Fungal Ecology 35 (2018) 116e126 Contents lists available at ScienceDirect Fungal Ecology journal homepage: www.elsevier.com/locate/funeco Developmental biology of Xyleborus bispinatus (Coleoptera: Curculionidae) reared on an artificial medium and fungal cultivation of 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, 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 beetles 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 avocado 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 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, Keywords: Raffaelea subalba and seven yeast species 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 Ascomycota (Ascomy- 2005). In this mutualistic interaction, ambrosia beetles proliferate cota: Sordariomycetes), in the orders Ophiostomatales 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: Ophiostomataceae) (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 fungus 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 Lauraceae, 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.