Fungal Ecology 35 (2018) 116e126

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Fungal Ecology

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Developmental biology of bispinatus (Coleoptera: ) reared on an artificial medium and fungal cultivation of symbiotic fungi in the '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 , 18905 SW 280th St, Homestead, 33031, FL, USA b Universidad Autonoma Chapingo, Km 38.5 Carretera Mexico - Texcoco, Chapingo, Mex, 56230, Mexico c Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria, Unidad Integral de Diagnostico, Servicios y Constatacion, Tecamac, 55740, Estado de Mexico, Mexico article info abstract

Article history: Survival of ambrosia relies on obligate nutritional relationships with fungal symbionts that are Received 10 January 2018 cultivated in tunnels excavated in the sapwood of their host trees. The dynamics of fungal associates, Received in revised form along with the developmental biology, and gallery construction of the ambrosia beetle Xyleborus bispi- 10 July 2018 natus were elaborated. One generation of this ambrosia beetle was reared in an artificial medium con- Accepted 12 July 2018 taining sawdust. The developmental time from egg to adult ranged from 22 to 24 d. The mean Available online 23 August 2018 total gallery length (14.4 cm and 13 tunnels) positively correlated with the number of adults. The most Corresponding Editor: Peter Biedermann prevalent fungal associates were 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, Keywords: Raffaelea subalba and seven yeast were also recovered. These results indicate flexibility in the Ambrosia beetle association between X. bispinatus and its symbiont species, and that this beetle may use more than one Ambrosia fungi Raffaelea species as a food source. These results are important, as X. bispinatus has been associated with Artificial rearing the transmission of Raffaelea lauricola, the causal agent of laurel wilt, a lethal disease affecting avocado Avocado trees. Fungal farming © 2018 Elsevier Ltd and British Mycological Society. All rights reserved. Laurel wilt Raffaelea species Scolytinae Symbiosis

1. Introduction and filamentous fungi in addition to ambrosia fungi (Haanstad and Norris, 1985). Through coevolution, a symbiotic nutritional relationship has Ambrosia beetles can have one or multiple ambrosia fungi arisen between ambrosia beetles (Coleoptera: Curculionidae: Sco- (Bateman et al., 2015; Kostovcik et al., 2015). Most fungal symbionts lytinae) and ambrosia fungi (Farrell et al., 2001; Mueller et al., of ambrosia beetles belong to the phylum (Ascomy- 2005). In this mutualistic interaction, ambrosia beetles proliferate cota: ), in the orders and Micro- in the low nutrient environment within the sapwood of their host ascales, and the species are classified in the anamorph genera trees (Bleiker et al., 2009), while concurrently ambrosia fungi Ambrosiella (Microascales: Ceratocystidaceae) and Raffaelea achieve their permanence and dissemination by being acquired and (Ophiostomatales: ) (Dreaden et al., 2014a; transported by beetles (Six, 2012). The establishment of the fungal Dreaden et al., 2014b; Mayers et al., 2015). Generally, these fungal garden is critically important to the reproductive success of each of symbionts are saprobes of stressed, dying, and dead trees (Beaver, the newly founded colonies (Biedermann et al., 2009, 2013). These 1989; Kirkendall et al., 1997). Exotic, invasive ambrosia beetle ambrosia gardens, however, contain a variety of bacteria, yeasts, species may carry plant pathogens among their symbionts, which may be very damaging pests of healthy trees (Hulcr et al., 2011). Ambrosia symbiont spores are carried by female beetles in a specialized cuticular pocket, termed a mycangium, the anatomical * Corresponding author. location of which varies among beetle species, for example, in the E-mail address: luisafcruz@ufl.edu (L.F. Cruz). https://doi.org/10.1016/j.funeco.2018.07.007 1754-5048/© 2018 Elsevier Ltd and British Mycological Society. All rights reserved. L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 117

