Rearing Xyleborus Volvulus (Coleoptera: Curculionidae) On

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

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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 solid consistency. After autoclaving, the tubes Subsequently, at least six other ambrosia beetle species have been reared containing Medium 2 were transferred to the laminar flow hood and artificially, including: Xyleborus dispar F. ( French and Roeper 1972), allowed to cool as before. Xyleborus pfeili Ratzeburg (Mizuno and Kajimura 2002, Mizuno and Kajimura 2009), Xyleborus affinis Eichhoff, Xyleborinus saxesenii Ratzeburg (Biedermann et al. 2009, Biedermann 2010), Xylosandrus Media Inoculation germanus (Biedermann et al. 2009, Castrillo et al. 2012), X. glabratus A subset of the tubes containing each of the three media was (Maner et al. 2013), and Euwallacea spp. (Cooperband et al. 2016). inoculated with an isolate of R. lauricola obtained from the Plant Here, we describe a series of studies that evaluate three artificial media Diagnostic Clinic at the Tropical Research and Education Center for rearing X. volvulus. The media incorporated sawdust obtained (denoted + RL). Conidia were harvested from cultures of the isolate from two of its major hosts, silkbay (Persea humilis Nash), a species by gently scraping the culture surface with a sterile plastic rod (Fisher endemic to central Florida that is frequently colonized by ambrosia bee- Scientific Catalog no. 23600896). The resulting suspension was tles following LW development (Kendra et al. 2012), and avocado, an transferred to a sterile 50 ml plastic centrifuge tube using a dispos- 2 3 4 important agricultural species in south Florida that is also threatened by able sterile pipette (5 ml). Serial dilutions (1, 10, 10 , 10 , and 10 ) LW. In addition, since R. lauricola has been isolated from X. volvulus were then plated on CSMA medium (cycloheximide, streptomycin, recovered from both of these hosts (Carrillo et al. 2012, Carrillo et al. malt, and agar), which is selective for species of Ophiostomatales, to 2014, Ploetz et al. 2017), we studied interactions of X. volvulus with determine the number of colony-forming units (CFUs) in the original 6 this fungus with the developed laboratory rearing methods. suspension; 500 µl of the original, which contained 8.2 × 10 CFUs, was added per tube of solidified medium. Tubes were loosely capped and maintained in a sterile environment for 10 d to allow fungal Materials and Methods growth on the medium. Ambrosia Beetles X. volvulus females were obtained from sections of logs (approx- Rearing Conditions imately 50 × 16 cm, length by diameter) collected from avocado Before introducing females into rearing tubes, four small holes were trees that were affected by LW and placed in emergence chambers made on the surface of the medium to facilitate the initiation of

Table 1. Recipes of the three types of artificial media using either avocado or silkbay sawdust for rearingX. volvulus

Ingredients Media type Manufacturer/Source

Type 1: AM1 or Type 2: AM2 or Type 3: AM3 or SM1 SM2 SM3

Sawdust (either avocado or 45 g 84 g 84 g Dried and stored sawdust as described in Materials and silkbay) Methods Granulated agar 12 g 12.6 g 12.6 g Difco Agar, Dickinson & Co., Sparks, MD Sucrose 6 g 2.1 g 2.1 g Fisher Scientific, Fair Lawn, NJ Starch 3 g 2.1 g 2.1 g Fisher Science Education, Nazareth, PA Yeast 3 g 2.1 g 2.1 g Fisher Science Education, Nazareth, PA Casein 3 g 4.2 g 4.2 g MP Biomedicals, LLC, Solon, OH Wesson’s salt mixture 0.6 g 0.52 g 0.52 g MP Biomedicals, LLC, Solon, OH Tetracycline 0.21 g 0.14 g 0.14 g Fisher Scientific, Fair Lawn, NJ Wheat germ oil 1.5 ml 1.05 ml 1.05 ml Frontier Scientific Services, Newark, DE Peanut oil - 1.05 ml 1.05 ml Ventura Foods, LLC, Brea, CA 95% ethanol 3 ml 2.1 ml 2.1 ml Decon Labs, Inc., King of Prussia, PA

Distilled H2O 370 ml 244 ml 540 ml

Type 1 medium was prepared using the ingredients and procedures described by Castrillo et al. (2011), except that avocado or silkbay sawdust was used. Type 2 and Type 3 media were prepared using the ingredients and procedures described by Biedermann et al. (2009), except that avocado or silkbay sawdust was used, and much more water was added into the Type 3 medium.

