OCTAVIO A. MENOCAL SANDOVAL

Transcription

1 ARTIFICIAL REARING OF XYLEBORUS VOLVULUS AND XYLEBORUS BISPINATUS AND VERTICAL DISTRIBUTION OF AMBROSIA BEETLES IN LAUREL WILT AFFECTED AVOCADO ORCHARDS By OCTAVIO A. MENOCAL SANDOVAL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA

2 2017 Octavio A. Menocal Sandoval 2

3 To the Almighty God and my parents 3

4 ACKNOWLEDGMENTS First of all, I would like to thank the Almighty God for helping and guiding me during my program. My deepest appreciation goes to Dr. Daniel Carrillo who provided me with this great opportunity to carry out this research. He did not only believe in me but also invested part of his time to show me how to be a better researcher. His scientific guidance, knowledge, comprehension, patience, and friendship have been invaluable. Thanks to Drs. Jonathan Crane and Paul Kendra for their professional advice and reviews on this thesis. I acknowledge Dr. James Colee (University of Florida Statistics Department) for his continuing support and time during my data analysis. My special gratitude goes towards Drs. Waldemar Klassen and Jorge Peña for their constant reviews to this document. Special recognition goes to Luisa Cruz, thank you for your friendship and valuable advices; also thank you for helping me during my data collection, data interpretation, and analysis. I would like to apologize that you had to work every single holiday and weekend with me. I would not have finished this research if it wasn t for you, thanks a lot from the bottom of my heart. Thanks to Edwin Gutiérrez for teaching me that the place or the time does not matter when it comes to a bad cup of coffee or the cheapest wine. Thanks to Raiza Castillo and Joe Martínez for teaching me that age doesn t matter when it comes to anime and video games. Thanks to Manuela Angel (Dios se lo pague) and Ramón Saucedo for their friendship and many invaluable memories. Thanks to Pablo Q-numi masca almohadas sopla velas Vargas for always having the time to read my work. I also give thanks to many friends: Leonardo Alvarez (Colombia), Rita Duncan (Chile-USA), José Moe Alegría (Mex-USA), Julio Mantilla and Teresa Narváez 4

5 (Ecuador), Ana Vargas and José Pérez (Cuba), Stephanie Suárez (Cuba-USA), Andrea Rubiverdu, Pablo Urbaneja and Georgina Sanahuja (Spain), Charlotte de Grave (Belgium), Wanda Montás (Dominican Republic), Simon Yeboah and William Heve (Ghana), Sabyan Faris and Imran Khan (Pakistan), Marielle Berto (Brazil), Mohammad Razzak (Bangladesh), Natalie Francis (Jamaica), Tina Dispenza and Julius Eason (USA), Deanroy Mbabazi (Uganda), Sonia Rocío, Lizeth Durán, Julia Meliton, Armando Garza (México), Guosheng Yao (China), and Tran Vo (Vietnam) for their friendship and support during my studies. Finally, last but not least, I would like to thank my parents Norma Sandoval and Octavio A. Menocal who always believed in me and encouraged me to pursue a degree in science. I can only offer an inadequate way of acknowledgment towards them. 5

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 8 LIST OF FIGURES... 9 ABSTRACT CHAPTER 1 LITERATURE REVIEW General Aspects of Ambrosia Beetles Host-Seeking Behavior Gallery Initiation and Fungal Inoculation Ambrosia Beetles as Pests Laurel Wilt Research Objectives Structure of the Thesis REARING XYLEBORUS VOLVULUS (COLEOPTERA: CURCULIONIDAE) ON MEDIA CONTAINING SAWDUST FROM AVOCADO OR SILKBAY, WITH OR WITHOUT RAFFAELEA LAURICOLA (OPHIOSTOMATALES: OPHIOSTOMATACEAE) Abstract Introduction Materials and Methods Ambrosia Beetles Artificial Media Media Inoculation Rearing Conditions Gallery Dissection Fungal Isolation Statistical Analysis Results Medium Medium Medium Recovery of R. lauricola and Other Fungi from X. volvulus Reared in Media Inoculated with R. lauricola Discussion Acknowledgments

7 3 XYLEBORUS BISPINATUS (COLEOPTERA: CURCULIONIDAE) REARED ON ARTIFICIAL MEDIA USING SAWDUST FROM AVOCADO OR SILKBAY IN PRESENCE OR ABSENCE OF THE LAUREL WILT PATHOGEN (RAFFAELEA LAURICOLA) Abstract Introduction Materials and Methods Ambrosia Beetles Artificial Media Media Inoculation Rearing Conditions Dissection of Colonies Fungal Isolation Statistical Analysis Results Medium Medium Medium Recovery of Other Fungi and R. lauricola from X. bispinatus Reared on Artificial Media Discussion Acknowledgments VERTICAL DISTRIBUTION OF IN-FLIGHT AMBROSIA BEETLES ASSOCIATED WITH LAUREL WILT AFFECTED IN AVOCADO ORCHARDS Abstract Introduction Materials and Methods Study Sites Beetle Trapping Statistical Analysis Results Discussion Acknowledgments CONCLUSIONS LIST OF REFERENCES BIOGRAPHICAL SKETCH

8 LIST OF TABLES Table page 2-1 Recipes of the three types of artificial media using either avocado or silkbay sawdust for rearing Xyleborus volvulus Biological parameters of Xyleborus 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 Raffaelea lauricola Frequency of recovery of Raffaelea lauricola from the main body parts of Xyleborus volvulus adults reared in artificial media previously inoculated with the fungus Fungal species isolated from subsamples of five Xyleborus volvulus beetles and their galleries collected from media based on sawdust of avocado or silkbay. The fungi were isolated from the head and pronotum or from the body lacking the head and pronotum Developmental stages and biological parameters of Xyleborus bispinatus on three artificial media based either on avocado or silkbay sawdust inoculated or not inoculated with Raffaelea lauricola Frequency and recovery of Raffaelea lauricola from females of Xyleborus bispinatus reared on artificial media previously inoculated with this fungus Fungal species isolated from 12 X. bispinatus beetles and their galleries collected either from avocado or silkbay based media. Fungi were isolated from the head and pronotum or from the body lacking the head and pronotum Mean number ± SE of ambrosia beetles caught at three different height levels in three avocado orchards in Homestead, Florida

9 LIST OF FIGURES Figure page 1-1 Adult female of Xyleborus glabratus Eichhoff (lateral view) Current distribution of LW in the commercial avocado growing area in south Florida. Purple dots represent incidence of diseased trees and the green lines represent avocado groves. Map credit: Charlotte de Grave & Don Pybas Adult female of (A) Xyleborus bispinatus Eichhoff and (B) Xyleborus volvulus Fabricius Number of Xyleborus volvulus females 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 Raffaelea lauricola*. Media contained sawdust of either avocado or silkbay. Bars represent the mean (± SE) number of brood (gray) and females (black) produced per foundress female. Columns with the same letters are not significantly different (P < 0.05) Number of Xyleborus bispinatus females and total brood produced by one female reared in one of three artificial media (A = medium 1; B = medium 2 and C = medium 3) prepared either of avocado or silkbay sawdust each inoculated or not inoculated with Raffaelea lauricola*. Bars represent the mean (± SE) number of females (black) and brood produced (gray) per female. Columns with the same letters are not significantly different (P < 0.05) Effect of three height levels on numbers of ambrosia beetles trapped. (Low = 0 2 m; Middle = 2 4 m; Top = 4 6 m). Bars represent the mean ± SE of ambrosia beetles caught per height level. Bars with the same letters in the same column are not significantly different (P < 0.05)

10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ARTIFICIAL REARING OF XYLEBORUS VOLVULUS AND XYLEBORUS BISPINATUS AND VERTICAL DISTRIBUTION OF AMBROSIA BEETLES IN LAUREL WILT AFFECTED AVOCADO ORCHARDS Chair: Daniel Carrillo Major: Entomology and Nematology By Octavio A. Menocal Sandoval May 2017 Two studies were conducted to evaluate three artificial media for rearing two ambrosia beetles, Xyleborus volvulus and Xyleborus bispinatus, reported as potential vectors of the Laurel Wilt disease in avocado. The media contained varying amounts of water and sawdust obtained from either avocado or silkbay (Persea humilis Nash), among other ingredients. The effect of the laurel wilt-pathogen (Raffaelea lauricola) on the biological parameters of both beetles was also evaluated. Of the three media, the one with the least amount of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Beetle reproduction on this medium was not affected by the type of sawdust or the presence of R. lauricola. On the other two media, there was a significant effect of sawdust type on beetle reproduction, and the presence of R. lauricola had a negative effect on brood production. There was limited colonization of R. lauricola within the mycangia of X. volvulus reared on media inoculated with the pathogen. Two of the media supported high reproduction of X. bispinatus, but the avocadobased medium was generally better than the silkbay-based medium. The presence of R. 10

