insects Article Xyleborus bispinatus Reared on Artificial Media in the Presence or Absence of the Laurel Wilt Pathogen (Raffaelea lauricola) Octavio Menocal 1,*, Luisa F. Cruz 1, Paul E. Kendra 2 ID , Jonathan H. Crane 1, Miriam F. Cooperband 3, Randy C. Ploetz 1 and Daniel Carrillo 1 1 Tropical Research & Education Center, University of Florida 18905 SW 280th St, Homestead, FL 33031, USA; luisafcruz@ufl.edu (L.F.C.); jhcr@ufl.edu (J.H.C.); kelly12@ufl.edu (R.C.P.); dancar@ufl.edu (D.C.) 2 Subtropical Horticulture Research Station, USDA-ARS, 13601 Old Cutler Rd., Miami, FL 33158, USA; [email protected] 3 Otis Laboratory, USDA-APHIS-PPQ-CPHST, 1398 W. Truck Road, Buzzards Bay, MA 02542, USA; [email protected] * Correspondence: omenocal18@ufl.edu; Tel.: +1-786-217-9284 Received: 12 January 2018; Accepted: 24 February 2018; Published: 28 February 2018 Abstract: Like other members of the tribe Xyleborini, Xyleborus bispinatus Eichhoff can cause economic damage in the Neotropics. X. bispinatus has been found to acquire the laurel wilt pathogen Raffaelea lauricola (T. C. Harr., Fraedrich & Aghayeva) when breeding in a host affected by the pathogen. Its role as a potential vector of R. lauricola is under investigation. The main objective of this study was to evaluate three artificial media, containing sawdust of avocado (Persea americana Mill.) and silkbay (Persea humilis Nash.), for rearing X. bispinatus under laboratory conditions. In addition, the media were inoculated with R. lauricola to evaluate its effect on the biology of X. bispinatus. There was a significant interaction between sawdust species and R. lauricola for all media. Two of the media supported the prolific reproduction of X. bispinatus, but the avocado-based medium was generally more effective than the silkbay-based medium, regardless whether or not it was inoculated with R. lauricola. R. lauricola had a neutral or positive effect on beetle reproduction. The pathogen was frequently recovered from beetle galleries, but only from a few individuals which were reared on inoculated media, and showed limited colonization of the beetle’s mycangia. Two media with lower water content were most effective for rearing X. bispinatus. Keywords: ambrosia beetle; ambrosia fungi; beetle–fungus symbiosis; Coleoptera; Curculionidae; Lauraceae; Persea americana; Persea humilis; Scolytinae; Xyleborini 1. Introduction Raffaelea lauricola (T. C. Harr., Fraedrich & Aghayeva; Ophiostomatales: Ophiostomataceae) is a fungal pathogen carried primarily by the redbay ambrosia Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) [1,2]. The pathogen is responsible for the vascular disease known as laurel wilt (LW), which affects American members of the Lauraceae family including redbay (Persea borbonia (L.) Spreng), swampbay (Persea palustris (Raf.) Sarg), silkbay (Persea humilis Nash), sassafras (Sassafras albidum (Nutall) Nees), pondspice (Litsea aestivalis (L.) Fernald), pondberry (Lindera melissifolia (Walter) Blume), and avocado (Persea americana Mill.) [1,3–6]. X. glabratus is rarely associated with LW-affected avocado trees in commercial plantings in south Florida [7,8]. Previously, the lateral transfer of R. lauricola to at least nine of the ambrosia beetle species, in addition to X. glabratus, was demonstrated in Florida [9,10]. Moreover, at least two of these Insects 2018, 9, 30; doi:10.3390/insects9010030 www.mdpi.com/journal/insects Insects 2018, 9, 30 2 of 13 Insects 2018, 9, x FOR PEER REVIEW 2 of 13 species, Xyleborus volvulus (F.) and Xyleborus ferrugineus (F.), were shown to experimentally transmit the pathogenspecies, Xyleborus to avocado volvulus and (F.) redbay and Xyleborus [9]. ferrugineus (F.), were shown to experimentally transmit Xyleborusthe pathogen bispinatus to avocado(Figure and redbay1) was [9]. reported in Florida for the first time by Atkinson et al. [ 11]. Xyleborus bispinatus (Figure 1) was reported in Florida for the first time by Atkinson et al. [11]. Previously, it had not been distinguished from X. ferrugineus, a smaller, morphologically similar Previously, it had not been distinguished from X. ferrugineus, a smaller, morphologically similar ambrosia beetle. Both species have broad distributions throughout South America, Central America, ambrosia beetle. Both species have broad distributions throughout South America, Central America, and inand the in southeasternthe southeastern United United States States [12 [12,13],,13], andand X. bispinatus hashas also also been been reported reported in Italy in Italy [14]. [14]. FigureFigure 1. Adult1. Adult female female of ofXyleborus Xyleborus bispinatus bispinatus EichhoffEichhoff (lateral (lateral view). view). Rabaglia et al. [15] stated that X. ferrugineus caused economic damage in