boring activity. Females of X. volvulus were collected from the emer- grinder (Pyres no. 7727-07). Then, 100 µl of each macerate solu- gence chambers and surface-disinfested in 70% ethanol for 5–7 s. tion were plated onto CSMA. In addition, fungal samples were col- Active and vigorous females were individually placed into each of 12 lected from galleries using sterile inoculation loops (Fisher Scientific tubes of each medium that was tested. Rearing tubes were then incu- Catalog no. 22170206) to scrape small portions of the tunnel where bated horizontally in plastic containers in a walk-in rearing room, in immature stages were found, and plated on CSMA medium. After complete darkness and at 25 ± 1°C, 75% RH. Tubes were inspected 7–10 d, the identity of colonies with the morphology of R. lauri- every 3 d for the appearance of lose medium on its surface, pushed cola (Harrington et al. 2010) was confirmed with two diagnostic out by females during tunneling (an indication of gallery formation), microsatellite markers, CHK and IFW (Dreaden et al. 2014). The and the sides of the tubes were examined for visible galleries. The number of CFUs of R. lauricola was calculated for each beetle, and number of days to the first occurrence of eggs, larvae, pupae, and the presence or absence of the fungus in galleries was determined. adults on the surface and in galleries were recorded, and the exper- In addition, fungal isolations from a subset of beetles and galleries iment was concluded after the uninterrupted development of two were identified by amplifying a portion of the nuclear large subunit generations. A 40-d generation time was used based on previous 28S ribosomal DNA (rDNA) using primers LR0R/LR5 (Vilgalys and findings by Maner et al. (2013) in similar studies on X. glabratus, Hester 1990). which could apply for other ambrosia beetles. Statistical Analysis Gallery Dissection The statistical software package SAS was used for all analyses. For After 40 d (i.e., first generation), the medium in each tube containing each medium type, the number of adult females and total brood pro- a beetle colony (brood) was dissected under a stereomicroscope in duction were compared between the sawdust species (avocado or a laminar flow hood. All developmental stages on the surface of the silkbay) with and without the inoculation with R. lauricola, for a medium were recorded before the medium was tapped out of the total of four treatments per medium. Each medium type was eval- tube into a Petri dish, and all beetle stages on the sides of the tubes uated separately and was considered a separate experiment. The were recorded. Finally, the medium was systematically cut into small effects of sawdust species and the presence of R. lauricola were pieces from the bottom to the top of the medium plug. Gallery tun- evaluated independently for each medium using two-way analysis of nels were opened carefully by removing the medium around them. variance (PROC GLIMMIX, SAS Institute 2010, v. 9.3). In addition, Eggs, larvae, pupae, and adults were removed, counted and placed brood size was compared between the first and second generations in separately into Petri dishes using a fine paintbrush. Adult mortality each medium type. Data were square-root transformed before anal- was also recorded. Twelve mature active females from each treat- ysis, and means were separated with Tukey’s HSD. ment were surface sterilized, and reared in new tubes (i.e., same treatment) prepared as earlier to produce a second generation. Results Fungal Isolation Medium 1 One mature female from each colony inoculated with R. lauricola The type of sawdust in medium 1 (Table 1) had no significant effect [first and second generation colonies in Media 1 and 3 (n = 24 for on the reproduction of X. volvulus (F = 0.03; df = 1, 95; P = 0.8521) each medium), and second generation in Medium 2 (n = 12), Table 3] (Fig. 1A). Brood sizes were generally greater on media not inocu- were surface-disinfested by immersing in 70% ethanol for 30 s, and lated with R. lauricola when compared with media inoculated with subsequently washed in sterile deionized water three times. The this pathogen, but these differences were not statistically significant head and pronotum were separated from the abdomen, and mac- (F = 2.42; df = 1, 95; P = 0.1222) (Fig. 1A). Similarly, female offspring erated separately in 200 µl of sterile water using a motorized tissue were greater on media without R. lauricola when compared with

Fig. 1. Number of X. volvulus female offsprings and total brood produced per single female founder cultured in one of three artificial media (a = medium 1; b = medium 2; and c = medium 3) either inoculated or not inoculated with R. lauricola. Media contained sawdust either of avocado or silkbay. Bars represent the mean (± SE) number of female offsprings (black) and total brood (light) produced per foundress. Error bars with the same letters are not significantly different P( < 0.05). AM1, Avocado medium 1; AM1 + RL, Avocado medium 1 inoculated with R. lauricola; SM1, Silkbay medium 1; SM1 + RL, Silkbay medium 1 inoculated with R. lauricola; AM2, Avocado medium 2; AM2 + RL, Avocado medium 2 inoculated with R. lauricola; SM2, Silkbay medium 2; SM2 + RL, Silkbay medium 2 inoculated with R. lauricola; AM3, Avocado medium 3; AM3 + RL, Avocado medium 3 inoculated with R. lauricola; SM3, Silkbay medium 3; SM3 + RL, Silkbay medium 3 inoculated with R. lauricola.

media inoculated with the pathogen; however, these differences were in the first (data not shown). Overall, the mean number of prog- not statistically significant (F = 1.76; df = 1, 95; P = 0.6301) (Fig. 1A). eny (± SE) in the first generation was 8.84 ± 1.60 and 15.88 ± 2.07 The number of male progeny per brood ranged from zero to in the second generation. The percentage of female founders that four. Males were present in 85 and 78% of AM1 and AM1 + RL established colonies in AM1 was 59 and 50 in the first and second broods, respectively, and in 77 and 64% of the SM1 and SM1 + RL generations, respectively. In contrast, the increase in brood establish- broods, respectively. Adult mortality in broods in the four variations ment by female founders between the first and second generations of Medium 1 increased in the following order: AM1 (16%) < SM1 increased from 25 to 92% in AM1 + RL, from 59 to 92% in SM1, (21%) < AM1 + RL (22%) < SM1 + RL (33%) (Table 2). and from 59 to 84% in SM1 + RL (data not shown). In all treatments, there was significantly more reproduction No eggs were visible in the galleries along the rearing tube walls. (F = 17.17; df = 1, 95; P < 0.0001) in the second generation than Larvae, pupae, and adults were first observed on Days 15, 19, and

Table 2. Biological parameters of X. volvulus populations reared in one of the three artificial media types, each containing sawdust of either avocado or silkbay, and each either inoculated or not inoculated with R. lauricola