11 lauricola had a neutral or enhancing effect on beetle reproduction. When X. bispinatus was reared on media inoculated with R. lauricola, there was limited mycangial colonization by the pathogen. One additional experiment was conducted to determine flight height of ambrosia beetles in laurel wilt affected avocado orchards. In this study, X. volvulus showed a preference for flying at 0 2 m and X. bispinatus at 0 4 m above the ground; other ambrosia beetles captured in this study did not show any preference for the different heights tested (0 6 m). From the first two studies, we can hypothesize that X. bispinatus is more likely to be a more efficient vector of R. lauricola than X. volvulus. These two ambrosia beetles interact with the main trunk and major scaffold limbs of avocado trees. Overall, these studies provide a better understanding of the role that these two species are playing as vectors of laurel wilt in avocado. 11

12 CHAPTER 1 LITERATURE REVIEW General Aspects of Ambrosia Beetles Ambrosia beetles belonging to the weevil tribe Xyleborini are fungal farmers and have symbiotic relationships with ambrosia fungi. Adult beetles carry fungal spores in special organs called mycangia (Fraedrich et al. 2008, Rabaglia et al. 2006). They breed in trunks and limbs of stressed and decaying trees. They bore through the bark of the tree and form galleries in the xylem wood. Ambrosia beetles usually do not feed on their host plant the excavated sawdust is not consumed but pushed out of the gallery in the form of frass instead, they cultivate fungi in the galleries that serve as food for them and their offspring (Wood 1977, Mayfield et al. 2008).Typically, ambrosia beetles have an obligate nutritional symbiosis with ambrosia fungi belonging to the order Ophiostomatales (Ascomycota) (Biedermann et al. 2009) and it has been suggested that this relationship is adapted to a particular fungal partner (Brace and Six 2015). Most of the ambrosia beetles share a unique characteristic: their life cycle is completed within their galleries (Rudinsky 1962). The eggs have an oval shape and are translucent and whitish. The larvae undergo three instars, are legless, and have a c- shaped form. Pupae are similar to the adults but white and motionless, and adults are typically between 2 3 mm long and dark brown. After boring in the host plant, ambrosia beetles reside in the galleries and oviposit over a prolonged period, which leads to overlapping generations (Mizuno and Kajimura 2002). Eggs, larvae, and pupae co-occur in the gallery system. In general, there is one male per colony that is flightless and usually do not leave the colony whereas females are present in great numbers (i.e., 12

13 female-biased sex ratio). The next generation of females mates with siblings and garner fungal spores in their mycangia before seeking for a new host. In some species young females may delay dispersal and remain in the natal gallery engaged in mutually beneficial behaviors maintaining the fungus garden, therefore showing a primitively eusocial behavior (Biedermann et al. 2012). Host-Seeking Behavior After complete sclerotization, mated females leave the natal gallery to search for a new host tree. Such emergence occurs when the right environmental conditions exist typically over a period of several hours (Rudinsky 1962). The goal of female s dispersion is to locate a good quality host tree in which to reproduce. During dispersal, they are subject to potentially adverse environmental conditions and predation. For instance, woodpeckers are the most important avian predator of bark beetles and may also attack ambrosia beetles. Other avian predators forage on beetles on the bark of standing or fallen trees as well as on beetles in-flight (Wegensteiner et al. 2015). These include bluebirds (Turdidae), jays (Corvidae), and tyrant flycatchers (Tyrannidae). Besides birds, other insects prey on ambrosia and bark beetles. The group of predators that prey on bark beetles and possibly on ambrosia beetles is diverse and includes other beetles (Coleoptera), true bugs (Hemiptera), flies (Diptera), snakeflies (Raphidioptera), and mites (Acari) (Wegensteiner et al. 2015). To reduce mortality by predators and avoid adverse environmental conditions ambrosia beetles have developed an efficient host-seeking behavior during dispersal that includes flying at particular times and heights. This behavior may be explained by a multi-step process 13

14 proposed by Kendra et al. (2014) for the redbay ambrosia beetle (RAB), Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae: Xyleborini) (Figure 1-1.), which may apply for other ambrosia beetles. Flight activity is initiated due to an interaction of environmental cues such as percent relative humidity, light intensity, and temperature (Chen et al. 2010). The peak flight activity of RAB begins in the late afternoon and early evening (Brar et al. 2012, Kendra et al. 2012). After females have left their previous host tree, they use long-range olfactory cues while in flight. Apparently, RAB does not utilize aggregation or sex pheromones to locate new hosts (Hanula et al. 2008). Females are attracted to natural volatiles emitted from Lauraceae trees (Hanula and Sullivan 2008, Kendra et al. 2011a). Four sesquiterpenes were identified that were correlated with attraction: α-cubebene, α-copaene, α-humulene, and calamenene (Kendra et al. 2014). In another study, the monoterpene eucalyptol was found to be an additional attractant for RAB (Kuhns et al. 2014). These findings suggest that RAB and possibly other ambrosia beetles use a variety of chemical cues to locate potential host trees (Kendra et al. 2014). As the RAB female approaches a new host, it may rely on visual cues such as a larger tree-diameter (Fraedrich et al. 2008). Preference for larger diameter trees could be related to the beetle fitness because a larger tree can support larger beetle galleries, which are positively correlated with the number of beetle offspring (Mizuno and Kajimura 2002). Little is known about shortrange cues, which could be a complex of different signals (gustatory, tactile, visual, olfactory, and chemosensory) that act synergistically. Besides, using chemoreceptors on the antennae, RAB may use receptors located on other parts of its body. According to Kendra et al. (2014), some females make shallow holes with their mandibles, 14

15 apparently sampling the potential host for suitability. Receptors located on the maxillary and labial palpi can detect chemical signals that may be positive (attractive) or negative (repellent) to the insect. Figure 1-1. Adult female of Xyleborus glabratus Eichhoff (lateral view). Gallery Initiation and Fungal Inoculation When a female ambrosia beetle lands on its host, it begins to bore perpendicularly to the surface. The gallery system consists primarily of an entrance hole, followed by the main tunnel that branches into secondary and tertiary tunnels (Brar et al. 2013, Mizuno and Kajimura 2002). During the gallery construction process, females inoculate symbiotic fungi carried in their mycangia on the walls of the growing tunnel. RAB and other Xyleborus species have mandibular mycangia. In contrast, females of Xyleborinus saxesenii Ratzeburg do not carry their fungi in the mycangia but in the gut (Biedermann et al. 2009). The developmental rate of ambrosia beetles depends on fungal growth (Rudinsky 1962) and the reproductive potential depends on the biomass of fungal symbiont present inside their galleries (Kajimura and Hijii 1994). Moreover, Castrillo et al. (2012) found that more rapid mycelial growth provides more food and could enhance brood production. The host tree species and its physiological state are likely to affect fungal growth (Brar et al. 2013). In addition to the internal 15

16 physical and chemical properties of the tree, the fungi are also affected by moisture content and temperature outside the tree (Rudinsky 1962). Ambrosia Beetles as Pests Ambrosia beetles were typically considered secondary pests of trees because they mostly target decaying or stressed trees (Wood 1982, Kühnholz et al. 2003). However, some exotic ambrosia beetle species are known to attack apparently healthy trees, and others have become important pests due to their association with plant pathogens. A good example of this is the exotic RAB, whose primary symbiont is Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), a fungal pathogen that causes vascular wilt to trees belonging to the Lauraceae (Fraedrich et al. 2008, Harrington et al. 2008). Both RAB and R. lauricola originated in Asia (Rabaglia 2006) and were introduced to Savannah, Georgia in 2002 probably on wood packing material (Fraedrich et al. 2008, Rabaglia et al. 2006). Raffaelea lauricola is the only known symbiont of ambrosia beetles that moves systematically within the host and causes its death by vascular wilt (Fraedrich et al. 2008). Initially, it was presumed that the fungus would grow in the xylem blocking the flow of nutrients and water inside the tree (Mayfield et al. 2008). However, subsequent studies suggest that the presence of R. lauricola triggers the formation of tyloses and secretion of resins by the host tree within the xylem vessels. This reaction, known as parenchymal tyloses, results in blockage of water movement, systemic wilt and tree death (Inch et al. 2011, 2012); all these are symptoms of the disease known as Laurel Wilt (LW). 16

