TY - JOUR
AU1 - Menocal,, Octavio
AU2 - Cruz, Luisa, F
AU3 - Kendra, Paul, E
AU4 - Crane, Jonathan, H
AU5 - Ploetz, Randy, C
AU6 - Carrillo,, Daniel
AB - 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 and R. 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 for X. volvulus. ambrosia beetle, ambrosia fungi, artificial rearing, beetle–fungus symbiosis, laurel wilt Laurel wilt (LW) is a lethal vascular disease of avocado (Persea americana Mill. [Laurales: Lauraceae]) and other woody species within the Lauraceae. The causal agent of laurel wilt is the fungal pathogen, Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae). The primary vector of R. lauricola in native ecosystems, including natural hammocks of the Florida Everglades, is the redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae: Xyleborini) (Kendra et al. 2014, Hughes et al. 2015). However, X. glabratus is rarely associated with laurel wilt-affected avocado trees in south Florida (Carrillo et al. 2012). Recently, R. lauricola was found in at least nine other ambrosia beetle species isolated from avocado (Carrillo et al. 2014, Ploetz et al. 2017). Two of the species, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) and Xyleborus bispinatus Eichhoff, were capable of transmitting R. lauricola to avocado trees under no-choice conditions (Carrillo et al. 2014). X. volvulus, a pantropical species that probably originated in the Neotropical realm, has become widely distributed throughout Florida, Central and South America, and the Caribbean (Wood 1982, Gohli et al. 2016). This beetle has a broad host range that includes species in 24 plant families including the Lauraceae (Atkinson 2016). Other ambrosia beetles that have been introduced to the New World are important pests (i.e., X. glabratus (Fraedrich et al. 2008, Hanula et al. 2008, Brar et al. 2013, Maner et al. 2013), Xylosandrus compactus (Eichhoff) [Coleoptera: Curculionidae] (Greco and Wright 2015), Euwallacea spp. (Carrillo et al. 2016, Cooperband et al. 2016), Xylosandrus germanus (Eichhoff), and Xylosandrus crassiusculus (Motschulsky) [Coleoptera: Curculionidae] (Castrillo et al. 2012, Ranger et al. 2016)). In Florida, X. volvulus occurs sympatrically with X. glabratus and breeds in hosts affected by LW (Kendra et al. 2011, Carrillo et al. 2012). Although X. volvulus has not been associated with economic damage to trees, its experimental transmission of R. lauricola to avocado (Carrillo et al. 2014) indicates that the association between the beetle and this pathogen warrants further investigation. Ambrosia beetles are difficult to study because of their cryptic life style. They bore through the bark of a host tree and form galleries within the xylem. Ambrosia beetles complete their life cycle in these galleries, where they actively cultivate symbiotic fungi that serve as their primary food (Rudinsky 1962, Farrell et al. 2001). The ambrosia beetle–fungal symbiosis is an area of active research. Recent studies revealed that Xyleborus species consistently carry not only multiple dominant fungal associates but also fungi from the environment, including plant pathogens and endophytes (Kostovcik et al. 2015). However, there is limited information regarding their biology, behavior, and the functional role of their symbiotic associations. Establishment of colonies of these insects would allow studies on their development, physiology, behavior, colony composition and size, and enable ambrosia beetle–fungus associations to be manipulated to improve understandings of this taxonomic group, and their direct and indirect effects on host trees. Artificial media have been used to mass rear insects, test compounds for physiological effects, and study insect nutrition and behavior (Vanderzant 1974). According to Singh (1977), ‘an artificial medium is an unfamiliar substrate, which has been formulated, synthesized, processed, and/or concocted by man, and on which an insect in captivity can develop through all or part of its life cycle.’ In the case of ambrosia beetles, culture conditions must be suitable for both the symbiotic fungi and the beetles (Maner et al. 2013). An effective medium requires an in-depth understanding of the insect’s biology, behavior, and physiology. Ideally, an artificial medium for ambrosia beetles should serve as a nutritional substrate for the fungal symbiont and support the economical production of large numbers of healthy insects that are similar to those living in the natural environment (Adeyeye and Blum 1988). Xyleborus ferrugineus F. was the first ambrosia beetle successfully reared on an artificial medium (Saunders and Knoke 1967). Subsequently, at least six other ambrosia beetle species have been reared artificially, including: Xyleborus dispar F. (French and Roeper 1972), Xyleborus pfeili Ratzeburg (Mizuno and Kajimura 2002, Mizuno and Kajimura 2009), Xyleborus affinis Eichhoff, Xyleborinus saxesenii Ratzeburg (Biedermann et al. 2009, Biedermann 2010), Xylosandrus germanus (Biedermann et al. 2009, Castrillo et al. 2012), X. glabratus (Maner et al. 2013), and Euwallacea spp. (Cooperband et al. 2016). Here, we describe a series of studies that evaluate three artificial media for rearing X. volvulus. The media incorporated sawdust obtained from two of its major hosts, silkbay (Persea humilis Nash), a species endemic to central Florida that is frequently colonized by ambrosia beetles following LW development (Kendra et al. 2012), and avocado, an important agricultural species in south Florida that is also threatened by LW. In addition, since R. lauricola has been isolated from X. volvulus recovered from both of these hosts (Carrillo et al. 2012, Carrillo et al. 2014, Ploetz et al. 2017), we studied interactions of X. volvulus with this fungus with the developed laboratory rearing methods. Materials and Methods Ambrosia Beetles X. volvulus females were obtained from sections of logs (approximately 50 × 16 cm, length by diameter) collected from avocado trees that were affected by LW and placed in emergence chambers [44-gallon (167 L) Brute® container (2643–60 Rubbermaid®) with a 2-quart Mason jar attached to a port on one of each side of the chamber, as described in Carrillo et al. (2012)]. Rolled moistened paper towels were placed inside the jars to collect beetles emerging from the logs and attracted to light. Fully sclerotized (dark brown) females were collected daily and identified as X. volvulus based on their morphological characteristics (Rabaglia et al. 2006). Artificial Media In February 2016, avocado logs were collected from an unsprayed avocado orchard in Miami-Dade County (25° 29’ 38” N 80° 28’ 53” W), and silkbay logs were collected from the Archbold Biological Station in Highlands County, FL (27° 10’ 50” N 81° 21’ 0” W). The logs were debarked and dried for 4 d in an industrial oven at 75°C and then cut into smaller pieces using a miter saw. A sander was used to create sawdust from the xylem-sapwood layer. The sawdust was sifted through a 12 mm sieve and stored at -18 ºC until it was used to prepare media. Three types of artificial media were evaluated (Table 1). Medium 1 with sawdust from avocado or silkbay (designated as AM1 and SM1, respectively) was prepared using the ingredients and procedures described by Castrillo et al. (2011). Medium 2 (either as AM2 or SM2) was prepared using different proportions of the same ingredients as proposed by Biedermann et al. (2009). Medium 3 (either as AM3 or SM3) was prepared with the same ingredients in the same quantities as in Medium 2, but more water was added to facilitate manipulation while transferring the medium into rearing tubes. All dry ingredients (sawdust, agar, sucrose, starch, yeast, casein, Wesson’s salt mixture, and tetracycline) were mixed in a 600 ml beaker. Then, with constant stirring, liquid ingredients were added in the following order: wheat germ oil, peanut oil, ethanol, and water. Homogenized media were autoclaved at 121°C for 30 min and were stirred to re-suspend settled ingredients; 15 ml was poured into 50 ml sterile plastic centrifuge tubes (Fisher Scientific Catalog no. 0644318, Suwanee, GA) that were loosely capped, tapped to remove air bubbles, and allowed to cool in the laminar flow hood for 24 h. Medium 2 was dispensed in the plastic tubes before autoclaving due to its more solid consistency. After autoclaving, the tubes containing Medium 2 were transferred to the laminar flow hood and allowed to cool as before. Table 1. Recipes of the three types of artificial media using either avocado or silkbay sawdust for rearing X. volvulus Ingredients . Media type . Manufacturer/Source . Type 1: AM1 or SM1 . Type 2: AM2 or SM2 . Type 3: AM3 or SM3 . Sawdust (either avocado or silkbay) 45 g 84 g 84 g Dried and stored sawdust as described in Materials and 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 Ingredients . Media type . Manufacturer/Source . Type 1: AM1 or SM1 . Type 2: AM2 or SM2 . Type 3: AM3 or SM3 . Sawdust (either avocado or silkbay) 45 g 84 g 84 g Dried and stored sawdust as described in Materials and 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. Open in new tab Table 1. Recipes of the three types of artificial media using either avocado or silkbay sawdust for rearing X. volvulus Ingredients . Media type . Manufacturer/Source . Type 1: AM1 or SM1 . Type 2: AM2 or SM2 . Type 3: AM3 or SM3 . Sawdust (either avocado or silkbay) 45 g 84 g 84 g Dried and stored sawdust as described in Materials and 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 Ingredients . Media type . Manufacturer/Source . Type 1: AM1 or SM1 . Type 2: AM2 or SM2 . Type 3: AM3 or SM3 . Sawdust (either avocado or silkbay) 45 g 84 g 84 g Dried and stored sawdust as described in Materials and 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. Open in new tab Media Inoculation A subset of the tubes containing each of the three media was inoculated with an isolate of R. lauricola obtained from the Plant Diagnostic Clinic at the Tropical Research and Education Center (denoted + RL). Conidia were harvested from cultures of the isolate by gently scraping the culture surface with a sterile plastic rod (Fisher Scientific Catalog no. 23600896). The resulting suspension was transferred to a sterile 50 ml plastic centrifuge tube using a disposable sterile pipette (5 ml). Serial dilutions (1, 10, 102, 103, and 104) were then plated on CSMA medium (cycloheximide, streptomycin, malt, and agar), which is selective for species of Ophiostomatales, to determine the number of colony-forming units (CFUs) in the original suspension; 500 µl of the original, which contained 8.2 × 106 CFUs, was added per tube of solidified medium. Tubes were loosely capped and maintained in a sterile environment for 10 d to allow fungal growth on the medium. Rearing Conditions Before introducing females into rearing tubes, four small holes were made on the surface of the medium to facilitate the initiation of boring activity. Females of X. volvulus were collected from the emergence chambers and surface-disinfested in 70% ethanol for 5–7 s. Active and vigorous females were individually placed into each of 12 tubes of each medium that was tested. Rearing tubes were then incubated horizontally in plastic containers in a walk-in rearing room, in complete darkness and at 25 ± 1°C, 75% RH. Tubes were inspected every 3 d for the appearance of lose medium on its surface, pushed out by females during tunneling (an indication of gallery formation), and the sides of the tubes were examined for visible galleries. The number of days to the first occurrence of eggs, larvae, pupae, and adults on the surface and in galleries were recorded, and the experiment was concluded after the uninterrupted development of two generations. A 40-d generation time was used based on previous findings by Maner et al. (2013) in similar studies on X. glabratus, which could apply for other ambrosia beetles. Gallery Dissection After 40 d (i.e., first generation), the medium in each tube containing a beetle colony (brood) was dissected under a stereomicroscope in a laminar flow hood. All developmental stages on the surface of the medium were recorded before the medium was tapped out of the tube into a Petri dish, and all beetle stages on the sides of the tubes were recorded. Finally, the medium was systematically cut into small pieces from the bottom to the top of the medium plug. Gallery tunnels were opened carefully by removing the medium around them. Eggs, larvae, pupae, and adults were removed, counted and placed separately into Petri dishes using a fine paintbrush. Adult mortality was also recorded. Twelve mature active females from each treatment were surface sterilized, and reared in new tubes (i.e., same treatment) prepared as earlier to produce a second generation. Fungal Isolation One mature female from each colony inoculated with R. lauricola [first and second generation colonies in Media 1 and 3 (n = 24 for each medium), and second generation in Medium 2 (n = 12), Table 3] were surface-disinfested by immersing in 70% ethanol for 30 s, and subsequently washed in sterile deionized water three times. The head and pronotum were separated from the abdomen, and macerated separately in 200 µl of sterile water using a motorized tissue grinder (Pyres no. 7727-07). Then, 100 µl of each macerate solution were plated onto CSMA. In addition, fungal samples were collected from galleries using sterile inoculation loops (Fisher Scientific Catalog no. 22170206) to scrape small portions of the tunnel where immature stages were found, and plated on CSMA medium. After 7–10 d, the identity of colonies with the morphology of R. lauricola (Harrington et al. 2010) was confirmed with two diagnostic microsatellite markers, CHK and IFW (Dreaden et al. 2014). The number of CFUs of R. lauricola was calculated for each beetle, and the presence or absence of the fungus in galleries was determined. In addition, fungal isolations from a subset of beetles and galleries were identified by amplifying a portion of the nuclear large subunit 