lowland areas of the RabagliaNeotropics. et However, al. [15] stated there are that noX. supporting ferrugineus datacaused for this economic statement. damageIn forest ecosystems in lowland of areascentral of the Neotropics.and southern However, Florida, there X. areferrugineus no supporting and X. bispinatus data for thisoccur statement. sympatrically In forest with ecosystemsX. glabratus, ofwith central and southernwhich they Florida, reproduceX. ferrugineus in LW-affectedand X. swampbay bispinatus anoccurd silkbay sympatrically [16,17]. Recently, with X. it glabratushas been, shown with which they reproduceshown that infemales LW-affected of X. bispinatus swampbay incorporate and silkbay R. lauricola [16,17 in]. their Recently, mycangia it has [10], been the storage shown organs shown that femalesfor ofsporesX. bispinatus of symbioticincorporate fungi [18].R. Although lauricola inthe their latter mycangia were not [distinguished10], the storage in X. organs ferrugineus for spores by of Carrillo et al. [9], it seems probable that the morphologically similar X. bispinatus can also transmit R. symbiotic fungi [18]. Although the latter were not distinguished in X. ferrugineus by Carrillo et al. [9], lauricola to avocado. it seems probable that the morphologically similar X. bispinatus can also transmit R. lauricola to avocado. Introduced pathogens have had significant detrimental effects on forest communities. Relevant Introducedexamples include pathogens Cryphonectria have hadparasitica significant (Murril) detrimental M. E. Barr, the effects causal on agent forest of communities.chestnut blight [19], Relevant examplesCronartium include ribicolaCryphonectria Fisch, the causal parasitica agent(Murril) of white M. pine E. Barr,blister the rust causal [20], agentand Ophiostoma of chestnut novo-ulmi blight [19], CronartiumBrasier, ribicolathe pathogenFisch, that the causalcauses the agent Dutch of whiteelm disease pine blister[21]. For rust an [20increasing], and Ophiostoma number of these novo-ulmi Brasier,diseases, the pathogen scolytine that beetles causes are vectors the Dutch of the elm causal disease agents [21 [22].]. For In angeneral, increasing there is number limited ofinformation these diseases, scolytineon the beetles behavior are and vectors biology of theof thes causale insects. agents However, [22]. In with general, the help there of isartificial limited media, information it may be on the behaviorpossible and to biology improve of our these understanding insects. However, of their withintera thections help with of host artificial plants media, and pathogens, it may be as possible well to improveas their our roles understanding in the epidemiology of their of interactions these diseases. with host plants and pathogens, as well as their roles The first ambrosia beetle species reared on an artificial medium was X. ferrugineus [23]. Since in the epidemiology of these diseases. then, agar- and sawdust-based media have been used to rear and study the biology of several other X. ferrugineus Theambrosia first ambrosiabeetles [24–28]. beetle species reared on an artificial medium was [23]. Since then, agar- andHere, sawdust-based we describe media a series have of beenstudies used in which to rear artificial and study media the were biology evaluated of several for otherrearing ambrosia X. beetlesbispinatus [24–28.]. The tested media incorporated sawdust from avocado or silkbay and were used to conduct Here,preliminary we describe studies on a X. series bispinatus of studies and its biological in which response artificial to R. media lauricola were. evaluated for rearing X. bispinatus. The tested media incorporated sawdust from avocado or silkbay and were used to conduct2. Materials preliminary and Methods studies on X. bispinatus and its biological response to R. lauricola. 2. Materials2.1. Artificial and Media Methods Sawdust from two hosts of X. bispinatus were evaluated. Logs of avocado were collected in 2.1. ArtificialFebruary Media 2016 from an unsprayed avocado orchard in Miami-Dade County, FL (25°29′38″ N; Sawdust80°28′53″ W), from andtwo logs of hosts silkbay of wereX. bispinatus collected inwere February evaluated. 2016 fromLogs the Archbold of avocado Biological were Station collected in Highlands County, FL (27°10′50″ N; 81°21′0″ W). The logs were only used when they were found in February 2016 from an unsprayed avocado orchard in Miami-Dade County, FL (25◦2903800 N; to be free of beetle infestation. Sawdust was produced as described by Castrillo et al. [29], and the 80◦2805300 W), and logs of silkbay were collected in February 2016 from the Archbold Biological Station rearing media were prepared as described by Menocal et al. [28]. in Highlands County, FL (27◦1005000 N; 81◦210000 W). The