Xyleborini, it may be mandibular, mesonotal or elytral (Six, 2003; the jar oriented outward from the main chamber. This was done by Hulcr and Stelinski, 2017), and in Xyleborus bispinatus it is gluing the threaded metal band into the hole and then screwing the mandibular. The spores are inoculated during tunnel excavation top of the jar into the metal band. A moistened paper towel was (Six, 2012). The immature stages develop feeding on fungi and the place into the jar. Infested avocado log segments were placed into new generation of adults fill their mycangia via feeding in the natal each container and its top was closed tightly with an opaque lid. galleries before dispersing (Li et al., 2018). These transmission After 1 week, dark brown females (fully sclerotized) emerged from processes strengthen the link and fidelity between the fungal the logs, and they were collected daily from the clear jars into symbionts and the beetles (Six, 2012). Although the mycangia which they had been attracted by the light. These females were ensure reliable transmission, the interaction of sympatric identified under a stereoscope without immobilization according to species co-occurring in the same host tree allows horizontal Rabaglia et al. (2006), and the X. bispinatus specimens were acquisition of new symbionts (Carrillo et al., 2014). retained for use in the experiments. Horizontal movement of symbionts plays an important role in the epidemiology of laurel wilt in avocado trees (Persea americana), 2.2. Artificial medium and rearing conditions a lethal disease of the , caused by the fungal pathogen Raffaelea lauricola (Ophiostomatales: Ophiostomataceae), and Sawdust was produced following the methodology described by mainly transmitted by the Asian redbay ambrosia beetle, Xyleborus Castrillo et al. (2011). Briefly, healthy avocado logs (without any glabratus (Curculionidae: Scolytinae) (Rabaglia et al., 2006; sign of beetle infestation) from an avocado orchard in Miami-Dade Fraedrich et al., 2008). However, X. glabratus has not been detected County (25 290 3800 N, 80 28’ 5300 W) were debarked and dried at breeding in avocado trees (Carrillo et al., 2012). Instead, nine other 75 C for 4 days. Sawdust was obtained from the xylem sapwood species of ambrosia beetles that breed in avocado, including X. layer using a sander. Large particles of sawdust were eliminated bispinatus, carry the pathogen and perhaps act as an alternative using a 12 mm sieve. Medium preparation was carried out ac- vector (Carrillo et al., 2014; Ploetz et al., 2017). cording to Castrillo et al. (2011). The medium consisted of 75 g of Despite the knowledge gained to date, regarding the interaction avocado sawdust, 20 g agar, 10 g sucrose, 5 g starch, 5 g casein, 5 g of X. bispinatus with R. lauricola (Carrillo et al., 2014; Saucedo et al., yeast, 1 g Wesson salt mixture, 0.35 g streptomycin, 5 mL 95% 2017; Menocal et al., 2018). There is a dearth of information on the ethanol, 2.5 mL wheat germ oil, and 500 mL distilled water. Solid basic biology of this beetle species and the mutualistic fungal as- ingredients were first homogenized, and then incorporated with sociations during its life cycle. Moreover, due to their cryptic life the liquid materials. The medium was autoclaved at 121 C and 15 style, the study of ambrosia beetles in their native environment is PSI for 30 min; mixed thoroughly to re-suspend settled ingredients, extremely difficult. Nevertheless, the development of artificial and 15 mL was poured into each 50 mL polypropylene tube under substrates has facilitated the study of this taxonomic group under aseptic conditions. The tubes were loosely capped and the medium laboratory conditions (Mizuno et al., 1997; Mizuno and Kajimura, was allowed to dry and solidify for 1 week under a laminar flow 2002, 2009; Biedermann et al., 2009; Castrillo et al., 2012; Maner hood (Castrillo et al., 2011). et al., 2013; Cooperband et al., 2016; Menocal et al., 2017). The Prior to infesting the medium in the tubes, the beetles were primary goal of the current study was to gain insight into the surface sterilized by immersing for 5 sec in 75% ethanol to eliminate biology of X. bispinatus and its fungal associates. The specific ob- surface contaminants. The medium was perforated with a sterile jectives were to: (1) elucidate the beetle's life cycle and its devel- needle to facilitate initial boring. One female was introduced into opmental stages, (2) determine the process of gallery construction, each of sixty tubes. Tubes with infested medium were incubated (3) survey the fungal repertoire associated with the galleries and horizontally at 25 ± 1 C under a LD 16:8 h photocycle for 38 d. with different developmental stages of X. bispinatus reared in an artificial medium, and (4) explore the possible existence of antag- 2.3. Medium dissection and fungal isolation from galleries onistic interactions of the X. bispinatus symbionts against R. lauricola. The findings of this study will establish whether the Fifty-five tubes containing successful colonies were processed in association of X. bispinatus with its fungal symbionts is species- this study. Dissection of the medium was performed twice a week specific or multipartite and flexible throughout its life cycle. for 5 weeks. Five tubes were randomly selected each time (i.e., 10 tubes per week) and dissected under the stereoscope. The medium 2. Materials and methods plugs were carefully drawn out of the tube and dissected along the gallery tunnels. Gallery length was measured, quantitative de- 2.1. Collection of beetles scriptions of the structures of the main and secondary galleries were recorded, and beetle developmental stages were located and X. bispinatus is a neotropical species, distributed throughout counted. For fungal isolation, samples from those gallery walls South America, Central America, and along southeastern North occupied by immature beetle stages, the gallery entrance, and the American coast (Fraedrich et al., 2008). Previously, X. bispinatus was extruded frass were acquired once a week with a sterile needle, and undistinguished from Xyleborus ferrugineus; however, these two streaked on plates of cycloheximide-streptomycin-malt agar species were later separated based on differences in morphological (CSMA) a semi-selective medium for ophiostomatoid fungi characters (Atkinson et al., 2013). For the current study, fully (Harrington et al., 2010), and potato dextrose agar amended with þ sclerotized X. bispinatus females were collected from avocado logs 0.2 g/L streptomycin (PDA ). These plates were incubated 7e10 d at showing signs of active infestation (i.e., excavated material room temperature. extruded from the beetle galleries). Logs were collected in January 2017 from an avocado grove in Homestead, Florida, USA (25 490 2.4. Symbiont isolation from beetles in different developmental 4500 N, 80 480 0900 W) and kept in emergence chambers, as stages described in Carrillo et al. (2012), i.e., Rubbermaid 2643-60 BRUTE 166 L containers made of opaque plastic each with two 8.3 cm Six adult females from the initial foundress group, five larvae, diameter holes cut into opposite sides one near to the top and the five tenerals, and five fully sclerotized females F1 progeny, were other near to the bottom of the container. Into each of the holes a collected when first observed while dissecting the colonized me- 0.9146 L clear Mason jar without a lid was fitted with the bottom of dium. Each individual obtained from a different colony was surface 118 L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 sterilized with 75% ethanol for one minute and subsequently and three dishes were used for each isolate each time. The exper- washed three times with sterile water. Heads and bodies of adult iment was performed for 13 isolates recovered from galleries and females, both foundresses and F1 offspring, were aseptically excised the beetle's developmental stages (Table 1). The isolate's suppres- and separately processed to isolate the symbionts associated with sive effect was determined by calculating the percentage of inhi- either the mycangium or the gut. Heads and bodies were individ- bition in radial growth according to Gaigole et al. (2011). ually macerated in 400 mL deionized sterile water; 100 mL sub- sample were plated on CSMA, and an additional 100 mL subsample þ 3. Results was plated on PDA . Plates were incubated at room temperature for 7e10 d, until fungal colonies were visible and distinguishable. 3.1. Life cycle and developmental stages of X. bispinatus

