., Harrington, T.C., Roeper, R.A., Fraedrich, S.W.,Biedermann, P.H.W., Castrillo, L.A., Reed, S.E., 2015. Three genera in the Cera-tocystidaceae are the respective symbionts of three independent lineages ofambrosia beetles with large, complex mycangia. Fungal Biol. 119, 1075e1092.

Menocal, O., Cruz, L.F., Kendra, P.E., Crane, J.H., Ploetz, R.C., Carrillo, D., 2017. RearingXyleborus volvulus (Coleoptera: Curculionidae) on media containing sawdustfrom avocado or silkbay, with or without Raffaelea lauricola (Ophiostomatales:ophiostomataceae). Environ. Entomol. 46, 1275e1283.

Menocal, O., Cruz, L.F., Kendra, P.E., Crane, J.H., Cooperband, M.F., Ploetz, R.C.,Carrillo, D., 2018. Xyleborus bispinatus reared on artificial media using sawdustfrom avocado or silkbay in presence or absence of the laurel wilt pathogen(Raffaelea lauricola). Insects 9, 30.

Mizuno, T., Kajimura, H., 2002. Reproduction of the ambrosia beetle, Xyleborus pfeili(Ratzeburg) (Col., Scolytidae), on semi-artificial diet. J. Appl. Entomol. 126,455e462.

Mizuno, T., Kajimura, H., 2009. Effects of ingredients and structure of semi-artificialdiet on the reproduction of an ambrosia beetle, Xyleborus pfeili (Ratzeburg)(Coleoptera: Curculionidae: scolytinae). Appl. Entomol. Zool 44, 363e370.

Mizuno, T., Fujiwara, S., Matsuda, M., 1997. Rearing of ambrosia beetle, Xyleboruspfeili (Ratzeburg) on artificial diet. Res. Bull. Plant Prot. Ser 33, 81e85.

Mueller, U.G., Gerardo, N.M., Aanen, D.K., Six, D.L., Schultz, T.R., 2005. The evolutionof agriculture in insects. Annu. Rev. Ecol. Evol. Syst. 36, 563e595.

Paul, N.C., Deng, J.X., Lee, J.H., Yu, S.H., 2013. New records of endophytic Paecilo-myces inflatus and Bionectria ochroleuca from chili pepper plants in Korea.Mycobiology 41, 18e24.

Peer, K., Taborsky, M., 2007. Delayed dispersal as a potential route to cooperativebreeding in ambrosia beetles. Behav. Ecol. Sociobiol. 61, 729e739.

Ploetz, R.C., Konkol, J., Narvaez, T., Duncan, R., Saucedo, J.R., Campbell, A., Mantilla, J.,Carrillo, D., Kendra, P.E., 2017. Presence and prevalence of Raffaelea lauricola,cause of laurel wilt, in different species of ambrosia beetles in Florida, USA.J. Econ. Entomol. 110, 347e354.

Rabaglia, R.J., Dole, S.A., Cognato, A.I., 2006. Review of american xyleborina (Cole-optera: Curculionidae: scolytinae) occurring North of Mexico, with an illus-trated key. Ann. Entomol. Soc. Am. 99, 1034e1056.

Roeper, R.A., Treeful, L.M., O'Brien, K.M., Foote, R.A., Bunce, M.A., 1980. Life history ofthe ambrosia beetle Xyleborus affinis (Coleoptera: scolytidae) from in vitroculture. Gt. Lakes Entomol. 13, 141e144.

Samaga, P.V., Rai, V.R., Rai, K.M.L., 2014. Bionectria ochroleuca NOTL33dan endo-phytic fungus from Nothapodytes foetida producing antimicrobial and freeradical scavenging metabolites. Ann. Microbiol. 64, 275.

Saucedo, J.R., Ploetz, R.C., Konkol, J.L., Angel, M., Mantilla, J., Menocal, O., Carrillo, D.,2017. Nutritional symbionts of a putative vector, Xyleborus bispinatus, of thelaurel wilt pathogen of avocado. Raffaelea lauricola. https://doi.org/10.1007/s13199-017-0514-3. Symbiosis.

Saunders, J.L., Knoke, J.K., 1967. Diets for rearing the ambrosia beetle Xyleborusferrugineus (Fabricius) in vitro. Science 157, 460e463.

Six, D.L., 2003. Bark beetle-fungus symbioses. In: Bourtzis, K., Miller, T.A. (Eds.),Insect Symbiosis. CRC Press, Boca Raton, pp. 97e114.

Six, D.L., 2012. Ecological and evolutionary determinants of bark beetleefungussymbioses. Insects 3, 339e366.

van der Walt, J.P., 1972. The yeast genus Ambrosiozyma gen. nov. (Ascomycetes).Mycopathol. Mycol. Appl. 46, 305e316.

Van der Walt, J.P., Scott, D.B., 1971. Saccharomycopsis synnaedendra, a new yeastfrom South African insect sources. Mycopathol. Mycol. Appl. 44, 101e106.

Vilgalys, R., Hester, M., 1990. Rapid genetic identification and mapping of enzy-matically amplified ribosomal DNA from several Cryptococcus species.J. Bacteriol. 172, 4238e4246.

White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing offungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H.,Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: a Guide to Methods and Applica-tion. Academic Press, San Diego, CA, pp. 315e322.