17 Other examples of ambrosia beetles attacking apparently healthy trees include Euwallacea nr. fornicatus Eichhoff on avocado (Persea americana Mill.) trees in Israel (Mendel et al. 2012), Xylosandrus germanus (Blandford) and Xylosandrus crassiusculus (Motschulsky) on healthy chestnut trees (Castanea mollisima Blume) (Oliver and Mannion 2001) and other species such as elm (Ulmus spp.), black walnut (Juglans nigra L.), and beech (Fagus spp.) and dogwood (Cornus spp.) (Weber and McPherson 1983, Ranger et al. 2010). Interestingly enough, Carrillo et al. (2014) documented that other ambrosia beetles besides RAB are carrying the laurel wilt pathogen and are probably vectors of this lethal disease in avocado orchard systems. Laurel Wilt Laurel Wilt has caused extensive mortality of redbay [Persea borbonia (L.) Spreng], swampbay [Persea palustris (Raf.) Sarg], silkbay (Persea humilis Nash) and other lauraceous trees in the southeastern USA. Avocado is the most important agricultural crop susceptible to LW. Moreover, avocado is after citrus (Citrus spp.; Sapindales: Rutaceae) and blueberry (Vaccinium hybrids) the largest fruit industry in Florida. Over 95% of the avocados are grown in Miami-Dade County, and around 85% of the fruit it is sold outside of Florida; thus bringing a significant amount of revenue to the state. The economic impact of avocado is about $100 million/year (Evans et al. 2010, E. A. Evans, personal communication). Since RAB was discovered in Georgia along with its natural symbiont, R. lauricola, much research has gone into trying to understand the biology of ambrosia beetles and to develop strategies to stop the spread of this disease. Although the 17

18 primary vector of LW in natural hammocks is RAB, it is rarely seen associated with laurel wilt-affected avocado trees. Despite this, since the original detection of LW in commercial avocado groves in 2012, the disease has spread throughout the commercial avocado production area in south Florida. Laurel wilt has been responsible for the death of about 20,000 trees commercial avocado tree (approximately 2% of the industry) (Figure 1-2). Figure 1-2. Current distribution of LW in the commercial avocado growing area in south Florida. Purple dots represent the incidence of diseased trees, and the green lines represent avocado groves. Map credit: Charlotte de Grave & Don Pybas. In the last five years of intensive trapping and sampling in avocado groves affected by laurel wilt, RAB has seldom been trapped and has never been observed 18

19 associated with laurel wilt-affected avocado logs collected from commercial avocado groves (Carrillo et al. 2012; Ploetz et al. 2017). By contrast, 14 species of resident ambrosia beetles have been recovered, and R. lauricola was common in two native ambrosia beetles, Xyleborus bispinatus Eichhoff (Figure 1-3A) and Xyleborus volvulus Fabricius (Figure. 1-3B), suggesting that alternative vectors are maybe playing a major role in the LW epidemic in avocado orchards. Scant information is available on their reproductive potential on native (i.e., silkbay and swampbay) and cultivated (i.e., avocado) hosts, and their flight behavior in laurel wilt affected avocado orchards is unknown. Figure 1-3. Adult female of (A) Xyleborus bispinatus Eichhoff and (B) Xyleborus volvulus Fabricius. The conspicuous interaction between these beetles, the pathogen, and avocado trees in south Florida suggests the need for more in-depth investigations into the biology of these potential vectors. The focus of this thesis is the biology of X. volvulus and X. bispinatus, their interaction with R. lauricola, and their flight behavior. 19

20 Research Objectives The aim of this thesis was to evaluate three artificial media consisting of sawdust of either avocado or silkbay for rearing Xyleborus volvulus and Xyleborus bispinatus and to conduct preliminary studies on the interaction between these two beetles and the fungal pathogen Raffaelea lauricola. In addition, the vertical distribution of ambrosia beetles in avocado orchards affected by laurel wilt was also investigated. Structure of the Thesis This thesis is divided into five chapters. Chapter 1 provides a general introduction on ambrosia beetles, their host-seeking behavior, their pest status, the laurel wilt disease, and the research problem and research objectives. Chapters 2 to 4 are presented as three separate articles formatted for submission to scientific peer review journals. Each of these chapters is, therefore, a stand-alone document. Chapter 2 Rearing Xyleborus volvulus (Coleoptera: Curculionidae) on media containing sawdust from avocado or silkbay, with or without Raffaelea lauricola (Ophiostomatales: Ophiostomataceae) explores the biology and reproductive potential of X. volvulus reared in three different artificial media. Aspects such as host effects and the presence of R. lauricola in the rearing media are discussed and provide a better understanding of the interaction of these beetles with the LW pathogen. Chapter 3 Xyleborus bispinatus (Coleoptera: Curculionidae) reared on artificial media using sawdust from avocado or silkbay in presence or absence of the laurel wilt pathogen (Raffaelea lauricola) followed the same methodologies used in Chapter 2 to study the effect of two hosts and the 20

21 presence of R. lauricola in the life cycle of X. bispinatus. Chapter 4 Vertical distribution of ambrosia beetles associated with laurel wilt-disease in avocado orchards explores differences in flight height of ambrosia beetles species and their possible interaction with the different structures of avocado trees. Abiotic and biotic factors influencing the flight behavior of ambrosia beetles are discussed. Chapter 5 presents a summary and conclusions of this research. 21

22 CHAPTER 2 REARING XYLEBORUS VOLVULUS (COLEOPTERA: CURCULIONIDAE) ON MEDIA CONTAINING SAWDUST FROM AVOCADO OR SILKBAY, WITH OR WITHOUT RAFFAELEA LAURICOLA (OPHIOSTOMATALES: OPHIOSTOMATACEAE) Abstract Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) is a pantropical ambrosia beetle species with a broad host range. Like other ambrosia beetles, X. volvulus lives in a mutualistic symbiotic relationship with Ophiostoma fungi that serve as its food source. Until recently, X. volvulus was not considered a pest, and none of its symbionts were considered pathogens of trees. However, recent reports of an association between X. volvulus and Raffaelea lauricola T.C. Harr. Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae) the cause of laurel wilt disease and its potential role as a vector of this lethal disease of avocado (Persea americana Mill.; Laurales: Lauraceae) merit further investigation. The objective of this study was to evaluate three artificial media containing sawdust obtained from either avocado or silkbay (Persea humilis Nash) for laboratory rearing of X. volvulus. The effect of R. lauricola in the rearing media on the beetle s reproduction was also evaluated. Of the three media, the one with the least content of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Beetle reproduction on this medium was not affected by the type of sawdust or the presence of R. lauricola. On the other two media, there was a significant effect of sawdust type on beetle reproduction, and the presence of R. lauricola had a negative effect on brood production. There was limited colonization of R. lauricola within the mycangia of X. volvulus reared on media inoculated with the pathogen. From galleries formed within the best medium, there was 22

23 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 rearing X. volvulus. Introduction 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), which is vectored by ambrosia beetles (Coleoptera: Curculionidae: Scolytinae: Xyleborini). The primary vector of the LW pathogen in native ecosystems, including natural hammocks of the Florida Everglades, is the redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Kendra et al. 2014, Hughes et al. 2015). However, X. glabratus is rarely associated with laurel wiltaffected avocado trees, yet the disease has spread throughout the avocado growing region in south Florida in the apparent absence of this important vector (Carrillo, unpublished data). Recently, R. lauricola was found in or on 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 and Xyleborus bispinatus Eichhoff, are capable of transmitting R. lauricola to avocado trees under greenhouse conditions (Carrillo et al. 2014). Xyleborus 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 with host species belonging to 24 plant families including the Lauraceae (Atkinson 2016). Unlike other important pest ambrosia beetles that have been 23

24 introduced to the New World [i.e. X. glabratus (Fraedrich et al. 2008, Hanula et al. 2008, Brar et al. 2013, Maner et al. 2013), Xylosandrus compactus (Eichhoff) (Greco and Wright 2015), Euwallacea fornicatus (Eichhoff) (Cooperband et al. 2016), Xylosandrus germanus (Eichhoff) and Xylosandrus crassiusculus (Motschulsky) (Castrillo et al. 2012, Ranger et al. 2016)], X. volvulus has not been documented to cause economic damage to trees. In Florida, X. volvulus occurs sympatrically with X. glabratus and breeds in hosts affected by LW (Kendra et al. 2011b, Carrillo et al. 2012), and has been documented as a potential vector of the LW pathogen in avocado (Carrillo et al. 2014). The association between X. volvulus and R. lauricola 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 share this unique characteristic of completing their life cycle within galleries in trees, 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). Despite the general interest in ambrosia beetles, there is limited information regarding their biology, behavior, and the functional role of their symbiotic associations. Establishment of beetle colonies would allow studies on development, physiology, behavior, colony composition and size, as well as allow the manipulation of ambrosia beetle-fungal associations that could improve our 24