2.5. Identification of isolates In the artificial medium, eggs were first observed at 4e7 d after colony initiation (DACI), with a maximum number at 11 DACI. Eggs Isolates were initially selected for subculturing based on colony were frequently found as clusters, with a maximum number of 24 morphology. A representative of each morphotype was transferred eggs located mostly at the distal ends of the secondary galleries þ to a fresh Petri dish containing either PDA or CSMA. After (Figs. 1 and 2D). Larvae (all instars) were first observed 8e10 DACI; obtaining pure fungal cultures from the different morphotypes d the maximum number was observed 18 DACI. They appeared in each from a single spore d DNA was isolated from mycelia and/or groups of up to 15 individuals mostly in the secondary galleries spores using a modified cetyl trimethyl-ammonium bromide (Figs. 1 and 2D). Pupae were first observed 15e17 DACI with a peak (CTAB) protocol (Doyle and Doyle, 1987). The fungal isolates were number at 25 DACI. They were distributed irregularly in main and typed by sequencing a section of the nuclear large subunit 28S ri- secondary galleries (Figs. 1 and 2D). Female tenerals were first bosomal DNA (rDNA) using primers LR0R/LR5 (Vilgalys and Hester, observed 18e21 DACI, with a maximum number at day 28, while 1990) and the small subunit rDNA with primers NS1/NS4 (White males were observed 22e24 DACI with a maximum number at 33 et al., 1990). PCR products were purified using ExoSAP-IT (Affime- days. Tenerals were distributed somewhat irregularly in main and trix, CA, USA) following the manufacturer's protocols and Sanger secondary galleries (Figs. 1 and 2D). Fully sclerotized F1 generation sequencing was performed by Eurofins genomics (Louisville, KY). female and male adults were observed at days 22e24 and 29e31 The NCBI Basic Local Alignment Search Tool (BLAST) was used to respectively, with a maximum number at 35 days for both (Fig. 1). identify the nucleotide sequences. 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. 2.6. Antagonistic assays of the X. bispinatus fungal symbionts Pupae were white and motionless. The first mature fertilized fe- versus R. lauricola male was recorded emerging through the surface of the medium during 24e25 DACI. By the end of the experiment (38 DACI), the In the present study, the pathogen R. lauricola was not detected male:female ratio was 1:6 and one of the five colonies did not in galleries and the different developmental stages of the beetle. To contain any males. The mean number of individuals per develop- determine if the absence of R. lauricola was related to the ability of mental stage ±SD at 38 d was as follows: 2.2 ± 2.16 larvae; 0.6 ± 1.3 any of the X. bispinatus fungal associates to inhibit the growth of the pupae; 2 ± 2.1 teneral males; 6.2 ± 4.5 teneral females; 0.4 ± 0.54 pathogen, a dual culture technique previously described by adult males; and 8 ± 7 adult females. No eggs were observed during Campanile et al. (2007) was deployed to test antagonistic activity. the dissection of the medium at 38 d. An isolate of R. lauricola was recovered from an infected avocado tree and identified with the primer sets previously reported by Dreaden et al. (2014a). Agar disks (5 mm diam) obtained from 2 3.2. Construction of the gallery system week-old pure fungal cultures of the symbionts and from the pathogen were transferred onto Petri dishes each containing either During the first week, each foundress female excavated the CSMA or the beetle rearing medium. Each disk was placed 3 cm main gallery in parallel to the walls of the tube, and, it reached an from a R. lauricola disk. These dishes were incubated at room average length of 1.48 cm after 1 week (Fig. 2A and B). Extension of temperature for 2 weeks. The experiment was repeated three times the length of the main gallery continued for 4 weeks to reach an

Table 1 Fungal isolates: identity and frequency of isolation from gallery entrances, tunnels and protruding material (frass) during the entire study (n ¼ 55).

Fungal species Closest relative GeneBank accession no. Frequency of isolation per location (n ¼ 25) (%)

LSU (% Similarity) SSU (% Similarity) Frass Entrance Tunnel

Raffaelea subfusca KR018422 (100) KJ909306 (99) 72 60 72 Raffaelea arxii KR018419 (99) EU170279 (99) 68 76 52 Raffaelea subalba KX267102 (100) KJ909304 (99) 0 16 32 Raffaelea sp. PL1001 KJ909293 (99) KJ909294 (99) 4 24 12 Candida nemodendra NG055146 (99) EU011709 (99) 32 32 28 Candida berthetii KY106320 (98) AB054883 (99) 36 56 48 Candida sp. NRRL Y-27127 EF550293 (98) EF550431 (99) 28 32 36 Saccharomycopsis synnaedendra EU057559 (99) EU057527 (98) 8 8 4 Ambrosiozyma monospora EU011590 (99) JQ698881 (99) 40 32 32 Ambrosiozyma ambrosiae EU011593 (99) EU011673 (99) 28 24 64 Alloascoidea spa JQ689066 (93) JQ698925 (92) 64 56 60 ochroleuca AY686634 (99) GU112755 (99) 36 32 32 Stilbocrea macrostomab GQ506004 (99) AY489693 (99)

a Closest match Alloascoidea africana. b Only recovered from foundresses gut. L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 119

excavated perpendicular to the surface of the medium, with an average length of 3.2 cm and up to 13 secondary galleries with a cumulative 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 tunnel entrances and on tunnel walls