28S ribosomal DNA (rDNA) using primers LR0R/LR5 (Vilgalys and Hester 1990). Statistical Analysis The statistical software package SAS was used for all analyses. For each medium type, the number of adult females and total brood production were compared between the sawdust species (avocado or silkbay) with and without the inoculation with R. lauricola, for a total of four treatments per medium. Each medium type was evaluated separately and was considered a separate experiment. The effects of sawdust species and the presence of R. lauricola were evaluated independently for each medium using two-way analysis of variance (PROC GLIMMIX, SAS Institute 2010, v. 9.3). In addition, brood size was compared between the first and second generations in each medium type. Data were square-root transformed before analysis, and means were separated with Tukey’s HSD. Results Medium 1 The type of sawdust in medium 1 (Table 1) had no significant effect on the reproduction of X. volvulus (F = 0.03; df = 1, 95; P = 0.8521) (Fig. 1A). Brood sizes were generally greater on media not inoculated with R. lauricola when compared with media inoculated with this pathogen, but these differences were not statistically significant (F = 2.42; df = 1, 95; P = 0.1222) (Fig. 1A). Similarly, female offspring were greater on media without R. lauricola when compared with media inoculated with the pathogen; however, these differences were not statistically significant (F = 1.76; df = 1, 95; P = 0.6301) (Fig. 1A). Fig. 1. Open in new tabDownload slide 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. Fig. 1. Open in new tabDownload slide 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. The number of male progeny per brood ranged from zero to four. Males were present in 85 and 78% of AM1 and AM1 + RL broods, respectively, and in 77 and 64% of the SM1 and SM1 + RL broods, respectively. Adult mortality in broods in the four variations of Medium 1 increased in the following order: AM1 (16%) < SM1 (21%) < AM1 + RL (22%) < SM1 + RL (33%) (Table 2). 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 females in brood . Adult mortality (%) . N with offspring (any stage) (%) . N with females (%) . N . Eggs . Larvae . Pupae . Male adults . Female adults . Brood (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 Mediuma . Average no. of offspring per tube after 40 d . (%) of females in brood . Adult mortality (%) . N with offspring (any stage) (%) . N with females (%) . N . Eggs . Larvae . Pupae . Male adults . Female adults . Brood (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. Open in new tab 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 females in brood . Adult mortality (%) . N with offspring (any stage) (%) . N with females (%) . N . Eggs . Larvae . Pupae . Male adults . Female adults . Brood (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 Mediuma . Average no. of offspring per tube after 40 d . (%) of females in brood . Adult mortality (%) . N with offspring (any stage) (%) . N with females (%) . N . Eggs . Larvae . Pupae . Male adults . Female adults . Brood (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. Open in new tab In all treatments, there was significantly more reproduction (F = 17.17; df = 1, 95; P < 0.0001) in the second generation than in the first (data not shown). Overall, the mean number of progeny (± SE) in the first generation was 8.84 ± 1.60 and 15.88 ± 2.07 in the second generation. The percentage of female founders that established colonies in AM1 was 59 and 50 in the first and second generations, respectively. In contrast, the increase in brood establishment by female founders between the first and second generations increased from 25 to 92% in AM1 + RL, from 59 to 92% in SM1, and from 59 to 84% in SM1 + RL (data not shown). No eggs were visible in the galleries along the rearing tube walls. Larvae, pupae, and adults were first observed on Days 15, 19, and 24, respectively, after the female founders had been introduced into AM1 and SM1. In AM1 + RL, larvae, pupae, and adults were observed at 18, 23, and 27 d after the females had been introduced, respectively. In SM1 + RL, larvae, pupae, and adults were observed later than in all other treatments, i.e., at 21, 27, and 31 d after the females had been introduced, respectively. Medium 2 There was a significant interaction between the species of sawdust and the presence of R. lauricola (F = 6.27; df = 3, 95; P = 0.0005); female production, but not total brood, was significantly lower in R. lauricola-inoculated medium containing avocado sawdust, whereas neither differed on silkbay sawdust medium, with or without R. lauricola (Fig. 1B). The size of the