Environmental Entomology, XX(X), 2017, 1–9 doi: 10.1093/ee/nvx151 Insect–Symbiont Interactions Research Rearing Xyleborus volvulus (Coleoptera: Curculionidae) on Media Containing Sawdust from Avocado or Silkbay, With or Without Raffaelea lauricola (Ophiostomatales: Ophiostomataceae) Octavio Menocal,1,3 Luisa F. Cruz,1 Paul E. Kendra,2 Jonathan H. Crane,1 Randy C. Ploetz,1 and Daniel Carrillo1 1Tropical Research & Education Center, University of Florida, 18905 SW 280th Street, Homestead, FL 33031-3314, 2USDA-ARS, Subtropical Horticulture Research Station, 13601 Old Cutler Road, Miami, FL 33158-1857, and 3Corresponding author, e-mail: [email protected] Subject Editor: Steve Perlman Received 8 June 2017; Editorial decision 20 August 2017 Abstract Like other ambrosia beetles, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) lives in a mutualistic symbiotic relationship with fungi that serve as food source. Until recently, X. volvulus was not considered a pest, and none of its symbionts were considered plant pathogens. However, recent reports of an association between X. volvulus and Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the cause of the laurel wilt disease of avocado (Persea americana Mill. [Laurales: Lauraceae]), and its potential role as vector of the pathogen merit further investigation. The objective of this study was to evaluate three artificial media containing sawdust obtained from avocado or silkbay (Persea humilis Nash) for laboratory rearing of X. volvulus. The effect of R. lauricola in the media on the beetle’s reproduction was also evaluated. Of the three media, the one with the lowest content of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Reproduction on this medium was not affected by the sawdust species or the presence of R. lauricola. On the other two media, there was a significant interaction between sawdust species andR. lauricola. The presence of R. lauricola generally had a negative effect on brood production. There was limited colonization of the mycangia of X. volvulus by R. lauricola on media inoculated with the pathogen. From galleries formed within the best medium, there was 50% recovery of R. lauricola, but recovery was much less from the other two media. Here, we report the best artificial substrate currently known forX. volvulus. Key words: ambrosia beetle, ambrosia fungi, artificial rearing, beetle–fungus symbiosis, laurel wilt Laurel wilt (LW) is a lethal vascular disease of avocado (Persea R. lauricola to avocado trees under no-choice conditions (Carrillo americana Mill. [Laurales: Lauraceae]) and other woody species et al. 2014). within the Lauraceae. The causal agent of laurel wilt is the fungal X. volvulus, a pantropical species that probably originated in pathogen, Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva the Neotropical realm, has become widely distributed throughout (Ophiostomatales: Ophiostomataceae). The primary vector of Florida, Central and South America, and the Caribbean (Wood 1982, R. lauricola in native ecosystems, including natural hammocks of Gohli et al. 2016). This beetle has a broad host range that includes the Florida Everglades, is the redbay ambrosia beetle, Xyleborus species in 24 plant families including the Lauraceae (Atkinson 2016). glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae: Other ambrosia beetles that have been introduced to the New World Xyleborini) (Kendra et al. 2014, Hughes et al. 2015). However, are important pests (i.e., X. glabratus (Fraedrich et al. 2008, Hanula X. glabratus is rarely associated with laurel wilt-affected avocado et al. 2008, Brar et al. 2013, Maner et al. 2013), Xylosandrus com- trees in south Florida (Carrillo et al. 2012). Recently, R. lauricola pactus (Eichhoff) [Coleoptera: Curculionidae] (Greco and Wright was found in at least nine other ambrosia beetle species isolated 2015), Euwallacea spp. (Carrillo et al. 2016, Cooperband et al. from avocado (Carrillo et al. 2014, Ploetz et al. 2017). Two of the 2016), Xylosandrus germanus (Eichhoff), and Xylosandrus cras- species, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) siusculus (Motschulsky) [Coleoptera: Curculionidae] (Castrillo and Xyleborus bispinatus Eichhoff, were capable of transmitting et al. 2012, Ranger et al. 2016)). In Florida, X. volvulus occurs © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected] 1 Downloaded from https://academic.oup.com/ee/article-abstract/doi/10.1093/ee/nvx151/4383829/Rearing-Xyleborus-volvulus-Coleoptera by University of Florida user on 12 October 2017 2 Environmental Entomology, 2017, Vol. XX, No. X sympatrically with X. glabratus and breeds in hosts affected by LW [44-gallon (167 L) Brute® container (2643–60 Rubbermaid®) with (Kendra et al. 2011, Carrillo et al. 2012). Although X. volvulus has a 2-quart Mason jar attached to a port on one of each side of the not been associated with economic damage to trees, its experimental chamber, as described in Carrillo et al. (2012)]. Rolled moistened transmission of R. lauricola to avocado (Carrillo et al. 2014) indi- paper towels were placed inside the jars to collect beetles emerging cates that the association between the beetle and this pathogen war- from the logs and attracted to light. Fully sclerotized (dark brown) rants further investigation. females were collected daily and identified as X. volvulus based on Ambrosia beetles are difficult to study because of their cryptic life their morphological characteristics (Rabaglia et al. 2006). style. They bore through the bark of a host tree and form galleries within the xylem. Ambrosia beetles complete their life cycle in these Artificial Media galleries, where they actively cultivate symbiotic fungi that serve as In February 2016, avocado logs were collected from an unsprayed their primary food (Rudinsky 1962, Farrell et al. 2001). avocado orchard in Miami-Dade County (25° 29’ 38” N 80° 28’ 53” The ambrosia beetle–fungal symbiosis is an area of active W), and silkbay logs were collected from the Archbold Biological research. Recent studies revealed that Xyleborus species consist- Station in Highlands County, FL (27° 10’ 50” N 81° 21’ 0” W). The ently carry not only multiple dominant fungal associates but also logs were debarked and dried for 4 d in an industrial oven at 75°C fungi from the environment, including plant pathogens and endo- and then cut into smaller pieces using a miter saw. A sander was used phytes (Kostovcik et al. 2015). However, there is limited information to create sawdust from the xylem-sapwood layer. The sawdust was regarding their biology, behavior, and the functional role of their sifted through a 12 mm sieve and stored at -18 ºC until it was used symbiotic associations. Establishment of colonies of these insects to prepare media. would allow studies on their development, physiology, behavior, Three types of artificial media were evaluated (Table 1). Medium colony composition and size, and enable ambrosia beetle–fungus 1 with sawdust from avocado or silkbay (designated as AM1 and associations to be manipulated to improve understandings of this SM1, respectively) was prepared using the ingredients and proce- taxonomic group, and their direct and indirect effects on host trees. dures described by Castrillo et al. (2011). Medium 2 (either as AM2 Artificial media have been used to mass rear insects, test com- or SM2) was prepared using different proportions of the same ingre- pounds for physiological effects, and study insect nutrition and dients as proposed by Biedermann et al. (2009). Medium 3 (either as behavior (Vanderzant 1974). According to Singh (1977), ‘an artifi- AM3 or SM3) was prepared with the same ingredients in the same cial medium is an unfamiliar substrate, which has been formulated, quantities as in Medium 2, but more water was added to facilitate synthesized, processed, and/or concocted by man, and on which an manipulation while transferring the medium into rearing tubes. insect in captivity can develop through all or part of its life cycle.’ All dry ingredients (sawdust, agar, sucrose, starch, yeast, casein, In the case of ambrosia beetles, culture conditions must be suitable Wesson’s salt mixture, and tetracycline) were mixed in a 600 ml for both the symbiotic fungi and the beetles (Maner et al. 2013). An beaker. Then, with constant stirring, liquid ingredients were added effective medium requires an in-depth understanding of the insect’s in the following order: wheat germ oil, peanut oil, ethanol, and biology, behavior, and physiology. Ideally, an artificial medium for water. Homogenized media were autoclaved at 121°C for 30 min ambrosia beetles should serve as a nutritional substrate for the fun- and were stirred to re-suspend settled ingredients; 15 ml was poured gal symbiont and support the economical production of large num- into 50 ml sterile plastic centrifuge tubes (Fisher Scientific Catalog bers of healthy insects that are similar to those living in the natural no. 0644318, Suwanee, GA) that were loosely capped, tapped to environment (Adeyeye and Blum 1988). remove air bubbles, and allowed to cool in the laminar flow hood Xyleborus ferrugineus F. was the first ambrosia beetle success- for 24 h. Medium 2 was dispensed in the plastic tubes before auto- fully reared on an artificial medium (Saunders and Knoke 1967). claving due to its more