Nucleotide sequencing of fungal isolates recovered from gallery walls, gallery entrances, and extruded frass (Table 1) revealed the presence 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 bionectraceous fungi (Stilbocrea macrostoma and Bionectria ochroleuca)(Table 1). Fig. 1. Developmental stages of X. bispinatus reared in avocado sawdust based artificial R. subfusca was the most abundant species recovered in the gallery medium at 25 C. Mean number of individuals in the different developmental stages recorded twice per week (n ¼ 5). tunnel in 72% of the tubes (n ¼ 25), followed by R. arxii found in 52% of the colonies. Among the yeast species, A. ambrosiae (64%) and C. berthetii (48%) were the most frequently isolated ones. All isolates average length of 3.2 cm. After 1 week, the female foundresses had were recovered from all three locations, with exception of started construction of secondary galleries near the middle or the R. subalba, which was found at the entrances and within the gal- end of the main tunnel. The aggregate length of the secondary leries, but not in the extruded frass material (Table 1). galleries increased gradually with brood size (Fig. 2A). Oviposition fi started with the construction of the rst secondary gallery, and 3.4. Symbiont species composition of fungal gardens during beetle e peak oviposition was observed after 1 2 weeks when the average development number of secondary galleries was three with a mean length of e 2.3 cm. During the larval and pupal periods, i.e., 3 4 weeks, the After 7 d, when the foundresses started laying eggs, R. arxii was foundresses continued to extend existing galleries and excavate the most frequent associate in the freshly excavated galleries, being e new ones, which measured 6.2 7.48 cm in length. The fastest present in four of five (4/5) colonies tested at this time, while growth of the gallery system coincided with the appearance of the R. subfusca was recovered at the low frequencies of 1/5. In contrast, e fi (F1) adults after 4 5 weeks when the galleries reached their nal neither R. subalba nor R. sp. PL1001 were detected (Fig. 3). Yeast- 2 ¼ sizes (Fig. 2B). A positive correlation (R 0.9792) was found be- like organisms found in the ambrosia gardens of 7 d included tween the number of adults and the cumulative length of second- A. ambrosiae (5/5) Alloascoidea sp. (4/5), C. berthetii (3/5), and ary galleries (Fig. 2C). By the end of the experiment at 38 DACI, the C. nemodendra (2/5) (Fig. 3). fi gallery system, in the arti cial medium, consisted of a main tunnel After 14 d, when larvae were the predominant developmental

Fig. 2. Xyleborus bispinatus gallery system construction in avocado sawdust based artificial medium. (A) Mean (n ¼ 5) length of primary and secondary galleries over time. (B) Gallery construction process related to brood development. (C) Mean number of adults plotted against gallery length. (D) Schematic representation of gallery construction weekly and location of beetle developmental stages: *egg clusters, : larvae groups, A pupae, tenerals, Cmature female. 120 L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126

Fig. 3. Frequency of isolations of the fungal associates recovered from the gallery tunnels once per week (n ¼ 5) during five weeks. stage in the galleries, R. arxii continued to be the most frequent also recovered at 21 d. Among them, A. ambrosiae was the pre- fungal associate, found in five of five galleries. R. subfusca was found dominant species being found in four of five galleries. at a higher frequency (4/5) than during the previous week. In After 4 weeks (28 DACI), when the number of tenerals peaked e contrast, R. subalba and R. sp. PL1001 were not detected at 14 d although larvae and pupae were still present e R. subfusca (Fig. 3). In addition to the yeast species recovered at 7 d, three continued to show the highest frequency (5/5), while R. arxii, which species: Candida sp. NRRL Y-27127 (2/5), S. synnaedendra (1/5) and had not been found in the third week, was found in 3/5 galleries, A. monospora (2/5) were now also observed growing in the tunnels. and R. subalba was found only in one of five galleries, while Raf- C. berthetii and Alloascoidea sp. were the most frequent yeast spe- faelea sp. PL1001 was not detected (Fig. 3). The composition of the cies (5/5) (Fig. 3). yeast community was similar to that at 21 d, although After 3 weeks (21 DACI), when pupae started to appear, R. arxii C. nemodendra was not recovered from the colonies at 28 d (Fig. 3). was not detected in the galleries. However, R. subfusca was found in In the last week of colony dissection (35 DACI), the population all galleries (5/5) and R. subalba was found for the first time, being consisted mainly of adults and the number of larvae and pupae had present in four of five galleries. However, Raffaelea sp. PL1001 was decrease. Now Raffaelea sp. PL1001 was recovered for the first time still not detected (Fig. 3). Also, B. ochroleuca was recovered for the from the galleries, while R. arxii was no longer detected. Similar first time from the galleries (2/5). With exception of C. berthetii and isolation frequencies were obtained for R. subfusca (3/5), R. subalba S. synnaedendra, all the yeast species that were found at 14 d were (3/5), and Raffaelea sp. PL1001 (3/5). The yeast species isolated at L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 121 this time included Candida sp. NRRL Y-27127 (4/5), A. monospora (3/ In agreement with the fungal composition of the galleries dur- 5), C. berthetii (1/5), Alloascoidea sp. (1/5) and B. ochroleuca.(Fig. 3). ing weeks two to three, isolates obtained from the larvae corre- sponded with three Raffaelea species: R. arxii with the highest 3.5. Fungal associates of X. bispinatus developmental stages frequency (4/5, 35.5 ± 9.1 CFU), R. subfusca (3/5, 43.3 ± 19.9) and R. subalba (2/5, 155 ± 5 CFU). In addition, four yeast species; R. arxii was the most frequent (6/6 beetles; Fig. 4) and abundant A. monospora (4/5, 64 ± 33.1 CFU), S. synnaedendra (2/5, (average ± SE/female head ¼ 578.3 ± 167.4 CFU) fungal associate in 34 ± 22 CFU), Candida sp. NRRL Y-27127 (1/5, 556 CFU), and the mycangia of mature foundresses. Other Raffaelea species, Alloascoidea sp. (1/5, 80 CFU) were also identified (Figs. 4 and 5 including R. subfusca (3/6, 109.3 ± 49.8 CFU) and R. subalba (4/6, respectively). 25.5 ± 10.5 CFU), were isolated from the beetle mycangia and gut, Three Raffaelea species were isolated from teneral females at respectively. Four species of yeast were also recovered: C. berthetii similar frequencies, i.e., R. subfusca (2/5, 458 ± 132 CFU), R. arxii (1/ (6/6; 176.3 ± 45.91 CFU) isolated from the mycangia, and 5, 8 CFU), R. subalba (2/5, 28 ± 24 CFU). There was no predominant S. synnaedendra (1/6; 8 CFU) and A. ambrosiae (1/6; 48 CFU) isolated species among the yeasts, Candida sp. NRRL Y-27127 (2/5, from the gut. A. monospora was isolated from both mycangia (1/6, 271 ± 101 CFU), A. ambrosiae (2/5, 8 CFU), A. monospora (1/5, 172 CFU) and the gut (1/6, 8 CFU). Another fungal species identified 335.5 CFU) and Alloascoidea sp. (1/5, 10) (Figs. 4 and 5). as S. macrostoma (4/6, 36.5 ± 13.26 CFU) was also found in the Like the fungal gardens in the fourth week, R. subfusca was the beetle gut (Figs. 4 and 5). most frequent (5/5) and abundant fungal associate