Mediuma Average no. of offspring per tube after 40 d (%) of Adult N with N with N females in mortality offspring (any females Eggs Larvae Pupae Male Female Brood brood (%) stage) (%) (%) adults adults (all stages combined)

AM1 0.04 3.29 0.50 0.88 11.04 15.75 70 16.1 13 (54) 11 (45) 24 AM1 + RL 0.04 0.92 0.04 0.71 9.21 10.92 84 22.7 14 (58) 14 (58) 24 SM1 0.29 3.08 0.33 0.88 9.08 13.67 66 21.3 18 (75) 18 (75) 24 SM1 + RL 0.08 2.38 0.29 0.75 5.58 9.08 61 33.6 17 (70) 15 (62) 24 AM2 0.63 0.71 0.29 0.42 5.50 7.54 73 16.2 10 (41) 10 (41) 24 AM2 + RL 0.00 0.08 0.00 0.00 1.21 1.29 94 82.8 3 (12) 2 (8) 24 SM2 0.54 0.71 0.08 0.54 8.00 9.88 81 17.6 20 (83) 20 (83) 24 SM2 + RL 0.42 1.71 0.08 0.54 6.71 9.46 71 14.4 15 (62) 11 (45) 24 AM3 0.75 1.88 0.38 0.79 7.33 11.13 66 37.4 15 (62) 14 (58) 24 AM3 + RL 0.13 0.63 0.17 0.25 2.67 3.83 70 37.1 11 (45) 8 (33) 24 SM3 0.04 0.50 0.00 0.21 2.67 3.42 78 39.1 9 (37) 7 (24) 24 SM3 + RL 0.00 0.67 0.21 0.13 1.29 2.29 56 58.8 6 (25) 3 (15) 24

AM1, Avocado medium 1; AM1 + RL, Avocado medium 1 inoculated with R. lauricola; SM1, Silkbay medium 1; SM1 + RL, Silkbay medium 1 inoculated with R. lauricola; AM2, Avocado medium 2; AM2 + RL, Avocado medium 2 inoculated with R. lauricola; SM2, Silkbay medium 2; SM2 + RL, Silkbay medium 2 inoculated with R. lauricola; AM3, Avocado medium 3; AM3 + RL, Avocado medium 3 inoculated with R. lauricola; SM3, Silkbay medium 3; SM3 + RL, Silkbay medium 3 inoculated with R. lauricola; N, number of batches of the medium. aNote that each medium was evaluated separately, and was considered a separate experiment.

24, respectively, after the female founders had been introduced adults were observed at 18, 25, and 31 d after female introduc- into AM1 and SM1. In AM1 + RL, larvae, pupae, and adults were tion. In AM2, larvae, pupae, and adults were seen at 20, 24, and observed at 18, 23, and 27 d after the females had been introduced, 29 d, respectively. In AM2 + RL, no larvae or pupae were observed, respectively. In SM1 + RL, larvae, pupae, and adults were observed although adults were seen at 33 d after female introduction. later than in all other treatments, i.e., at 21, 27, and 31 d after the females had been introduced, respectively. Medium 3 There was a significant interaction (F = 3.91; df = 3, 95; P = Medium 2 0.0103) between sawdust species and R. lauricola. The presence of There was a significant interaction between the species of sawdust R. lauricola in both media (i.e., avocado and silkbay) had a neutral and the presence of R. lauricola (F = 6.27; df = 3, 95; P = 0.0005); or negative effect on female offspring and brood production com- female production, but not total brood, was significantly lower pared with media without the pathogen, although this effect was in R. lauricola-inoculated medium containing avocado sawdust, not significant (Fig. 1C). The number of males per brood ranged whereas neither differed on silkbay sawdust medium, with or with- from zero to three. Males were present in 73% of the broods in out R. lauricola (Fig. 1B). The size of the brood produced declined in AM3, 54% in AM3 + RL, 44% in SM3, and 33% in SM3 + RL. the following order: SM2 > SM2 + RL > AM2 > AM2 + RL (Fig. 1B). Adult mortality was similar in AM3 (38%), SM3 (39%), and AM3 The number of males per brood ranged from zero to two. Males were + RL (37%), but mortality was greater in SM3 + RL (58%) (Table present in 60% of the broods in SM2 and SM2 + RL, and in 90% of 2). the broods in AM2 (data not shown). No males were observed in the There was no significant difference in brood size between the first AM2 + RL broods. Adult mortality was similar in SM2 (17%), AM2 and second generation (F = 0.17; df = 1, 95; P = 0.6785) (data not (16%), and SM2+RL (14%). However, much adult mortality (82%) shown). The mean number of progeny produced (± SE) in the first was observed in AM2 + RL (Table 2). generation was 6.17 ± 1.57 compared with 4.17 ± 0.62 in the sec- The average brood size produced in the first genera- ond generation. The percentages of female founders that established tion (6.40 ± 1.32) was less than that in the second generation broods in AM3 were 66 and 50 in the first and second generations, (7.69 ± 1.23), although this difference was not statistically signif- respectively. In contrast, in AM3 + RL, the percentages of female icant (F = 1.08; df = 1, 95; P = 0.3005) (data not shown). The founders that established colonies increased from 25 to 66 from the percentage of female founders that established colonies increased first to the second generation. The percentages of brood establish- from the first to the second generation in non-inoculated media, ment increased from 33 to 41 in SM3, and from 8 to 41 in SM3 + but decreased in media inoculated with R. lauricola. Brood estab- RL from the first to the second generation. lishment increased from 66 to 100% in the SM2, but decreased No eggs were seen in galleries along the rearing tube walls. from 66 to 58% in SM2 + RL. Similarly, brood establishment Larvae, pupae, and adults were first observed at 19, 25, and 30 d, increased from 33 to 50% in AM2 and decreased from 16 to 8% respectively, after the introduction of the female founder in AM3. In in AM2 + RL. SM3, larvae, pupae, and adults were first observed 18, 24, and 29 As was the case with Medium 1, no eggs were visible along d, respectively, after female founder introduction. In AM3 + RL and galleries in the rearing tube walls. Larvae, pupae, and adults were SM3 + RL, larvae and pupae were not observed along the rearing observed at 19, 24, and 30 d, respectively, after the female founder tube walls, although adults were seen at 35 and 40 d after female had been introduced into SM2. With SM2 + RL, larvae, pupae, and introduction, respectively.