25 understanding of this taxonomic group, and their direct and indirect effects on host trees. Artificial media have been used for mass rearing of insects, testing compounds for physiological effects and studying 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. The ultimate criterion for judging the quality of an artificial medium for ambrosia beetles is that it serves as a substrate that is nutritional for the symbiont and that it supports 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, more than 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), Xylosandrus germanus (Biedermann et al. 2009, Castrillo et al. 2012), X. glabratus (Maner et al. 2013), and Euwallacea fornicatus (Cooperband et al. 2016). Here we describe a series of studies that evaluate three artificial media for rearing X. volvulus. These media incorporate sawdust obtained from 25

26 two of its major hosts, silkbay (Persea humilis Nash), a species endemic to central Florida that is frequently colonized by ambrosia beetles following LW (Kendra et al. 2012), and avocado, an important agricultural species in south Florida that is also threatened by LW. In addition, since X. volvulus has been reported in association with R. lauricola in both of these hosts, we utilized the laboratory rearing methods to conduct preliminary studies on the interactions of X. volvulus with this fungus. Materials and Methods Ambrosia Beetles Xyleborus volvulus females were obtained from sections of infested logs (approximately cm, length diameter) collected from avocado orchards and placed in emergence chambers [44-gallon (167 L) Brute container ( 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 Avocado and silkbay logs were collected in February 2016 from an unsprayed avocado orchard in Miami-Dade County (N 25 29' 38" W 80 28' 53") and the Archibald Biological Station in Highlands County, FL (N 27 10' 50" W 81 21' 0"), respectively. The logs were debarked and dried for four days 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 26

27 the xylem-sapwood layer. The sawdust was sifted through a 12 mm sieve and stored at 18 ºC until the time of media preparation. Three types of artificial media were evaluated. 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. The ingredients of the three types of media are provided in Table 2-1. 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 immediately transferred to a laminar flow hood, where the media was stirred to re-suspend settled ingredients, and poured into 50 ml sterile plastic centrifuge tubes (Fisher Scientific Catalog No , Suwanee, Georgia), with 15 ml of media per tube. The tubes were loosely capped, tapped to remove air bubbles, and allowed to cool in the laminar flow hood for 24 h. Medium 2 was packed into 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 above. 27

28 Media Inoculation A subset of the tubes containing each of the three media was inoculated with R. lauricola (referred to as the medium type + RL, i.e., AM1 + RL). A spore solution was made from a R. lauricola culture obtained from the Plant Diagnostic Clinic at the Tropical Research and Education Center. In the laminar flow hood, 3 ml of sterile deionized water was added to a Petri dish with a fully-grown culture of R. lauricola. The surface of the plate was gently scraped using a sterile plastic rod (Fisher Scientific Catalog No ). The resulting spore solution was transferred to a sterile 50 ml plastic centrifuge tube using a disposable sterile pipette (5 ml). Serial dilutions (1, 10, 10 2, 10 3, and 10 4 ) were then plated on CSMA media (cycloheximide, streptomycin, malt, and agar) which is selective for species of Ophiostomatales to determine the number of colony-forming units (CFUs) in the original solution. Each medium was inoculated with 500 µl of the initial solution, which contained CFUs. Tubes were loosely capped and maintained in a sterile environment for 10 days to allow fungal growth on the medium. Rearing Conditions Female X. volvulus collected from the emergence chambers were dipped into a 70% ethanol solution for 5-7 seconds to remove fungi and bacteria present on their exoskeletons. Twelve active and vigorous females were each individually placed into a rearing tube containing each of the media tested. Before introducing each female into a rearing tube, four small holes were made on the surface of the medium to facilitate the initiation of boring activity. The rearing tubes were placed horizontally in plastic containers in a walk-in rearing room under controlled conditions of 25 ± 1 C, 75% RH, 28

29 and in complete darkness. Each tube was inspected every three days by removing the lid and examining the surface of the rearing medium and the sides of the tubes to detect visible galleries. Data collection consisted of recording the number of days to the first occurrence of eggs, larvae, pupae and adults on the surface and in galleries. Gallery formation was indicated by the appearance of loose medium on the surface, being pushed out by females during tunneling. The experiment was concluded after successfully recording the uninterrupted development of two complete generations. Gallery Dissection After 40 days, the medium in each tube containing a beetle colony (brood) was dissected under a stereomicroscope in a laminar flow hood. First, all developmental stages on the surface of the medium were recorded. Thereafter, the medium was tapped out of the tube into a Petri dish, and all beetle stages on the sides of the tubes were recorded. The medium was systematically cut into small pieces starting at the bottom of the medium plug and then proceeding upwards. Gallery tunnels were opened carefully by removing the medium around them. Eggs, larvae, pupae and adults were removed and placed separately into Petri dishes using a fine paintbrush. Adult mortality was also recorded. One mature active female from each treatment was selected, surface sterilized, and reared in the same medium to produce a second generation of adults. Fungal Isolation One mature female from each R. lauricola inoculated colony was surfacedisinfested by immersing in 70% ethanol for 30 seconds and subsequently washed in 29

30 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 ). 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 ) to scrape small portions of the tunnel where immature stages were found and plated the scraping onto CSMA medium. After 7-10 days, colonies showing R. lauricola morphology (Harrington et al. 2010) were identified 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. Each medium was evaluated separately and was considered a separate experiment. The effects of sawdust type and the presence of R. lauricola on the numbers of adult females, and the total brood produced were evaluated using two-way analysis of variance (ANOVA) (PROC GLIMMIX, SAS Institute 2010, v. 9.3). Data were square root transformed before analysis. Tukey s HSD was used for mean separation. 30

31 Results Medium 1 The type of sawdust in medium 1 (Table 2-1) had no significant effect on the reproduction of X. volvulus (F = 0.03; df = 1, 95; P = ) (Figure. 2-1A). Brood sizes were generally greater on media not inoculated with R. lauricola as compared to media inoculated with this pathogen, but these differences were not statistically significant (F = 2.42; df = 1, 95; P = ) (Figure. 2-1A). 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 2-2). In all treatments, there was significantly more reproduction (F = 17.17, df = 1, 95; P < ) 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 ± 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. By contrast, the increase in brood establishment by female founders between the first and second generations was a change 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 days after the females had been introduced, 31

32 respectively. In SM1 + RL, larvae, pupae, and adults were observed later than in all other treatments, i.e., at 21, 27 and 31 days after the females had been introduced, respectively. Medium 2 The type of sawdust in medium 2 had a significant effect on brood production (F = 11.70; df = 1, 95; P = ) (Figure. 2-1B). Brood size per female founder was significantly greater in silkbay (mean ± SE, n = 48) than in avocado. The presence of R. lauricola also had a significant effect on brood production (F = 5.28; df = 1, 95; P = ) (Figure. 2-1B). Moreover, there was a significant interaction between the species of sawdust and the presence of R. lauricola (F = 6.27; df = 3, 95; P = ), indicating that the effect of sawdust species on reducing brood production was greater in R. lauricola inoculated media containing avocado sawdust than in the medium containing silkbay sawdust (Figure. 2-1B). The size of the brood produced declined in the following order: SM2 > SM2 + RL > AM2 > AM2 + RL (Figure. 2-1B). 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 2-2). The average brood size produced in the first generation was less than that the second generation, although this difference was not statistically significant (F = 1.08; df = 1, 95; P = ) (data not shown). The mean ± SE brood sizes produced were 6.40 ± 1.32 and 7.69 ± 1.23 in the first and second generation, respectively. The percentage 32

33 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 tubes walls. Larvae, pupae, and adults were observed at 19, 24, and 30 days, respectively, after the female founder had been introduced into SM2. With SM2 + RL, larvae, pupae, and adults were observed at 18, 25, and 31 days after female introduction. In AM2, larvae, pupae, and adults were seen at 20, 24, and 29 days, respectively. In AM2 + RL, no larvae or pupae were observed, although adults were seen at 33 days after female introduction. Medium 3 Foundress females produced significantly more progeny on the avocado-based medium than on silkbay-based medium (F = 5.72; df = 1, 95; P = ) (Figure. 2-1C). Inoculation with R. lauricola had a significant effect on brood production in both variations of medium 3 (F = 4.47; df = 1, 95; P = ). Moreover, there was a significant interaction (F = 3.91; df = 3, 95; P = ) between sawdust species and R. lauricola, indicating that the presence of R. lauricola in both media had a negative effect on brood production, but this effect was greater in avocado than in silkbay (Figure. 2-1C). 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 33