Fig. 4. Frequency of fungal species isolated from the different beetle developmental stages. 122 L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126

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

(645.4 ± 184.5 CFU) in the mycangia of the F1 generation females, fungal associate grew until its mycelia merged with R. lauricola while R. arxii was only detected in one of five F1 females with mycelia. Only Raffaelea sp. PL1001 showed an antagonistic inter- 158 CFU. In addition, four species of yeast were identified among action with R. lauricola on CSMA, as demonstrated by the inhibition the mycangial isolates at high frequencies, i.e., C. berthetii (5/ of R. lauricola hyphal growth (Fig. 6). On the artificial rearing me- 5,149.6 ± 36.4 CFU), Candida sp. NRRL Y-27127 (5/5, dium, the interaction between R. lauricola and Raffaelea sp. PL1001 260.8 ± 137.4 CFU), Alloascoidea sp. (5/5, 78 ± 11.71 CFU), and showed inconsistent results, i.e., only one out of nine plates A. ambrosiae (2/5, 14 ± 10 CFU). In addition, C. berthetii (5/5, exhibited inhibition of R. lauricola growth. The mean (n ¼ 9) per- 224 ± 143.54 CFU) and Candida sp. NRRL Y-27127 (5/5, centage of inhibition in radial growth ± SE on CSMA was 812 ± 534.1 CFU) were also recovered from the beetle gut (Figs. 4 41.25 ± 0.13%. and 5). 4. Discussion 3.6. Antagonistic assays of X. bispinatus fungal associates versus R. lauricola 4.1. Life cycle and developmental stages of X. bispinatus

The in vitro antagonism assays on plates either of CSMA or The life cycle of one generation of X. bispinatus was investigated artificial rearing medium showed that for most of the isolates by infesting an artificial medium based on avocado sawdust and by recovered from the beetle developmental stages and the galleries, rearing the F1 progeny from the egg to the adult under laboratory there was no antagonism against R. lauricola, i.e., each X. bispinatus conditions. The developmental times for larvae, pupae, and adults L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126 123