brood produced declined in the following order: SM2 > SM2 + RL > AM2 > AM2 + RL (Fig. 1B). The number of males per brood ranged from zero to two. Males were present in 60% of the broods in SM2 and SM2 + RL, and in 90% of the broods in AM2 (data not shown). No males were observed in the AM2 + RL broods. Adult mortality was similar in SM2 (17%), AM2 (16%), and SM2+RL (14%). However, much adult mortality (82%) was observed in AM2 + RL (Table 2). The average brood size produced in the first generation (6.40 ± 1.32) was less than that in the second generation (7.69 ± 1.23), although this difference was not statistically significant (F = 1.08; df = 1, 95; P = 0.3005) (data not shown). The percentage of female founders that established colonies increased from the first to the second generation in non-inoculated media, but decreased in media inoculated with R. lauricola. Brood establishment increased from 66 to 100% in the SM2, but decreased from 66 to 58% in SM2 + RL. Similarly, brood establishment increased from 33 to 50% in AM2 and decreased from 16 to 8% in AM2 + RL. As was the case with Medium 1, no eggs were visible along galleries in the rearing tube walls. Larvae, pupae, and adults were observed at 19, 24, and 30 d, respectively, after the female founder had been introduced into SM2. With SM2 + RL, larvae, pupae, and adults were observed at 18, 25, and 31 d after female introduction. In AM2, larvae, pupae, and adults were seen at 20, 24, and 29 d, respectively. In AM2 + RL, no larvae or pupae were observed, although adults were seen at 33 d after female introduction. Medium 3 There was a significant interaction (F = 3.91; df = 3, 95; P = 0.0103) between sawdust species and R. lauricola. The presence of R. lauricola in both media (i.e., avocado and silkbay) had a neutral or negative effect on female offspring and brood production compared with media without the pathogen, although this effect was not significant (Fig. 1C). The number of males per brood ranged from zero to three. Males were present in 73% of the broods in AM3, 54% in AM3 + RL, 44% in SM3, and 33% in SM3 + RL. Adult mortality was similar in AM3 (38%), SM3 (39%), and AM3 + RL (37%), but mortality was greater in SM3 + RL (58%) (Table 2). There was no significant difference in brood size between the first and second generation (F = 0.17; df = 1, 95; P = 0.6785) (data not shown). The mean number of progeny produced (± SE) in the first generation was 6.17 ± 1.57 compared with 4.17 ± 0.62 in the second generation. The percentages of female founders that established broods in AM3 were 66 and 50 in the first and second generations, respectively. In contrast, in AM3 + RL, the percentages of female founders that established colonies increased from 25 to 66 from the first to the second generation. The percentages of brood establishment increased from 33 to 41 in SM3, and from 8 to 41 in SM3 + RL from the first to the second generation. No eggs were seen in galleries along the rearing tube walls. Larvae, pupae, and adults were first observed at 19, 25, and 30 d, respectively, after the introduction of the female founder in AM3. In SM3, larvae, pupae, and adults were first observed 18, 24, and 29 d, respectively, after female founder introduction. In AM3 + RL and SM3 + RL, larvae and pupae were not observed along the rearing tube walls, although adults were seen at 35 and 40 d after female introduction, respectively. Recovery of R. lauricola and Other Fungi From X. volvulus Reared in Media Inoculated With R. lauricola R. lauricola was recovered at low frequencies from adult females reared on all three media (Table 3). The fungus was recovered from 13 and 10 beetle galleries in AM1 + RL and SM1 + RL, respectively, but was not recovered from any galleries in AM2 + RL and SM2 + RL. In AM3 + RL and SM3 + RL, the pathogen was recovered from one and six beetle galleries, respectively. Six and 10 other fungi were isolated from colonies inoculated or not inoculated with R. lauricola, respectively (Table 4). R. arxii was the most frequent and abundant fungus detected in heads, bodies, and galleries of X. volvulus, both in avocado and silkbay media. Interestingly, other Raffaelea species (R. fusca T.C. Harr., Aghayeva & Fraedrich, R. subalba T.C. Harr., Aghayeva & Fraedrich, and R. subfusca T.C. Harr., Aghayeva & Fraedrich) were isolated only from non-inoculated media. Candida spp. were isolated only from beetle bodies and galleries, but never from beetle heads. Other fungi were found only in heads (Alloascoidea africana comb. nov. [Saccharomycetales: Alloascideaceae], Ambrosiozyma monospora Saito [Saccharomycetales: incertae sedis], and Zygozyma oligophaga Van der Walt & Arx [Saccharomycetales: Lipomycetaceae]), or galleries (Leucosphaerina arxii Malloch [Hypocreales: Bionectriaceae], Pichia manshurica Saito [Saccharomycetales: Pichiaceae], and Saccharomycopsis microspore (L. R. Batra) Kurtzman [Saccharomycetales: Saccharomycopsidaceae]) (Table 4). Table 3. Frequency of recovery of R. lauricola from the main body parts of X. volvulus adults reared in artificial media previously inoculated with the fungus Host . Medium typea . Mean no. of CFUs per head and pronotum . Frequency n/N . Mean no. of CFUs per body lacking head and pronotum . Frequency n/N . Avocado Medium 1 15 1/24 6 3/24 Medium 2 348 1/12 17 1/12 Medium 3 0 0/24 0 0/24 Silkbay Medium 1 4 1/24 36 2/24 Medium 2 0 0/12 5 1/12 Medium 3 8.7 3/24 16 1/24 Host . Medium typea . Mean no. of CFUs per head and pronotum . Frequency n/N . Mean no. of CFUs per body lacking head and pronotum . Frequency n/N . Avocado Medium 1 15 1/24 6 3/24 Medium 2 348 1/12 17 1/12 Medium 3 0 0/24 0 0/24 Silkbay Medium 1 4 1/24 36 2/24 Medium 2 0 0/12 5 1/12 Medium 3 8.7 3/24 16 1/24 n, number of beetles positive for the presence of R. lauricola; N, number of beetles tested; CFU, colony-forming units of R. lauricola. aNote that each medium was evaluated separately, and was considered a separate experiment. Open in new tab Table 3. Frequency of recovery of R. lauricola from the main body parts of X. volvulus adults reared in artificial media previously inoculated with the fungus Host . Medium typea . Mean no. of CFUs per head and pronotum . Frequency n/N . Mean no. of CFUs per body lacking head and pronotum . Frequency n/N . Avocado Medium 1 15 1/24 6 3/24 Medium 2 348 1/12 17 1/12 Medium 3 0 0/24 0 0/24 Silkbay Medium 1 4 1/24 36 2/24 Medium 2 0 0/12 5 1/12 Medium 3 8.7 3/24 16 1/24 Host . Medium typea . Mean no. of CFUs per head and pronotum . Frequency n/N . Mean no. of CFUs per body lacking head and pronotum . Frequency n/N . Avocado Medium 1 15 1/24 6 3/24 Medium 2 348 1/12 17 1/12 Medium 3 0 0/24 0 0/24 Silkbay Medium 1 4 1/24 36 2/24 Medium 2 0 0/12 5 1/12 Medium 3 8.7 3/24 16 1/24 n, number of beetles positive for the presence of R. lauricola; N, number of beetles tested; CFU, colony-forming units of R. lauricola. aNote that each medium was evaluated separately, and was considered a separate experiment. Open in new tab Table 4. Fungal species isolated from subsamples of five X. 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 pronotum . Bodya . Gallery . Head and pronotum . Bodya . Gallery . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Media inoculated with R. lauricola Alloascoidea africana 1/5 42 0/5 0 2/5 3/5 9.7 0/5 0 0/5 Ambrosiozyma monospora 1/5 49 0/5 0 2/5 0/5 0 0/5 0 0/5 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 inoculated with R. lauricola Alloascoidea africana 1/5 11 0/5 0 2/5 1/5 4 0/5 0 0/5 Candida berthetii 0/5 0 1/5 2356 0/5 0/5 0 0/5 0 0/5 Candida nemodendra 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 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 Treatment . Isolate ID . Medium containing avocado sawdust . Medium containing silkbay sawdust . Head and pronotum . Bodya . Gallery . Head and pronotum . Bodya . Gallery . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Media inoculated with R. lauricola Alloascoidea africana 1/5 42 0/5 0 2/5 3/5 9.7 0/5 0 0/5 Ambrosiozyma monospora 1/5 49 0/5 0 2/5 0/5 0 0/5 0 0/5 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 inoculated with R. lauricola Alloascoidea africana 1/5 11 0/5 0 2/5 1/5 4 0/5 0 0/5 Candida berthetii 0/5 0 1/5 2356 0/5 0/5 0 0/5 0 0/5 Candida nemodendra 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 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. Open in new tab Table 4. Fungal species isolated from subsamples of five X. 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 pronotum . Bodya . Gallery . Head and pronotum . Bodya . Gallery . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Media inoculated with R. lauricola Alloascoidea africana 1/5 42 0/5 0 2/5 3/5 9.7 0/5 0 0/5 Ambrosiozyma monospora 1/5 49 0/5 0 2/5 0/5 0 0/5 0 0/5 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 inoculated with R. lauricola Alloascoidea africana 1/5 11 0/5 0 2/5 1/5 4 0/5 0 0/5 Candida berthetii 0/5 0 1/5 2356 0/5 0/5 0 0/5 0 0/5 Candida nemodendra 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 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 Treatment . Isolate ID . Medium containing avocado sawdust . Medium containing silkbay sawdust . Head and pronotum . Bodya . Gallery . Head and pronotum . Bodya . Gallery . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Avg. CFU/ Beetle . Freq. n/N . Media inoculated with R. lauricola Alloascoidea africana 1/5 42 0/5 0 2/5 3/5 9.7 0/5 0 0/5 Ambrosiozyma monospora 1/5 49 0/5 0 2/5 0/5 0 0/5 0 0/5 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 inoculated with R. lauricola Alloascoidea africana 1/5 11 0/5 0 2/5 1/5 4 0/5 0 0/5 Candida berthetii 0/5 0 1/5 2356 0/5 0/5 0 0/5 0 0/5 Candida nemodendra 0/5 0 0/5 0 0/5 0/5 0 0/5 0 1/5 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. Open in new tab Discussion Ambrosia beetles live in nutritional symbioses with ambrosia fungi, which are most often species within the order Ophiostomatales (Ascomycota) (Biedermann et al. 2009). These fungi are cultivated in galleries made by the beetles in the sapwood or heartwood of stressed trees (Kirkendall et al. 2015). Initially, it was presumed that ambrosia beetles were associated with a single dominant fungal species (Batra 1963). However, these insects are now known to have multiple fungal associates (Gebhardt et al. 2004, Harrington et al. 2010). Scott and Du Toit (1970) reported that the dominant symbiont of Xyleborus torquatus (a synonym of X. volvulus) was R. arxii, which was repeatedly isolated from galleries excavated in Schefflera (Cussonia) umbellifera (Sond.) Baill. (Apiales: Araliaceae) in the Dukuduku Forest, Natal, South Africa. In this study, R. arxii was the most abundant and frequently recovered symbiont from X. volvulus heads, bodies, and galleries. However, in the present study, other Raffaelea species (R. fusca, R. subalba, and R. subfusca) and several yeast species were also frequently associated with X. volvulus. Our results corroborate that R. arxii may be the primary symbiont of X. volvulus. Previously, R. lauricola was recovered from 20 of 63 individuals of X. volvulus (32%) from LW-affected native Persea spp. and 13 of 217 individuals (6%) from LW-affected avocado trees (Ploetz et al. 2017). In the present studies, we inoculated the rearing media with R. lauricola. In general, media inoculated with the pathogen resulted in less progeny than non-inoculated media; however, these results were not significant except on female production in AM2 versus AM2 + RL. R. lauricola was recovered from bodies of few individuals (four of 60 total on both avocado and silkbay sawdust, 6.7%) and showed very limited colonization of the mycangium (two of 60 total on avocado, 3.3%, and four of 60 total on silkbay, 6.7%). These results suggest that R. lauricola does not fulfill the nutritional requirements of X. volvulus. Foundress females that were used in this work were healthy adults with mycangia that were presumably colonized with their primary or other symbiont(s). Mycangia have specialized glandular cells that secrete substances that protect fungal cells from desiccation, regulate fungal species composition, provide nourishment for fungal propagules, and determine the form of fungal growth in the mycangium (Batra 1963, Happ et al. 1971, Six 2003, Yuceer et al. 2011). The infrequent recovery of R. lauricola from mycangia and bodies of X. volvulus in the present work may reflect the adaptation of other ambrosial fungi in this beetle species. The displacement of these primary symbionts by R. lauricola might not be expected, especially if the individual had already established a nutritional mutualism with its primary, adapted symbiont. That R. lauricola was not recovered from galleries in media inoculated with the pathogen also suggests females of X. volvulus do not cultivate R. lauricola, even in substrates that were previously colonized with the fungus. In summary, suboptimal reproduction on the inoculated media indicated that R. lauricola is probably a poor nutritional partner for X. volvulus. How it disrupts the beetle’s normal nutritional cycle is unclear, as it was generally absent in both the beetle and the +RL rearing media. Little is known about the nutrition of ambrosia beetles by their fungal associates. Freeman et al. (2016) reported that larvae of Euwallacea nr. fornicatus fed on Paracremonium pembeum sp. nov. did not complete their life cycle, whereas those reared on Fusarium euwallaceae and Graphium euwallaceae completed development. Similarly, Paecilomyces variotii Bainier negatively impacted larvae and adults of X. saxesenii, whereas the beetle’s primary nutritional symbiont, Raffaelea sulfurea (L. R. Batra), had positive effects (Biedermann et al. 2012). In addition, R. lauricola had been shown to negatively impact the reproduction of X. crassiusculus (Ott 2007). Recently, Saucedo et al. (2017) reported that four different species of Raffaelea, including R. lauricola, supported the development of a single generation of X. bispinatus. The latter experiments were initiated 