34 + RL. Adult mortality was similar in AM3 (38%), SM3 (39%) and AM3 + RL (37%), but mortality was greater in SM3 + RL (58%) (Table 2-2). There was no significant difference in brood size between the first and second generation (F = 0.17; df = 1, 95; P = ) (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. By 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 tubes walls. Larvae, pupae, and adults were first observed at 19, 25, and 30 days, respectively, after the introduction of the female founder in AM3. In SM3 larvae, pupae, and adults were first observed 18, 24, and 29 days, 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 days after female introduction, respectively. Recovery of R. lauricola and Other Fungi from X. volvulus Reared in Media Inoculated with R. lauricola Raffaelea lauricola was recovered at low frequencies from adult females reared on all three media (Table 2-3). 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. With AM3 + RL and SM3 + RL, one and six X. 34

35 volvulus galleries were colonized by R. lauricola, respectively. Other fungi and yeast isolated from heads, bodies, and galleries of X. volvulus reared in avocado and silkbay media are presented in Table 2-4. Six and ten other fungi were isolated from colonies inoculated or not inoculated with R. lauricola, respectively. Raffaelea 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 fungal species 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 2-4). Discussion Ambrosia beetles live in nutritional symbiosis with ambrosia fungi, typically 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). Scott and Du Toit (1970) reported that the dominant symbiont of Xyleborus torquatus (a synonym of X. 35

36 volvulus) was Raffaelea 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, other Raffaelea species (R. fusca, R. subalba, and R. subfusca) and several yeasts species were also frequently associated with X. volvulus. Our results indicate that R. arxii might be the primary symbiont of X. volvulus, but like other ambrosia beetles, this species has multiple fungal associates (Gebhardt et al. 2004, Harrington et al. 2010). Xyleborus volvulus and other ambrosia beetles were reported to carry R. lauricola (Carrillo et al. 2014). In our experiments, we inoculated the rearing media with R. lauricola. In all treatments, the inoculated media resulted in less progeny than noninoculated media. These results suggest that R. lauricola does not fulfill the nutritional requirements of X. volvulus and could antagonize with the nutritional symbionts of this beetle. Raffaelea lauricola was recovered from few individuals and showed very limited colonization of the mycangium. This result might be due to fungal regulation within the mycangium (Yuceer et al. 2011). Some studies have reported that 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). 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 36

37 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 more than the type of sawdust had an effect 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. Xyleborus 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 days and from 19 to 27 days after the female had been introduced into the medium, respectively. During our experiment, adults were first seen inside the galleries at 24 days after female introduction. These adults remained inside the galleries for several days (~1 week). During this period it seems probable that mating occurred between sibling females and males, and adults engaged in colony maintenance activities (i.e., cleaning) before leaving the gallery. Often in this study, at the time of dissection (i.e., 40 days 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 and probably some of them were produced by the new generation of females. The reproductive 37

38 potential of X. volvulus was similar to that of X. glabratus reared in two artificial media (Maner et al. 2013). Using a different approach, Brar et al. (2013) reared X. glabratus on bolts of three tree species (i.e., avocado, redbay, and swampbay) and found small reproductive rates. For instance, each bolt was originally infested with 20 female beetles, but an average of only 1.1 females was obtained from each bolt. These results suggest that sawdust based media are better than bolts for establishing laboratory colonies of ambrosia beetles. Interactions between ambrosia beetles and symbiotic fungi are difficult to investigate under natural conditions. The rearing of ambrosia beetles in artificial substrates may facilitate manipulation of various fungi to study larval feeding habits and explore their nutritional quality in different hosts. 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. However, developing artificial substrates for ambrosia beetles is challenging because the substrate must adequately resemble the natural habitat of the beetles and their symbiotic fungi. 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 R. lauricola is not a nutritional symbiont of X. volvulus, 38

39 but in some cases, the association with this pathogen could be commensalistic, not affecting the beetle's reproduction. Acknowledgments This study partially fulfills the requirements for the MS degree, University of Florida, by Octavio A. Menocal. The authors 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. The authors also thank Jose Alegría, Julio Mantilla, and Manuela Angel for their help. This research was funded by FDACS-SCBG grant to Daniel Carrillo. 39

40 Table 2-1. Recipes of the three types of artificial media using either avocado or silkbay sawdust for rearing Xyleborus volvulus. Media types Ingredients 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 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 H 2 O 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 40

41 Table 2-2. Biological parameters of Xyleborus 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 Raffaelea lauricola. Media* Medium 1 Medium 2 Treatments Average no. offspring per tube after 40 days Eggs Larvae Pupae Male Adults Female Adults Brood (All stages combined) Females in brood (%) Adult mortality (%) N with offspring (any stage) (%) N with females (%) AM % 16.1% 13 (54%) 11 (45%) 24 AM1 + RL % 22.7% 14 (58%) 14 (58%) 24 SM % 21.3% 18 (75%) 18 (75%) 24 SM1 + RL % 33.6% 17 (70%) 15 (62%) 24 AM % 16.2% 10 (41%) 10 (41%) 24 AM2 + RL % 82.8% 3 (12%) 2 (8%) 24 SM % 17.6% 20 (83%) 20 (83%) 24 SM2 + RL % 14.4% 15 (62%) 11 (45%) 24 AM % 37.4% 15 (62%) 14 (58%) 24 AM3 + RL % 37.1% 11 (45%) 8 (33%) 24 Medium 3 SM % 39.1% 9 (37%) 7 (24%) 24 SM3 + RL % 58.8% 6 (25%) 3 (15%) 24 AM1 = Avocado medium 1; AM1 + RL = Avocado medium 1 inoculated with Raffaelea lauricola; SM1 = Silkbay medium 1; SM1 + RL = Silkbay medium 1 inoculated with Raffaelea lauricola; AM2 = Avocado medium 1; AM2 + RL = Avocado medium 2 inoculated with Raffaelea lauricola; SM2 = Silkbay medium 2; SM2 + RL = Silkbay medium 2 inoculated with Raffaelea lauricola; AM3 = Avocado medium 3; AM3 + RL = Avocado medium 3 inoculated with Raffaelea lauricola; SM3 = Silkbay medium 3; SM3 + RL = Silkbay medium 3 inoculated with Raffaelea lauricola. N = number of batches of the medium. * Note that each medium was evaluated separately, and was considered a separate experiment. N 41

42 Table 2-3. Frequency of recovery of Raffaelea lauricola from the main body parts of Xyleborus volvulus adults reared in artificial media previously inoculated with the fungus. Host Mean no. of CFUs Mean no. of CFUs per Medium Frequency Frequency per head & body lacking head & type* n/n n/n pronotum pronotum Medium /24 6 3/24 Avocado Medium / /12 Medium 3 0 0/24 0 0/24 Medium 1 4 1/ /24 Silkbay Medium 2 0 0/12 5 1/12 Medium / /24 n: number of beetles positive for the presence of Raffaelea lauricola. N: Number of beetles tested. CFU: colony-forming units of Raffaelea lauricola. * Note that each medium was evaluated separately, and was considered a separate experiment. 42

43 Table 2-4. Fungal species isolated from subsamples of five Xyleborus volvulus beetles and their galleries collected from media based on sawdust of avocado or silkbay. The fungi were isolated from the head and pronotum or from the body lacking the head and pronotum. Medium containing avocado sawdust Medium containing silkbay sawdust Treatment Isolate ID Head and pronotum Body 1 Gallery Head and pronotum Body 1 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 Alloascoidea africana 1/5 42 0/5 0 2/5 3/ /5 0 0/5 Media inoculated with R. lauricola Media not inoculated with R. lauricola 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 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 Raffaelea arxii 3/ / /5 5/ /5 0 5/5 Raffaelea lauricola 1/ /5 0 2/5 1/ /5 0 2/5 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 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 Raffaelea arxii 2/ /5 42 3/5 4/ / /5 Raffaelea fusca 1/5 17 0/5 0 0/5 0/5 0 0/5 0 1/5 Raffaelea lauricola 0/5 0 0/5 0 0/5 0/5 0 0/5 0 0/5 Raffaelea subalba 2/ /5 72 3/5 0/5 0 0/5 0 0/5 Raffaelea subfusca 1/ /5 0 1/5 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 1 Body lacked the head and the 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. 43