Recovery of R. lauricola and Other Fungi From and 10 other fungi were isolated from colonies inoculated or not X. volvulus Reared in Media Inoculated With inoculated with R. lauricola, respectively (Table 4). R. arxii was R. lauricola the most frequent and abundant fungus detected in heads, bodies, R. lauricola was recovered at low frequencies from adult females and galleries of X. volvulus, both in avocado and silkbay media. reared on all three media (Table 3). The fungus was recovered Interestingly, other Raffaelea species (R. fusca T.C. Harr., Aghayeva from 13 and 10 beetle galleries in AM1 + RL and SM1 + RL, & Fraedrich, R. subalba T.C. Harr., Aghayeva & Fraedrich, and respectively, but was not recovered from any galleries in AM2 + R. subfusca T.C. Harr., Aghayeva & Fraedrich) were isolated only RL and SM2 + RL. In AM3 + RL and SM3 + RL, the pathogen from non-inoculated media. Candida spp. were isolated only from was recovered from one and six beetle galleries, respectively. Six beetle bodies and galleries, but never from beetle heads. Other fungi were found only in heads (Alloascoidea africana comb. nov. Table 3. Frequency of recovery of R. lauricola from the main body [Saccharomycetales: Alloascideaceae], Ambrosiozyma monospora parts of X. volvulus adults reared in artificial media previously Saito [Saccharomycetales: incertae sedis], and Zygozyma oligophaga inoculated with the fungus Van der Walt & Arx [Saccharomycetales: Lipomycetaceae]), or galleries (Leucosphaerina arxii Malloch [Hypocreales: Bionectriaceae], Host Medium Mean no. Frequency Mean no. Frequency Pichia manshurica Saito [Saccharomycetales: Pichiaceae], typea of CFUs n/N of CFUs n/N and Saccharomycopsis microspore (L. R. Batra) Kurtzman per head and per body pronotum lacking [Saccharomycetales: Saccharomycopsidaceae]) (Table 4). head and pronotum Discussion Avocado Medium 1 15 1/24 6 3/24 Ambrosia beetles live in nutritional symbioses with ambrosia fungi, Medium 2 348 1/12 17 1/12 which are most often species within the order Ophiostomatales Medium 3 0 0/24 0 0/24 (Ascomycota) (Biedermann et al. 2009). These fungi are cultivated in Silkbay Medium 1 4 1/24 36 2/24 Medium 2 0 0/12 5 1/12 galleries made by the beetles in the sapwood or heartwood of stressed Medium 3 8.7 3/24 16 1/24 trees (Kirkendall et al. 2015). Initially, it was presumed that ambrosia beetles were associated with a single dominant fungal species (Batra n, number of beetles positive for the presence of R. lauricola; N, number of 1963). However, these insects are now known to have multiple fungal beetles tested; CFU, colony-forming units of R. lauricola. associates (Gebhardt et al. 2004, Harrington et al. 2010). aNote that each medium was evaluated separately, and was considered a Scott and Du Toit (1970) reported that the dominant symbiont separate experiment. of Xyleborus torquatus (a synonym of X. volvulus) was R. arxii,

Table 4. Fungal species isolated from subsamples of fiveX. volvulus beetles and their galleries collected from medium 2 based on sawdust of avocado or silkbay

Treatment Isolate ID Medium containing avocado sawdust Medium containing silkbay sawdust

Head and Bodya Gallery Head and Bodya Gallery pronotum pronotum

Freq. Avg. CFU/ Freq. Avg. CFU/ Freq. Freq. Avg. CFU/ Freq. Avg. CFU/ Freq. n/N Beetle n/N Beetle n/N n/N Beetle n/N Beetle n/N