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

Fig. 6. Test of antagonism against the Raffaelea lauricola on CSMA after two weeks of 4.2. Gallery construction, expansion, and housekeeping incubation at room temperature. (A) R. lauricola control plate. (B) Control plate microscopic view of hyphal growth at the edge of the colony. (C) Inhibition effect of Raffaelea sp. PL1001 on Raffaelea lauricola. (D) Microscopic view of hyphal growth on The development of the brood depends on the growth of sym- the assay agar plate (C). biotic fungi in the galleries (Beaver, 1989). The foundress oviposits only when the nutritional symbionts have been established in the tunnels (Peer and Taborsky, 2007). In the present study, females were 8e10, 15e17, 18e21 DACI, respectively, which coincide with started to oviposit immediately after finishing the construction of the times reported by Menocal et al. (2018) and Saucedo et al. the main galleries, i.e., by the end of the first week; at this time, (2017). The same artificial medium was used in these three sepa- fungal growth was evident as a dark layer on the walls of the gal- rate investigations, which corroborates the reproducibility of re- leries. However, eggs were laid by the foundresses only until the sults in this substrate. In the present study, the first fully sclerotized beginning of the fourth week. The placing of eggs at the distant adult was seen in the various replicates at 22e24 DACI. Similar time ends of the galleries may be an adaptation to assure the optimal use intervals for the development of the adult stage have been of both food resources and space. The foundress appears to main- observed for other artificially reared Scolytinae species, such as tain the main gallery clear to allow its use as a corridor for the Xyleborus volvulus (Menocal et al., 2017), X. ferrugineus (Saunders management of the ambrosia gardens in the main and secondary and Knoke, 1967), saxesenii (Biedermann et al., 2009), galleries and the care of eggs and larvae. (Cooperband et al., 2016), Xylosandrus muti- Once fungal symbionts were established, the colonies started to latus (Kajimura and Hijii, 1994), Xyleborus pfeili (Mizuno and expand. In X. mutilatus and X. pfeili there was a direct correlation Kajimura, 2002, 2009), and X. glabratus, in logs (Brar et al., 2013). between the number of offspring and the length of the gallery In our study teneral females eclosed first (21 DACI) followed by system (Kajimura and Hijii, 1994), which suggests that the length of teneral males (24 DACI). This female-male sequence has also been the gallery system is a factor on which the amount of food available documented in X. saxesenii (Biedermann, 2010). Although, the for the mother beetle and her brood depends, i.e., space available opposite order of eclosion of the sexes was found in Xyleborus affinis for cultivating fungi (Kajimura and Hijii, 1994; Mizuno and (Roeper et al., 1980). The earlier development of either the male or Kajimura, 2002). Likewise, in the present study, a positive corre- the female has been associated with factors that increase the lation between gallery length and brood size was observed during probability of mating (Castrillo et al., 2012). Certain differences in the initial 3 weeks of the colony, and during this period only the development between the sexes are intrinsic to each beetle species foundress was involved in tunnel excavation. After 3 weeks, as including synchrony of male and female development, male life oviposition decreased, the brood size stayed relatively steady. After span, and male insemination capacity (Castrillo et al., 2012). 4 weeks, a positive correlation between the number of new adults Biedermann (2010) hypothesized that the delay of hatching of and gallery size was observed, and indeed the greatest growth of males in X. saxesenii allowed females to reach sexual maturity the gallery system was recorded. The enlargement in gallery size before males. If such a relative delay in the hatch of male eggs were may imply an increase in the production of ambrosia fungi, which found to occur also in X. bispinatus, it would explain why females would facilitate the acquisition of fungi for the new generation of were found to eclose sooner than males in the present study. females and the later hatched individuals in the brood. A similar By 38 d the average of brood production ± SE (n ¼ 5) was behavior has been observed in X. pfeili (Mizuno and Kajimura, 19 ± 5.3 individuals, similar to the findings that Menocal et al. 2002) and X. saxesenii, the latter of which exhibits partial overlap ± (2018) reported in their experiments, i.e., (22.42 1.72). Brood of the parental and F1 generations (Biedermann et al., 2012). The F1 production seems to be species-specific and dependent on the females delay their dispersal and cooperate in the care of the am- composition of the medium, as has been documented in studies brosia gardens and the brood, suggesting a high level of sociality that tested the effect of medium constituents on offspring number (Biedermann et al., 2013). In the present study, eggs were absent at of different beetle species (Saunders and Knoke, 1967; Mizuno and the last dissection time on 38 DACI even though mature fecund Kajimura, 2002; Biedermann et al., 2009; Castrillo et al., 2012; females were still present, but which had not oviposited. This could Maner et al., 2013; Menocal et al., 2017, 2018). be caused by deterioration of the artificial medium. 124 L.F. Cruz et al. / Fungal Ecology 35 (2018) 116e126