44 Figure 2-1. Number of Xyleborus volvulus females 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 Raffaelea lauricola*. Media contained sawdust of either avocado or silkbay. Bars represent the mean (± SE) number of brood (gray) and females (black) produced per foundress female. Columns with the same letters are not significantly different (P < 0.05) 44

45 *AM1 = Avocado medium 1; AM1 + RL = Avocado medium 1 inoculated with Raffaelea lauricola; SM1 = Silkbay medium 1; SM1 + RL = Silkbay medium 1 inoculated with Raffaelea lauricola; AM2 = Avocado medium 1; AM2 + RL = Avocado medium 2 inoculated with Raffaelea lauricola; SM2 = Silkbay medium 2; SM2 + RL = Silkbay medium 2 inoculated with Raffaelea lauricola; AM3 = Avocado medium 3; AM3 + RL = Avocado medium 3 inoculated with Raffaelea lauricola; SM3 = Silkbay medium 3; SM3 + RL = Silkbay medium 3 inoculated with Raffaelea lauricola. 45

46 CHAPTER 3 XYLEBORUS BISPINATUS (COLEOPTERA: CURCULIONIDAE) REARED ON ARTIFICIAL MEDIA USING SAWDUST FROM AVOCADO OR SILKBAY IN PRESENCE OR ABSENCE OF THE LAUREL WILT PATHOGEN (RAFFAELEA LAURICOLA) Abstract Xyleborus bispinatus Eichhoff (Coleoptera: Curculionidae) was reported in Florida for the first time in Previously, it was unrecognized and not distinguished from the morphologically similar Xyleborus ferrugineus (F.). Like other members of the tribe Xyleborini, X. ferrugineus (and possibly X. bispinatus) can cause economic damage in lowland areas of the Neotropics. In addition, when breeding in a host infected with laurel wilt, X. bispinatus has been found to incorporate the laurel wilt pathogen, Raffaelea lauricola T. C Harr., Fraedrich & Aghayeva; Ophiostomatales: Ophiostomataceae) into its mycangia and may potentially function as a vector of the disease in avocado (Persea americana Mill.; Laurales: Lauraceae). The main objective of this study was to evaluate three artificial media containing sawdust from avocado or silkbay (Persea humilis Nash) for rearing X. bispinatus under laboratory conditions. In addition, the media was inoculated with R. lauricola to evaluate its effect on the biological parameters of X. bispinatus. Two of the media supported prolific reproduction of X. bispinatus, but the avocado-based medium was better than the silkbay-based medium. The presence of R. lauricola had a neutral or enhancing effect on beetle reproduction. However, the pathogen was recovered from few individuals reared in inoculated media and showed limited colonization of the beetle's mycangia. Raffaelea lauricola was frequently recovered from beetle galleries. The two media with lower water content were best for rearing X. bispinatus under laboratory conditions. 46

47 Introduction Raffaelea lauricola (T. C Harr., Fraedrich & Aghayeva; Ophiostomatales: Ophiostomataceae), is a fungal pathogen carried primarily by the redbay ambrosia beetle (Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) (Fraedrich et al. 2008, Harrington et al. 2008). The pathogen is responsible for the disease known as Laurel Wilt (LW) that has affected many 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.) (Mayfield 2007, Fraedrich et al. 2008, Fraedrich et al. 2011, Kendra et al. 2014). Nonetheless, X. glabratus is rarely associated with laurel wilt affected avocado trees in commercial plantings (Carrillo et al. 2012), and subsequent studies (Carrillo et al. 2014) documented the lateral transfer of R. lauricola to other ambrosia beetles species in Florida. Two of those species, Xyleborus bispinatus Eichhoff and Xyleborus volvulus Fabricius were also capable of transmitting R. lauricola to avocado trees under greenhouse conditions (Carrillo et al. 2014). Xyleborus bispinatus was reported in Florida for the first time by Atkinson et al. (2013). Previously, it was unrecognized and undistinguished from Xyleborus ferrugineus (F.), a smaller, morphologically very similar ambrosia beetle. Both species have broad distributions throughout South America, Central America, the Gulf Coast, southeastern USA (Atkinson and Peck 1994, Kirkendall and Jordal 2006) and X. bispinatus has also been reported in Italy (Faccoli et al. 2016). Rabaglia et al. (2006) stated that like other members of the tribe Xyleborini, X. ferrugineus (and possibly X. bispinatus) can cause economic damage in lowland areas of the Neotropics. However, there is no additional 47

48 documentation that can confirm this statement. In forest ecosystems of central and southern Florida, X. bispinatus occurs sympatrically with X. glabratus and commonly breeds in native hosts (i.e., swampbay, silkbay) affected by laurel wilt (Kendra et al. 2015, 2016). Recently, it has been shown that females of X. bispinatus can incorporate R. lauricola into their mycangia, the storage organs for spores of symbiotic fungi (Carrillo et al. 2014, Ploetz et al. 2017). Carrillo et al. (2014) suggested that X. bispinatus may be a potential vector of the LW pathogen in avocado groves, in the apparent absence of X. glabratus. The introduction of exotic pathogens has typically had a significant detrimental effect on forest communities. Relevant examples include Cryphonectria parasitica (Murril) M. E. Barr, the causal agent of chestnut blight (Stephenson 1986); Cronartium ribicola Fisch, the causal agent of white pine blister rust (Sotrer et al. 1999), and Ophiostoma novo-ulmi Brasier, the pathogen that causes the Dutch elm disease (Brasier 1991). In many cases, scolytine beetles are the vectors of these pathogens. However, due to their cryptic life styles, there is limited information on the behavior, biology and functional role of their symbiotic associations. The use of artificial media may help improve our understanding of the interactions among host plants, pathogens, and vectors. The first ambrosia beetle species reared on agar and sawdust based medium was X. ferrugineus (Saunders and Knoke 1967). Since then, artificial media have been used to rear and study the biology of different ambrosia beetles. Here we describe a series of studies evaluating three artificial media for rearing X. bispinatus using sawdust 48

49 from avocado and silkbay. The rearing method was also used to conduct preliminary studies on X. bispinatus and its biological response to R. lauricola. Materials and Methods Ambrosia Beetles Xyleborus bispinatus females were obtained from laboratory stock colonies originally collected from avocado orchards in Homestead, FL. Fully sclerotized (i.e., dark brown) females were quickly surface sterilized with 70% ethanol to prevent microbes other than its symbiotic fungus from colonizing the media (Cooperband et al. 2016). Artificial Media Sawdust from two main hosts of X. bispinatus was evaluated. Avocado logs were collected in February 2016 from an unsprayed avocado orchard in Miami-Dade County (25 29'38" N 80 28'53" W). Silkbay logs were collected in February 2016 from the Archibald Biological Station in Highlands County (27 10' 50" N; 81 21' 0 " W), FL. Logs were carefully inspected to detect any possible beetle infestation and used only when were considered pest-free. Sawdust was obtained as reported in Castrillo et al. (2011) and stored at 18 C until media preparation. Artificial media were prepared as described in Chapter 2. Briefly, three types of artificial media [Medium 1, either avocado or silkbay (from now on AM1 and SM1) following the procedures described in Castrillo et al. (2011); Medium 2 (AM2 and SM2) described by Biedermann et al. (2009) and Medium 3 (AM3 and SM3), which contained the same ingredients as in Medium 2, but with more water to facilitate pouring the 49

50 medium into the rearing tubes (Fisher Scientific Catalog No , Suwanee, Georgia). Twenty-four rearing tubes were filled with each medium. Media Inoculation Twelve tubes of each medium were inoculated with R. lauricola (referred to as the medium type + RL, e.g., AM1 + RL). A spore solution from an R. lauricola culture was serially diluted (1, 10, 10 2, 10 3, and 10 4 ) to determine the number of colony-forming units (CFUs) in the solution. Each medium was then inoculated with 500 µl ( CFU) from the initial solution. Tubes were maintained in a sterile environment for ten days to allow fungal growth on the medium. Rearing Conditions Rearing conditions were described in Chapter 2. Briefly, twelve active and fully sclerotized X. bispinatus females were selected and individually placed into rearing tubes with each media combination. The beetles were reared in a walk-in rearing room under controlled conditions (25 ± 1 C, 75% RH, and complete darkness). Colonies were visually evaluated every 2 3 days by removing the lid and observing the surface of the medium and the visible galleries on the sides of the tubes. The days of the earliest observed life stages, i.e., eggs, larvae, pupae, and adults on the surface and galleries were recorded. Gallery formation was evident by the appearance of excavated material and frass on the media surface during tunneling activities. The experiment concluded when two complete generations of X. bispinatus were recorded. 50