Media inocu- Alloascoidea africana 1/5 42 0/5 0 2/5 3/5 9.7 0/5 0 0/5 lated with Ambrosiozyma monospora 1/5 49 0/5 0 2/5 0/5 0 0/5 0 0/5 R. lauricola Candida berthetii 0/5 0 1/5 10 0/5 0/5 0 0/5 0 0/5 Candida laemsonensis 0/5 0 1/5 376 1/5 0/5 0 0/5 0 0/5 Leucosphaerina arxii 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 R. arxii 3/5 360.3 2/5 216 4/5 5/5 422 0/5 0 5/5 R. lauricola 1/5 348 0/5 0 2/5 1/5 5.5 0/5 0 2/5 Media not Alloascoidea africana 1/5 11 0/5 0 2/5 1/5 4 0/5 0 0/5 inocu- Candida berthetii 0/5 0 1/5 2356 0/5 0/5 0 0/5 0 0/5 lated with Candida nemodendra 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 R. lauricola Pichia manshurica 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 R. arxii 2/5 207 1/5 42 3/5 4/5 368.8 3/5 143 4/5 R. fusca 1/5 17 0/5 0 0/5 0/5 0 0/5 0 1/5 R. lauricola 0/5 0 0/5 0 0/5 0/5 0 0/5 0 0/5 R. subalba 2/5 843 1/5 72 3/5 0/5 0 0/5 0 0/5 R. subfusca 1/5 928 0/5 0 1/5 1/5 680 1/5 22 0/5 Saccharomycopsis microspora 0/5 0 0/5 0 1/5 0/5 0 0/5 0 0/5 Zygozyma oligophaga 0/5 0 0/5 0 0/5 1/5 17 0/5 0 0/5

The fungi were isolated from the head and pronotum or from the body lacking the head and pronotum. Freq., frequency of detecting the fungal species; Avg., average; n, the number of either beetle body parts or the number of galleries that were positive for a given fungal species; N, the number of specimens examined, or number of galleries assayed for the presence of various species of fungi. aBody lacked the head and the pronotum.

which was repeatedly isolated from galleries excavated in Schefflera no clear differences on the suitability of avocado and silkbay saw- (Cussonia) umbellifera (Sond.) Baill. (Apiales: Araliaceae) in the dust to rear X. volvulus. In the medium that provided the best rearing Dukuduku Forest, Natal, South Africa. In this study, R. arxii was the conditions, the beetle bred similarly in avocado and silkbay sawdust. most abundant and frequently recovered symbiont from X. volvu- In the two other media, which differed only by their water content, lus heads, bodies, and galleries. However, in the present study, other we obtained contrasting results in regards to the type of sawdust Raffaelea species (R. fusca, R. subalba, and R. subfusca) and several used. Our results suggest that the water content in the rearing sub- yeast species were also frequently associated with X. volvulus. Our strate had a greater effect than the type of sawdust on the ability results corroborate that R. arxii may be the primary symbiont of of X. volvulus to produce offspring. Interestingly, natural breeding X. volvulus. of this beetle species occurs in avocado trees showing early signs of Previously, R. lauricola was recovered from 20 of 63 individu- decline, when the wood retains substantial water content. A range als of X. volvulus (32%) from LW-affected native Persea spp. and of water contents in these media should be tested to determine how 13 of 217 individuals (6%) from LW-affected avocado trees (Ploetz water content affects both fungal growth and beetle development. et al. 2017). In the present studies, we inoculated the rearing media X. volvulus females started excavating in the media soon after with R. lauricola. In general, media inoculated with the pathogen they were introduced into the rearing tubes. Female tunneling resulted in less progeny than non-inoculated media; however, these activity normally occurred along the wall of the rearing tube mak- results were not significant except on female production in AM2 ing observations possible without disturbing or altering the media. versus AM2 + RL. R. lauricola was recovered from bodies of few Larvae and pupae were seen at different times depending on the individuals (four of 60 total on both avocado and silkbay sawdust, treatment, ranging from 15 to 21 d and from 19 to 27 d after the 6.7%) and showed very limited colonization of the mycangium (two female had been introduced into the various media. Adults were first of 60 total on avocado, 3.3%, and four of 60 total on silkbay, 6.7%). seen in galleries 24 d after female introduction, and they remained in These results suggest that R. lauricola does not fulfill the nutritional galleries for ~1 wk. During this period, it seems probable that mating requirements of X. volvulus. occurred between sibling females and males, and adults engaged in Foundress females that were used in this work were healthy colony maintenance activities (i.e., cleaning). Often in this study, at adults with mycangia that were presumably colonized with their the time of dissection (i.e., 40 d after the female’s introduction), all primary or other symbiont(s). Mycangia have specialized glandular stages were found concurrently in the colony suggesting overlapping cells that secrete substances that protect fungal cells from desicca- generations. Eggs and larvae were frequently clumped between the tion, regulate fungal species composition, provide nourishment for main and the secondary galleries, some of which were probably pro- fungal propagules, and determine the form of fungal growth in the duced by the new generation of females. mycangium (Batra 1963, Happ et al. 1971, Six 2003, Yuceer et al. The reproductive potential of X. volvulus was similar to that of 2011). The infrequent recovery of R. lauricola from mycangia and X. glabratus reared in two artificial sawdust-based media (Maner bodies of X. volvulus in the present work may reflect the adaptation et al. 2013). Using a different approach, Brar et al. (2013) found of other ambrosial fungi in this beetle species. The displacement of lower rates of X. glabratus reproduction on bolts of three tree spe- these primary symbionts by R. lauricola might not be expected, espe- cies (i.e., avocado, redbay, and swampbay) (only 1.1 females devel- cially if the individual had already established a nutritional mutual- oped on bolts originally infested with 20 female beetles). Thus, ism with its primary, adapted symbiont. That R. lauricola was not sawdust-based media appear to be better than bolts for establishing recovered from galleries in media inoculated with the pathogen also laboratory colonies of ambrosia beetles. suggests females of X. volvulus do not cultivate R. lauricola, even in Although the development of artificial substrates for ambrosia substrates that were previously colonized with the fungus. In sum- beetles is challenging, these media can be quite useful. Artificial sub- mary, suboptimal reproduction on the inoculated media indicated strates facilitate the otherwise difficult study of larval feeding habits that R. lauricola is probably a poor nutritional partner for X. volvu- on, and the nutritional quality of, different fungi. Artificial media lus. How it disrupts the beetle’s normal nutritional cycle is unclear, as may be used to study the social behavior of ambrosia beetles and it was generally absent in both the beetle and the +RL rearing media. to study their sexual dimorphism, which could provide characters Little is known about the nutrition of ambrosia beetles by their to separate closely related species. Artificial media may also be used fungal associates. Freeman et al. (2016) reported that larvae of to investigate intra- and inter-specific competition and to evaluate Euwallacea nr. fornicatus fed on Paracremonium pembeum sp. nov. potential management tactics. did not complete their life cycle, whereas those reared on Fusarium In summary, this study demonstrated that X. volvulus can be euwallaceae and Graphium euwallaceae completed development. reared on artificial substrates made with sawdust obtained from Similarly, Paecilomyces variotii Bainier negatively impacted larvae avocado or silkbay. Based on our results, Medium 1 previously pro- and adults of X. saxesenii, whereas the beetle’s primary nutritional posed by Castrillo et al. (2012) was superior for rearing X. volvulus. symbiont, Raffaelea sulfurea (L. R. Batra), had positive effects Females in this medium developed faster, survived longer, and pro- (Biedermann et al. 2012). In addition, R. lauricola had been shown duced more progeny. Our results suggest that probably R. lauricola to negatively impact the reproduction of X. crassiusculus (Ott 2007). is not a nutritional symbiont (i.e., does not fulfill the requirements) Recently, Saucedo et al. (2017) reported that four different species of of X. volvulus, but in some cases, the association with this patho- Raffaelea, including R. lauricola, supported the development of a sin- gen could be commensalistic, not affecting the beetle’s reproduction. gle generation of X. bispinatus. The latter experiments were initiated More research is needed to confirm this hypothesis. with newly eclosed females that had been exposed to only one species of Raffaelea (no-choice forcing of symbiosis). Whether X. bispinatus would have propagated successfully on the four species of Raffaelea if Acknowledgments previously colonized females had been used (as in the present studies) This study partially fulfills the requirements for the MS degree, University of is not clear. Better understandings are needed for these interactions. Florida, by O. A. M. We thank James Colee (UF-IFAS-Statistics Department) In artificial rearings, the type of sawdust used in the medium may for his help with the statistical analysis, Waldemar Klassen and Jorge E. Peña affect ambrosia beetle reproduction (Castrillo et al. 2012). We found (University of Florida) for suggestions to improve the manuscript. We also