4.3. Symbiont dynamics in the fungal gardens and in the beetle changes in humidity, in the availability of certain nutrients, or the developmental stages presence of an antagonistic organism, it is possible that other symbionts are able to proliferate and avoid the aposymbiotic state Ambrosia beetles are known to have an obligate nutritional and failure in the establishment of the new colony. Moreover, relationship with ambrosia fungi mostly belonging to the poly- multiple symbiont species also may decrease the competition for phyletic genera Ambrosiella, Raffaelea and Dryadomyces (Dreaden food resources among the various beetle developmental stages et al., 2014a, 2014b; Mayers et al., 2015). A community of ambro- even if these beetle stages do not prefer a particular symbiont sia symbiont species within the mycangia and the cultivation of species. In multipartite symbiosis in which what seems to be a more than one nutritional symbiont species is now evident in single niche is shared, the optimal conditions for each species may numerous ambrosia beetle species (Kostovcik et al., 2015). In this vary and the performance of a given symbiont member is con- study, we were able to recover four species of Raffaelea. Two of strained by environmental gradients (Six, 2012). The relative which were predominant in the fungal gardens, i.e., R. arxii and dominance of fungal species in this experiment could have been R. subalba. R. arxii is the most frequent associate in the mycangia of shaped by the conditions of the medium per se. For instance, X. bispinatus foundresses and subsequently in the fresh fungal Castrillo et al. (2012) found that the type of sawdust in the artificial gardens during the first weeks of gallery construction. Moreover, media has an effect on growth rate and mycelial density of different R. arxii is the most frequent nutritional resource used by larvae. It is strains of Ambrosiella hartigii, a symbiont of X. germanus. Thus, the thought that the larva is the stage with the greatest food re- rapid growth in the medium of R. arxii and subsequent decline in quirements. Therefore, the most dominant fungal symbiont in the occurrence and the proliferation of R. subfusca could be galleries during the larval growth period is likely to be crucial for conditioned by the medium i.e., depletion of certain nutrients or the development of the beetles (Beaver, 1989). R. arxii seemed to change in humidity, and not driven by a developmental stage- depopulate progressively with the development of the gallery and specific requirement. the appearance of the adult F1 progeny of X. bispinatus. In contrast, R. subfusca the other prevalent species, whose fraction in the gar- 4.4. Antagonistic assays of the X. bispinatus fungal symbionts dens increased progressively during the course of the experiment, versus R. lauricola was the most frequent and abundant associate in the mycangia of the mature F1 generation females. R. subalba, the only Raffaelea sp. R. lauricola was not recovered from either the beetles at different isolated from the foundress guts, appeared in the galleries in the developmental stages (n ¼ 21) or from the galleries at the different second week and was found at relative low frequencies until the dissection times (n ¼ 25). To determine a possible antagonistic ef- end of the experiment. R. subalba was recovered from larvae and fect of any of the isolates recovered from the experiment, we per- tenerals but not from F1 generation females. Raffaelea sp. PL1001 formed an antagonistic assay on CSMA and on the rearing media. was only found in the galleries at the last dissection time i.e., 38 The assay demonstrated that only Raffaelea sp. PL1001 was able to DACI. The distinctively great dominance of R. subfusca, R. arxii, and inhibit the growth of R. lauricola. However, Raffaelea sp. PL1001 was R. subalba in the gardens and in the different developmental stages only found in the galleries 38 DAIC. Due to its low frequency, the during this experiment may suggest a preference or a nutritional presence of Raffaelea sp. PL1001 does not seem to be a determinant requirement of a particular beetle developmental stage for a spe- factor to explain the absence of R. lauricola. One possibility is that cific species of Raffaelea as has been observed in other species. R. lauricola was not present in the population used for the experi- Euwallacea nr. fornicatus harbors three symbionts: Graphium ment or that if present, R. lauricola was undetected in the various euwallaceae, Acremonium pembeum, and Fusarium euwallaceae. samples taken from galleries and beetles due to its low frequency Freeman et al. (2016) reported that G. euwallaceae was the pre- and scarcity. Nevertheless, R. lauricola was previously found in a dominant symbiont in the initial stages of gallery development and great percentage of the X. bispinatus tested for the pathogen (Ploetz used as a primary food source for larval development, whereas et al., 2017). Native symbionts should be better adapted to their F. euwallaceae was the food source for adult stages (Freeman et al., beetle host and consequently carried in larger numbers in their 2016). Even though, in our experiments the mature stages, foun- mycangia allowing them to establish more efficiently in the gal- dresses and F1 females, did not exhibit the same symbiont species leries under the experimental conditions used in this study. In their dominance, our data may still suggest a certain level of food pref- study, Menocal et al. (2018) found low colonization of the mycangia erence. This might be explored by rearing a second generation in when the medium was inoculated with R. lauricola contrary to the order to determine whether the foundresses are able to control the findings of Saucedo et al. (2017). Mature females containing the growth of a given fungal species in the galleries regardless of the native symbionts were used by Menocal et al. (2018), while relative abundance of various symbiont species in their mycangia. Saucedo et al. (2017) performed their study with newly eclosed Saucedo et al. (2017) tested the role of R. arxii, R. subfusca, and females ensuring the exclusive presence of R. lauricola in their R. subalba, as nutritional symbionts of X. bispinatus. Their findings mycangia. It is worth mentioning that R. lauricola, when present, indicate that males of X. bispinatus were able to complete their life seems not to alter the community of X. bispinatus fungal symbionts, cycle when fed solely on one of these symbionts species, indicating as observed by Menocal et al. (2018). In their study, media inocu- the lack of fidelity in this mutualistic interaction, or flexibility in the lated and non-inoculated with R. lauricola exhibit similar fungal beetle's association with its repertoire of symbionts (Saucedo et al., symbiont constituents, which accords with the current study. 