51 Dissection of Colonies Beetle s colonies were destructively sampled to quantify all developmental stages after 40 days of female introduction. Each colony was dissected under a stereomicroscope in an aseptic environment. All developmental stages on the surface of the medium were quantified. Afterward, the medium was tapped out onto a Petri dish and all beetle stages on the sides of the tubes recorded. Once all beetle stages were removed from the surface and sides of the plug, the medium was systematically chipped away into small bits starting at the bottom of the medium. Galleries were dissected carefully by removing the medium around them to allow eggs, larvae, pupae, and adults within to be collected and quantified. Adult mortality was recorded. To produce the second generation, one fully sclerotized female from each treatment was surface sterilized and reared in the same medium. Fungal Isolation One fully sclerotized female from each colony inoculated with R. lauricola was surface-disinfested with 70% ethanol for 30 sec and rinsed three times with sterile deionized water. The head and body were separated, and both parts were macerated separately in 200 µl of sterile water using a motorized tissue grinder (Pyres no ). Next, 100 µl of inoculum (sterile water + conidia from the tissue) were plated on CSMA medium (cycloheximide, streptomycin, malt, and agar). Additionally, fungal samples were taken from galleries where immature beetle stages resided by scraping a small portion of the tunnel using a sterile inoculation loop (Fisher Scientific Catalog No ) and plated on CSMA. After seven days, colonies with the appearance of R. lauricola on this medium (Harrington et al. 2010) were identified with two diagnostic 51

52 microsatellite markers, CHK and IFW (Dreaden et al. 2014). The presence/absence of the fungus in galleries was determined, and the number of CFUs of R. lauricola was calculated for individual beetles. Additionally, fungal isolations from a subset of beetles and galleries were identified using the same procedures presented in Chapter 2 for X. volvulus. Statistical Analysis Two-way analysis of variance (ANOVA, PROC GLIMMIX, SAS Institute 2010, v. 9.3) was used to detect the effect of sawdust and the presence of R. lauricola on the number of females and total brood production. Each medium was analyzed independently and thus considered a separate experiment. Data were square root transformed before analysis to meet the normality assumption. Means separation tests used Tukey s HSD. Results Medium 1 Foundress females of X. bispinatus had higher reproductive output on avocado than on silkbay based medium 1 (F = 52.14; df = 1, 95; P < ) (Figure. 3-1A). The effect of R. lauricola on brood production was not statistically significant (F = 3.66; df = 1, 95; P = ). However, reproduction of X. bispinatus was greater on avocado and silkbay media 1 that had been inoculated with R. lauricola. The interaction between sawdust type and R. lauricola was significant (F = 18.60; df = 3, 95; P < ), suggesting that the effect of R. lauricola was less on avocado than silkbay (Figure. 3-1A). The number of males per brood ranged from zero to four. Males were present in 52

53 91% of AM1 + RL; 88% of AM1; 79% of SM1 + RL, and 58% of SM1. Adult mortality in colonies was low in all treatments, and decreased in the following order: AM1 (7%) > AM1 + RL (6%) > SM1 (5%) > SM1 + RL (3%) (Table 3-1). In general, there was significantly less reproduction (F = 5.04; df = 1, 95; P < ) in the second generation than in the first (data not shown). The mean ± SE number of progeny in the first generation was ± 1.05 versus ± 1.46 in the second generation. Foundress females established colonies in all treatments for the first and second generations. No eggs were observed in galleries along the rearing tube walls. Larvae, pupae, and adults were first observed on days 12, 15 and 21, respectively, after female introduction in AM1 + RL. In AM1, larvae, pupae, and adults were observed at 15, 18 and 24 days after female introduction, respectively. In SM1 + RL, larvae, pupae, and adults were observed at 18, 23 and 29 days after the female introduction and in SM1 were seen at 15, 21 and 27 respectively. Medium 2 The sawdust type in medium 2 had no significant effect on brood production (F = 0.63; df = 1, 95; P = ) (Figure. 3-1B). Inoculation with R. lauricola had a significant effect on brood production (F = 6.25; df = 1, 95; P = ) and there was a significant interaction between sawdust species and the presence of R. lauricola (F = 13.41; df = 1, 95; P < ) (Figure. 3-1B). Interestingly, in avocado the reproduction of X. bispinatus was not significantly different in inoculated or not inoculated media, but reproduction greatly increased with the presence of R. lauricola in silkbay media (Figure. 3-1B). The number of males per brood ranged from zero to two. Males were 53

54 seen in 88% of the colonies in SM2 + RL; 88% of AM2; 83% of AM2 + RL and 71% of SM2. As with medium 1, adult mortality was low for all treatments; AM2 (6.6%), AM2 + RL (5.1%), SM2 + RL (3.6%) and SM2 (2.9%) (Table 3-1). In all treatments, there were significantly more progeny (F = 68.98; df = 1, 95; P < ) in the first generation than in the second generation. The mean ± SE of brood was ± 1.39 versus ± 1.62 in the first and second generation, respectively. The percentage of foundress females that established colonies in SM2 + RL, AM2, and SM2, decreased from 100 to 96 from the first to the second generation. In AM2 + RL, foundress females established 100% of colonies for both generations (data not shown). No eggs were visible along galleries in the rearing tubes walls. Larvae, pupae, and new adults were observed at 12, 16, and 21 days after female founder had been introduced into SM2 + RL. In AM2, larvae, pupae, and adults were observed at 16, 20, and 25 days after female introduction. In the case of AM2 + RL, larvae, pupae, and adults were seen at 18, 23, and 29 after female introduction. In SM2, larvae, pupae, and adults were visible at 16, 21, and 25 days after the female founder was introduced. Medium 3 Foundress females bred significantly less in silkbay sawdust than in avocado sawdust (F = 22.54; df = 1, 95; P < ) (Figure. 3-1C). The effect of the presence of R. lauricola in the medium was not significant on beetle productivity (F = 0.01; df = 1, 95; P = ). However, the interaction effect between R. lauricola and sawdust type was significant (F = 7.57; df = 1, 95; P = ), suggesting that avocado sawdust was better than silkbay (Figure. 3-1C). The number of males per colony ranged from zero to three. Males were present in 79% of the colonies in AM3, 66% in AM3 + RL, 70% and 54

55 66% in SM3 and SM3 + RL, respectively. Adult mortality was similar in AM3 (5.2%), SM3 (7.8%), and SM3 + RL (4.5%). By contrast, mortality was greater in AM3 + RL (12.9%) (Table 3-1). There was a significant difference (F = 16.64; df = 1, 95; P < ) in brood size between first and second generation (data not shown). The mean ± SE of brood produced in the first generation was ± 0.66 versus 7.65 ± 0.71 in the second generation. The percentage of foundress females that established colonies in AM3 was 100 and 83 in the first and second generations, respectively. In AM3 + RL, 100% of females established colonies in both generations. In SM3, 85% of females established colonies in both generations. When using SM3 + RL, the percentage of colony establishment decreased from 100 to 91%. As was the case with media 1 and 2, no eggs were visible in galleries along the rearing tubes walls. Larvae, pupae, and new adults were first observed at 17, 21, and 25 days (respectively) after the introduction of the female founder in AM3. In AM3 + RL, larvae, pupae, and new adults were first observed 19, 22, and 27 days after female introduction. In SM3 and SM3 + RL, larvae, pupae and new adults were seen at 20, 25, 30 days and at 18, 24, and 28 days after the female foundress was introduced. Recovery of Other Fungi and R. lauricola from X. bispinatus Reared on Artificial Media Adult females reared on inoculated media were found carrying low frequencies of R. lauricola. However, R. lauricola was more frequently recovered from females reared on silkbay than on avocado based media (Table 3-2). Typically, R. lauricola was recovered more frequently from heads than bodies (Table 3-2). The fungus was 55