thank Jose Alegría, Julio Mantilla, Manuela Angel, and Ana Vargas for their Gebhardt, H., D. Begerow, and F. Oberwinkler. 2004. Identification of the help. This research was funded by FDACS-SCBG 021757 grant and NIFA ambrosia fungus of Xyleborus monographus and X. dryographus grant 2015-51181-24257 to D. C. (Coleoptera: Curculionidae, Scolytinae). Mycol. Progr. 3: 95–102. Gohli, J., T. Selvarajah, L. R. Kirkendall, and B. H. Jordal. 2016. Globally distributed Xyleborus species reveal recurrent intercontinental dispersal References Cited in a landscape of ancient worldwide distributions. Evol. Biol. 16: 37. doi: Adeyeye, O. A., and M. S. Blum. 1988. A cowpea artificial diet for Noctuid 10.1186/s12862-016-0610-7. larvae. Insect. Sci. Applic. 9: 609–611. Greco, E. B., and M. G. Wright. 2015. Ecology, biology, and management Atkinson, T. H. 2016. Bark and ambrosia beetles. http://www.barkbeetles. of Xylosandrus compactus (Coleoptera: Curculionidae: Scolytinae) with info/. Accessed December 2016. emphasis on coffee in Hawaii. J. Integr. Manag. 6: 7. doi: 10.1093/jipm/ Batra, L. R. 1963. Ecology of ambrosia fungi and their dissemination by bee- pmv007. tles. Trans. Kansas. Acad. Sci. 66: 213–236. Hanula, J. L., A. E. Mayfield III, S. W. Fraedrich, and R. J. Rabaglia. 2008. Biedermann, P. H. W., K. D. Klepzig, and M. Taborsky. 2009. Fungus culti- Biology and host association of the redbay ambrosia beetle (Coleoptera: vation by ambrosia beetles: behavior and laboratory breeding success in Curculionidae: Scolytinae), exotic vector of laurel wilt killing redbay trees three Xyleborine species. Environ. Entomol. 38: 1096–1105. in the southern United States. J. Econ. Entomol. 101: 1276–1286. Biedermann, P. H. 2010. Observations on sex ratio and behavior of males in Happ, G. M., C. M. Happ, and S. J. Barras. 1971. Fine structure of the pro- Xyleborinus saxesenii Ratzeburg (Scolytinae, Coleoptera). ZooKeys 56: thoracic mycangium, a chamber for the culture of symbiotic fungi, in the 253–267. southern pine beetle, Dendroctonus frontalis. Tissue and Cell 3: 295–308. Biedermann, P. H. W., K. D. Klepzig, M. Taborsky, and D. L. Six. 2012. Abundance Harrington, T. C., D. N. Aghayeva, and S. W. Fraedrich. 2010. New combi- and dynamics of filamentous fungi in the complex ambrosia gardens of the nations in Raffaelea, Ambrosiella, and Hyalorhinocladiella, and four new primitively eusocial beetle Xyleborinus saxesenii Ratzeburg (Coleoptera: species from the redbay ambrosia beetle, Xyleborus glabratus. Mycotaxon Curculionidae, Scolytinae). FEMS Microbiol. Ecol. 83: 711–723. 111: 337–361. Brar, G. S., J. L. Capinera, P. E. Kendra, S. McLean, and J. E. Peña. 2013. Hughes, M. A., J. A. Smith, R. C. Ploetz, P. E. Kendra, A. E. Mayfield III, J. Life cycle, development, and culture of Xyleborus glabratus (Coleoptera: L. Hanula, J. Julcr, L. L. Stelinski, S. Cameron, J. J. Riggins, et al. 2015. Curculionidae: Scolytinae). Fla. Entomol. 96: 1158–1167. Recovery plan for laurel wilt on redbay and other forest species caused Carrillo, D., R. E. Duncan, and J. E. Peña. 2012. Ambrosia beetles (Coleoptera: by Raffaelea lauricola and disseminated by Xyleborus glabratus. Plant. Curculionidae: Scolytinae) that breed in avocado wood in Florida. Fla. Health. Prog. 16: 173–210. Entomol. 95: 573–579. Kendra, P. E., J. S. Sanchez, W. S. Montgomery, K. E. Okins, J. Niogret, J. Carrillo, D., R. E. Duncan, J. N. Ploetz, A. F. Campbell, R. C. Ploetz, and E. Peña, N. D. Epsky, and R. R. Heath. 2011. Diversity of Scolytinae J. E. Peña. 2014. Lateral transfer of a phytopathogenic symbiont among (Coleoptera: Curculionidae) attracted to avocado, lychee, and essential oil native and exotic ambrosia beetles. Plant Pathol. 63: 54–62. lures. Fla. Entomol. 94: 123–130. Carrillo, D., L. F. Cruz, P. E. Kendra, T. I. Narvaez, W. S. Montgomery, Kendra, P. E., W. S. Montgomery, J. Niogret, M. A. Deyrup, L. Guillén, and A. Monterroso, C. De Grave, and M. F. Cooperband. 2016. Distribution, N. D. Epsky. 2012. Xyleborus glabratus, X. affinis, and X. ferrugineus pest status and fungal associates of Euwallacea nr. fornicatus in Florida (Coleoptera: Curculionidae; Scollytinae): electroantennogram responses avocado groves. Insects 7: 55. doi:10.3390/insects7040055. to host-based attractants and temporal patterns in host-seeking flight. Castrillo, L. A., M. H. Griggs, C. M. Ranger, M. E. Reding, and J. Environ. Entomol. 