2017). Interestingly, slightly larger broods were obtained by col- onies fed on R. arxii followed by R. subfusca and R. subalba although 4.5. Yeasts and bionectraceous fungi associated with fungal gardens these differences were not statistically significant (Saucedo et al., and beetle developmental stages 2017). Functional redundancy in the symbiont repertoire may result in In addition to the Raffaelea spp., seven species of yeast were an advantage by increasing beetle fitness. According to Biedermann recovered from galleries and different developmental stages of the et al. (2013), the presence of multiple symbiont species ensures the beetle. These included C. berthetii, Candida sp. NRRL Y-27127, survival of the under dissimilar conditions. Thus, if there is C. nemodendra, A. ambrosiae, A. monospora, S. synnaedendra and, any accidental loss of a symbiont species during dispersal, or the Alloascoidea sp. With the exception of C. berthetii, which was only conditions are less suitable for one of the symbiont species e.g., found 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 regular Acknowledgements pattern for their appearance in the galleries and their presence seemed to depend on what randomly a certain beetle develop- Thanks to Waldemar Klassen and Jorge E. Pena~ (University of mental stage was carrying at a given dissection time. According to Florida) for suggestions to improve the manuscript. Thanks to Julio the CBS-database from the Westerdijk Fungal Biodiversity Institute, Mantilla, Jose Alegría, and Rita E. Duncan for experimental set-up. C. berthetii was found associated with the galleries constructed by This research was funded by NIFA grant 2015-51181-24257 to bark and ambrosia beetles species in various host-plants, including Daniel Carrillo. the tunnels of X. volvulus in Cussonia umbellifera (Araliaceae), Platypus externedentatus in Macaranga capensis (Euphorbiaceae), References and P. externedentatus in Ficus sycomorus (Moraceae) (west- erdijkinstitute.nl/Collections). Similarly, C. nemodendra was initially Atkinson, T.H., Carrillo, D., Duncan, R.E., Pena,~ J.E., 2013. Occurrence of Xyleborus isolated from tunnels of pin-borer beetles, Xyleborus aemulus, in bispinatus (Coleoptera: Curculionidae: scolytinae) Eichhoff in southern Florida. Zootaxa 3669, 96e100. South Africa (westerdijkinstitute.nl/Collections). Candida sp. NRRL Bateman, C., Kendra, P.E., Rabaglia, R., Hulcr, J., 2015. Fungal symbionts in three Y-27127 was identified among the mycangial fungi of X. glabratus exotic ambrosia beetles, Xylosandrus amputatus, Xyleborinus andrewesi, and (Harrington and Fraedrich, 2010). The genus Ambrosiozyma is well- Dryoxylon onoharaense (Coleoptera: Curculionidae: scolytinae: Xyleborini) in Florida. Symbiosis 66, 141e148. known to be associated with the galleries and/or different devel- Beaver, R.A., 1989. -fungus relationships in the bark and ambrosia beetles. In: opmental stages of beetles in the groups Platypodidae or Scoly- Wilding, N., Collins, N.M., Hammind, P.M., Webber, J.F. (Eds.), Insect-fungus toidea, as observed in samples obtained in South Africa, USA, and Interactions. Academic, London, United Kingdom, pp. 121e143. Biedermann, P.H.W., 2010. Observations on sex ratio and behavior of males in Japan (van der Walt, 1972). S. synnaedendra has been isolated from Xyleborinus saxesenii Ratzeburg (Scolytinae, Coleoptera). ZooKeys 56, 253e267. the tunnels of P. externedentatus in South Africa (Van Der Walt and Biedermann, P.H.W., Klepzig, K.D., Taborsky, M., 2009. Fungus cultivation by am- Scott, 1971). The genus Alloascoidea was recently distinguished brosia beetles: behavior and laboratory breeding success in three xyleborine species. Environ. Entomol. 38, 1096e1105. from Ascoidea (Kurtzman and Robnett, 2013); the members of Biedermann, P.H.W., Klepzig, K.D., Taborsky, M., Six, D.L., 2013. Abundance and Ascoidea are all known to be associated with decaying , bark dynamics of filamentous fungi in the complex ambrosia gardens of the primi- beetles, and insect galleries in trees (de Hoog and Smith, 2011). In tively eusocial beetle Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculioni- e spite of their ubiquity and abundance, no studies have been con- dae, Scolytinae). FEMS Microbiol. Ecol. 83, 711 723. Biedermann, P.H.W., Peer, K., Taborsky, M., 2012. Female dispersal and reproduction ducted to determine their role in the beetle-ambrosia fungi in the ambrosia beetle Xyleborinus saxesenii Ratzeburg (Coleoptera; Scolytinae). mutualistic system of these two genera. Yeast species have been Mitt. Dtsch. Ges. Allg. Angew. Entomol 18, 231e235. considered to be non-specific, opportunistic, commensals or para- Blackwell, M., 2017. Yeasts in insects and other invertebrates. In: Buzzini, P., fi Lachance, M.A., Yurkov, A. (Eds.), Yeasts in Natural Ecosystems: Diversity. sites, due to their non-speci c relationship with different species of Springer, Cham, pp. 397e433. bark or ambrosia beetles (Blackwell, 2017). It is not known if other Bleiker, K.P., Potter, S.E., Lauzon, C.R., Six, D., 2009. Transport of fungal symbionts by genera of yeasts besides Ambrosiozyma, which are considered true mountain pine beetles. Can. Entomol. 141, 503e514. Brar, G.S., Capinera, J.L., Kendra, P.E., McLean, S., Pena,~ J.E., 2013. Life cycle, devel- ambrosia symbionts, are functionally associated with ambrosia opment, and culture of (Coleoptera: Curculionidae: scoly- beetles (Hulcr and Stelinski, 2017). Hypothetical functions have tinae). Fla. Entomol. 96, 1158e1167. been established based on their enzymatic profiles and on their Campanile, G., Ruscelli, A., Luisi, N., 2007. Antagonistic activity of endophytic fungi towards Diplodia corticola assessed by in vitro and in planta test. Eur. J. Plant production of volatiles (Davis, 2015), such as mediation of Pathol. 117, 237e246. competitive interactions, production of semiochemicals, defenses Carrillo, D., Duncan, R.E., Pena,~ J.E., 2012. Ambrosia beetles (Coleoptera: Curculio- against plant toxins, and nutritional supplements or nutrient nidae: scolytinae) that breed in avocado wood in Florida. Fla. Entomol. 95, 573e579. cycling (Davis, 2015). Carrillo, D., Duncan, R.E., Ploetz, J.N., Campbell, A.F., Ploetz, R.C., Pena,~ J.E., 2014. In addition to the Raffaelea and yeast species, two bio- Lateral transfer of a phytopathogenic symbiont among native and exotic am- nectraceous fungi (Ascomycota: : ), brosia beetles. Plant Pathol. 63, 54e62. were identified. These were B. ochroleuca and S. macrostoma. There 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- is little information regarding these two fungi. They seem likely to neum (Ascomycota: Hypocreales) against adult Xylosandrus germanus (Coleop- have been introduced accidentally in the beetle-gallery system. tera: Curculionidae) and impact on brood. Biol. Contr. 58, 121e126. Both of them are known to occur on living plant material. Castrillo, L.A., Griggs, M.H., Vandenberg, J.D., 2012. Brood production by Xylosandrus fi germanus (Coleoptera: Curculionidae) and growth of its fungal symbiont on B. ochroleuca has been identi ed as a plant endophyte in various artificial diet based on sawdust of different tree species. Environ. Entomol. 41, species (Paul et al., 2013). This species can also produce metabolites 822e827. with antibacterial and antifungal activity (Samaga et al., 2014) and Cooperband, M.F., Stouthamer, R., Carrillo, D., Eskalen, A., Thibault, T., Cosse, A.A., Castrillo, L.A., Vandenberg, J.D., Rugman-Jones, P.F., 2016. Biology of two be entomopathogenic (Guesmi-Jouini et al., 2014). members of the Euwallacea fornicatus species complex (Coleoptera: Curculio- To summarize, we reported the developmental biology of nidae: scolytinae), recently invasive in the USA, reared on an ambrosia beetle X. bispinatus and documented its fungal symbionts. Based on our artificial diet. Agric. For. Entomol 18, 223e237. Davis, T.S., 2015. 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