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 previously colonized females had been used (as in the present studies) is not clear. Better understandings are needed for these interactions. In artificial rearings, the type of sawdust used in the medium may affect ambrosia beetle reproduction (Castrillo et al. 2012). We found no clear differences on the suitability of avocado and silkbay sawdust to rear X. volvulus. In the medium that provided the best rearing conditions, the beetle bred similarly in avocado and silkbay sawdust. In the two other media, which differed only by their water content, we obtained contrasting results in regards to the type of sawdust used. Our results suggest that the water content in the rearing substrate had a greater effect than the type of sawdust on the ability of X. volvulus to produce offspring. Interestingly, natural breeding of this beetle species occurs in avocado trees showing early signs of decline, when the wood retains substantial water content. A range of water contents in these media should be tested to determine how water content affects both fungal growth and beetle development. X. volvulus females started excavating in the media soon after they were introduced into the rearing tubes. Female tunneling activity normally occurred along the wall of the rearing tube making observations possible without disturbing or altering the media. Larvae and pupae were seen at different times depending on the treatment, ranging from 15 to 21 d and from 19 to 27 d after the female had been introduced into the various media. Adults were first seen in galleries 24 d after female introduction, and they remained in galleries for ~1 wk. During this period, it seems probable that mating occurred between sibling females and males, and adults engaged in colony maintenance activities (i.e., cleaning). Often in this study, at the time of dissection (i.e., 40 d after the female’s introduction), all stages were found concurrently in the colony suggesting overlapping generations. Eggs and larvae were frequently clumped between the main and the secondary galleries, some of which were probably produced by the new generation of females. The reproductive potential of X. volvulus was similar to that of X. glabratus reared in two artificial sawdust-based media (Maner et al. 2013). Using a different approach, Brar et al. (2013) found lower rates of X. glabratus reproduction on bolts of three tree species (i.e., avocado, redbay, and swampbay) (only 1.1 females developed on bolts originally infested with 20 female beetles). Thus, sawdust-based media appear to be better than bolts for establishing laboratory colonies of ambrosia beetles. Although the development of artificial substrates for ambrosia beetles is challenging, these media can be quite useful. Artificial substrates facilitate the otherwise difficult study of larval feeding habits on, and the nutritional quality of, different fungi. Artificial media may be used to study the social behavior of ambrosia beetles and to study their sexual dimorphism, which could provide characters to separate closely related species. Artificial media may also be used to investigate intra- and inter-specific competition and to evaluate potential management tactics. In summary, this study demonstrated that X. volvulus can be reared on artificial substrates made with sawdust obtained from avocado or silkbay. Based on our results, Medium 1 previously proposed by Castrillo et al. (2012) was superior for rearing X. volvulus. Females in this medium developed faster, survived longer, and produced more progeny. Our results suggest that probably R. lauricola is not a nutritional symbiont (i.e., does not fulfill the requirements) of X. volvulus, but in some cases, the association with this pathogen could be commensalistic, not affecting the beetle’s reproduction. More research is needed to confirm this hypothesis. Acknowledgments This study partially fulfills the requirements for the MS degree, University of Florida, by O. A. M. We thank James Colee (UF-IFAS-Statistics Department) for his help with the statistical analysis, Waldemar Klassen and Jorge E. Peña (University of Florida) for suggestions to improve the manuscript. 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TI - Rearing Xyleborus volvulus (Coleoptera: Curculionidae) on Media Containing Sawdust from Avocado or Silkbay, With or Without Raffaelea lauricola (Ophiostomatales: Ophiostomataceae)
JF - Environmental Entomology
DO - 10.1093/ee/nvx151
DA - 2017-12-08
UR - https://www.deepdyve.com/lp/oxford-university-press/rearing-xyleborus-volvulus-coleoptera-curculionidae-on-media-sSvvMj4H0M
SP - 1275
VL - 46
IS - 6
DP - DeepDyve
ER -