41: 1597–1605. D. Vandenberg. 2011. Virulence of commercial strains of Beauveria bassi- Kendra, P. E., W. S. Montgomery, J. Niogret, G. E. Pruett, A. E. Mayfield ana and Metarhizium brunneum (Ascomycota: Hypocreales) against adult III, M. MacKenzie, M. A. Deyrup, G. R. Bauchan, R. C. Ploetz, and N. Xylosandrus germanus (Coleoptera: Curculionidae) and impact on brood. D. Epsky. 2014. North American Lauraceae: Terpenoid emissions, relative Biol. Control 58: 121–126. attraction and boring preferences of redbay ambrosia beetle, Xyleborus Castrillo, L. A., M. H. Griggs, and J. D. Vandenberg. 2012. Brood production glabratus (Coleoptera: Curculionidae: Scolytinae). PLoS One 9: e102086. by Xylosandrus germanus (Coleoptera: Curculionidae) and growth of its Kirkendall, L., P. H. Biedermann, and B. H. Jordal. 2015. Evolution and diver- fungal symbiont on artificial diet based on sawdust of different tree spe- sity of bark and ambrosia beetles, pp. 85–156. In F. E. Vega and R. W. cies. Environ. Entomol. 41: 822–827. Hofstetter (eds.), Bark beetles: biology and ecology of native and invasive Cooperband, M. F., R. Stouthamer, D. Carrillo, A. Eskalen, T. Thibault, A. species. Elsevier, San Diego. A. Cossé, L. A. Castrillo, J. D. Vandenberg, and P. F. Rugman-Jones. 2016. Kostovcik, M., C. C. Bateman, M. Kolarik, L. L. Stelinski, B. H. Jordal, and Biology of two members of the Euwallacea fornicatus species complex J. Hulcr. 2015. The ambrosia symbiosis is specific in some species and (Coleoptera: Curculionidae: Scolytinae), recently invasive in the U.S.A, promiscuous in others: evidence from community pyrosequencing. ISME reared on an ambrosia beetle artificial diet. Agr. Forest. Entomol. 18: J. 9: 126–138. 223–237. Maner, M. L., J. L. Hanula, and K. Braman. 2013. Rearing redbay ambrosia Dreaden, T. J., J. M. Davis, C. L. Harmon, R. C. Ploetz, A. J. Palmateer, P. beetle, Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae), on S. Soltis, and J. A. Smith. 2014. Development of multilocus PCR assays semi-artificial media. Fla. Entomol. 96: 1042–1051. for Raffaelea lauricola, causal agent of laurel wilt disease. Plant Dis. 98: Mizuno, T., and H. Kajimura. 2002. Reproduction of the ambrosia beetle, 379–383. Xyleborus pfeili (Ratzeburg) (Col., Scolytidae), on semi-artificial diet. Farrell, B. D., A. S. Sequeira, B. C. O’Meara, B. B. Normark, J. H. Chung, and J. Appl. Entomol. 126: 455–462. B. H. Jordal. 2001. The evolution of agriculture in beetles (Curculionidae: Mizuno, T., and H. Kajimura. 2009. Effects of ingredients and structure of Scolytinae and Platypodinae). Evolution 55: 2011–2027. semi-artificial diet on the reproduction of an ambrosia beetle,Xyleborus Fraedrich, S. W., T. C. Harrington, R. J. Rabaglia, M. D. Ulyshen, A. pfeili (Ratzeburg) (Coleoptera: Curculionidae: Scolytinae). Appl. Entomol. E. Mayfield III, J. L. Hanula, J. M. Eickwort, and D. R. Miller. 2008. A fun- Zool. 44: 363–370. gal symbiont of redbay ambrosia beetle causes a lethal wilt in redbay and Ott, E. P. 2007. Chemical ecology, fungal interactions and forest stand cor- other Lauraceae in the southern United States. Plant. Dis. 92: 215–224. relations of the exotic Asian ambrosia beetle, Xylosandrus crassiusculus Freeman, S., M. Sharon, M. Dori-Bachash, M. Maymon, E. Belausov, Y. Maoz, (Motschulsky) (Curculionidae). M.S. thesis. Louisiana State University, O. Margalit, A. Protasov, and Z. Mendel. 2016. Symbiotic association Louisiana. of three fungal species throughout the life cycle of the ambrosia beetle Ploetz, R. C., J. L. Konkol, T. Narvaez, R. E. Duncan, R. J. Saucedo, Euwallacea nr. fornicatus. Symbiosis. 68: 115–128. A. Campbell, J. Mantilla, D. Carrillo, and P. E. Kendra. 2017. Presence French, J. R. J., and R. A. Roeper. 1972. In vitro culture of the ambrosia bee- and prevalence of Raffaelea lauricola, cause of laurel wilt, in different spe- tle Xyleborus dispar (Coleoptera: Scolytidae) with its symbiotic fungus, cies of ambrosia beetle in Florida, USA. J. Econ. Entomol. doi: 10.1093/ Ambrosiella hartigii. Ann. Entomol. Soc. Amer. 65: 719–721. jee/tow292.

Rabaglia, R. J., S. A. Dole, and A. I. Cognato. 2006. Review of American Singh, P. 1977. Artificial diets for insects, mites, and spiders. IFI/Plenum, New Xyleborina (Coleoptera: Curculionidae: Scolytinae) occurring North of York. Mexico, with an illustrated key. Ann. Entomol. Soc. Am. 99: 1034–1056. Six, D. L. 2003. Bark beetle–fungus symbioses, pp. 99–116. In K. Bourtzis and Ranger, C. M., M. E. Reding, P. B. Schultz, J. B. Oliver, S. D. Frank, K. M. Addesso, T. A. Miller (eds.), Insect symbiosis. CRC Press, Boca Raton. J. H. Chong, B. Sampson, C. Werle, S. Gill, et al. 2016. Biology, ecology, and Vanderzant, E. S. 1974. Development, significance, and application of artificial management of nonnative ambrosia beetles (Coleoptera: Curculionidae: diets for insects. Annu. Rev. Entomol. 18: 139–160. Scolytinae) in ornamental plant nurseries. J. Integr. Pest. Manag. 7: 1–23. Vilgalys, R., and M. Hester. 1990. Rapid genetic identification and mapping of Rudinsky, J. A. 1962. Ecology of Scolytidae. Annu. Rev. Entomol. 7: 327–348. enzymatically amplified ribosomal DNA from several Cryptococcus spe- SAS Institute. 2010. SAS Version 9.3. SAS Institute, Cary, NC. cies. J. Bacteriol. 172: 4238–4246. Saucedo, J., R. Ploetz, J. Konkol, M. Angel, J. Mantilla, O. Menocal, and D, Carrillo. Wood, S. L. 1982. The bark and ambrosia beetles of North and Central 2017. Nutritional symbionts of a putative vector, Xyleborus bispinatus, of the America (Coleoptera: Scolytidae), a taxonomic monograph. Great. Basin. laurel wilt pathogen of avocado, Raffaelea lauricola. Symbiosis (in press). Nat. Mem. 6: 1–1359. Saunders, J. L., and J. K. Knoke. 1967. Diets for rearing the ambrosia beetle Yuceer, C., C. Y. Hsu, N. Erbilgin, and K. D. Klepzig. 2011. Ultrastructure Xyleborus ferrugineus (Fabricius) in vitro. Science 157: 460–463. of the mycangium of the southern pine beetle Dendroctonus frontalis Scott, D. B., and J. W. Du Toit. 1970. Three new Raffaelea species. Trans. Br. (Coleoptera: Curculionidae, Scolytinae): complex morphology for com- Mycol. Soc. 55: 181–186. plex interactions. Acta Zool-Stockholm 92: 216–224.

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