56 recovered from 11 of 24 and 22 of 24 beetle galleries in AM1 + RL and SM1 + RL, respectively. In AM2 + RL and SM2 + RL, R. lauricola was recovered from 14 of 24 and 15 of 24 beetle s galleries. In AM3 + RL and SM3 + RL, the fungus colonized 14 of 24 and 21 of 24 beetle s galleries. Besides R. lauricola other fungal species were recovered from the heads, bodies, and galleries of X. bispinatus reared in avocado and silkbay media (Table 3-3). Five other fungal species were isolated from colonies inoculated with R. lauricola and six from colonies not inoculated with R. lauricola. Raffaelea subalba (T. C. Harr., Aghayeva & Fraedrich) was the most abundant and frequent fungus in heads and bodies for avocado and silkbay media. However, in galleries, the most frequent fungus was Raffaelea subfusca (T. C. Harr., Aghayeva & Fraedrich). Raffaelea arxii T. C. Harr., Aghayeva & Fraedrich was isolated at a low frequency and abundance. Candida multigemmis (Buhagiar) S. A Mey & Yarrow (Saccharomycetales) was isolated from all treatments but only from beetle heads. Alloascoidea africana comb. nov. (Saccharomycetales: Alloascideaceae) and Phaeoacremonium inflatipes W. Gams, Crous & M. J. Wingf (Diaporthales: Togniniaceae) were only isolated from beetles galleries (Table 3-3). Discussion Interactions among plants and insects have resulted in enhanced rates of insect diversity, but in some cases, insects and plants are not the only key players in such interactions. Several insect-plant interactions also include microbial associates, and some include fungal pathogens (Paine et al. 1997). Ambrosia beetles rely on fungal symbionts to fulfill their nutritional requirements (Farrell et al. 2001, Mueller et al. 2005). 56

57 However, the associations among symbiotic fungi and ambrosia beetles remain severely understudied. Reproduction and survival of ambrosia beetles relate to growth and quality of fungal symbionts in the colony (Beaver 1989). Fungal growth and therefore the reproductive potential of X. bispinatus in this study may have been influenced by the amount of water, sucrose, casein, yeast and starch in the media. Robinson et al. (2011) found that the sugar content in wood is a significant factor determining fungal colonization by Ophiostoma piceae Munch (Ophiostomatales: Ophiostomataceae). In addition, Abraham et al. (1993) found that nitrogen availability affected the growth of O. piceae. Like most Ophiostoma species, O. piceae utilizes organic nitrogen, i.e., ammonia and not nitrate as nitrogen source (Käarik 1960). Nitrogen is typically scarce in wood (Merrill and Cowling 1966, French and Roeper 1972), ranging between 0.01 and 0.1% of the dry weight. Other ingredients such as yeast and starch have been reported to be important for ambrosia beetle reproduction. Mizuno and Kajimura (2009) reported more progeny of Xyleborus pfeili in media containing more yeast and starch than media with less of these components. In this study, the media containing more yeast and starch also provided better reproduction of X. bispinatus. However, Maner et al. (2013) stated that increasing levels of yeast and starch did not enhance reproduction of X. glabratus. Interestingly, female mortality increased in the medium with greater levels of these nutrients, yet when females survived, they produced more brood (Maner et al. 2013). Overall, this and other studies suggest that ambrosia beetles require certain amounts of yeast and starch in the rearing media, but an excess of these substances might result in reduced fitness. Similarly, the amount of water in the media 57

58 could affect symbiotic fungal growth and therefore the beetle s fitness. In this study, two media with the same type and amount of ingredients except for the water content provided contrasting results regarding X. bispinatus reproduction. Females reared in the medium with higher water content yielded less progeny. In addition, antibiotics have been added to the rearing media to prevent contamination by fungi other than those inoculated by the beetle (Batra 1985). In this study, several symbiotic fungi were found associated with X. bispinatus. Three Raffaelea species (R. arxii, R. subalba, R. subfusca) were frequently associated with galleries, heads, and bodies, suggesting that they are nutritional symbionts of X. bispinatus. Moreover, the indiscriminate association of X. bispinatus with these three species suggests that this beetle does not rely on a single fungus to fulfill its nutritional requirements. Additional experiments evaluating the effect of varying amounts of the main ingredients of the rearing media on these fungi are needed to further improve the rearing conditions of X. bispinatus. Currently, we recommend the artificial media described by Castrillo et al. (2012) and Biedermann et al. (2009) to rear X. bispinatus. This is the first study that documents the effect of R. lauricola on the reproductive potential of X. bispinatus. It has been documented that other ambrosia beetles including X. bispinatus carry R. lauricola the nutritional symbiont of X. glabratus (Harrington et al. 2008, Maner et al. 2013) in their mandibular mycangia (Carrillo et al. 2014). In our experiments, R. lauricola showed low colonization of the mycangium of X. bispinatus, whereas other Raffaelea species were more frequent and abundant. It could be hypothesized that in X. bispinatus the low colonization of the mycangium by R. lauricola is related to mycangial selectivity. How ambrosia beetles maintain an association with 58

59 specific symbionts is not well understood, but Bleiker et al. (2009) suggest that glandular cells located at the mycangium may play a critical role selecting fungal species. Although R. lauricola was recovered in low frequency, the presence of R. lauricola in the rearing media had a neutral or enhancing effect on the reproductive potential of X. bispinatus. The overall good performance of the beetle in media inoculated and not inoculated with the pathogen may indicate that this pathogen could have a commensalistic or synergistic association with X. bispinatus. Future research is warranted to evaluate if R. lauricola is capable of supporting the development of X. bispinatus in the absence of its other fungal symbionts. In our study, the sawdust of two host tree species affected the development and reproduction of X. bispinatus. These results are comparable to other studies of ambrosia beetles reared artificially in different types of sawdust (Castrillo et al. 2012, Brar et al. 2013). There is also evidence of differences in host suitability for ambrosia beetles using field collected logs as the rearing substrate. Brar et al. (2013) reared X. glabratus on avocado, redbay, and swampbay logs. They found that swampbay was a better host for X. glabratus than the other two species. Results of this study suggest that avocado is a better host for X. bispinatus than silkbay. A great percentage (83 to 100% depending on the medium) of females successfully established colonies. Foundress females initiated tunneling activities almost immediately after introduction into the rearing tubes. Active tunneling was evident by the appearance of copious frass pushed out of the gallery through the entry hole. Frass production continued throughout the life cycle of the beetle and was a sign of colony health. Approximately five days after female introduction, the first galleries 59

60 were observed along the rearing tube walls together with fungal growth on the surface of the rearing media. Two weeks after female introduction, the first immature stages were observed in visible galleries or on the surface of the rearing media. The first new adults were observed approximately 3 weeks after female introduction. These adults remained inside the galleries for approximately 1 week presumably mating with sibling males and helping in colony maintenance. During the fourth week after female introduction, adults started to emerge from the rearing medium and sometimes exhibited an aggressive behavior chewing underneath the lid or through the plastic walls of the tube. This behavior was typical after 35 days of female introduction when the colonies were crowded, and probably the media had started to deteriorate. At the time of dissection (day 40), new adults and the original female were found concurrently with other developmental stages; new adults were dark-brown and very active whereas the foundress female was typically dark-black and scarcely moving. The performance of X. bispinatus was similar to other ambrosia beetles that were previously reared on artificial media (Mizuno and Kajimura 2002, Castrillo et al. 2012, Maner et al. 2013, Cooperband et al. 2016). In our study, few females, likely unmated females, produced offspring composed of males only; this observation suggests that like other ambrosia beetles X. bispinatus possess haplo-diploid reproduction where males are haploid derived from unfertilized eggs and females are diploid derived from fertilized eggs (Peer and Taborsky 2005). However, approximately 5% of broods did not contain males during two consecutive generations. It is unclear whether the females did not produce males or males died prematurely and were degraded by other microorganisms. However, male-less broods have also been reported in other ambrosia beetles including 60

61 Xylossandrus compactus (Entwhistle 1964), Xylosandrus germanus (Peer & Taborsky 2004), Xyleborinus saxesenii (Biedermann 2010), and Euwallacea fornicatus (Cooperband et al. 2016). Xyleborus bispinatus, as other ambrosia beetles, also exhibits female-biased sex ratios as a consequence of local mate competition (LMC) (Hamilton 1967). Hamilton (1967) stated eight principles that define LMC, i.e., the females greatly outnumber the males; development is gregarious; reproduction is arrhenotokous; males are disinclined to emigrate from the colony; every colony has at least one male; males hatch sooner than females and can mate multiple times; and mating occurs soon after female eclosion; and females can store sperm after mating. The results of this study verify that X. bispinatus meets the first three principles of LMC, but the fourth principle (one male in every brood) was not met. We could not fully confirm the other four principles of LMC for X. bispinatus. The methods developed here may facilitate the study of LMC principles and other aspects of the biology of X. bispinatus such as the interactions with symbiotic fungi. Moreover, laboratory colonies can be used to understand the processes influencing R. lauricola acquisition by X. bispinatus and the persistence of this association. Laboratory rearing of X. bispinatus on artificial media may greatly facilitate behavioral, physiological, and ecological studies of this important ambrosia beetle. Acknowledgments Thanks to Julio Mantilla, Jose Alegría, and Manuela Angel for experiment set-up and data collection. Thanks to James Colee (UF-IFAS-Statistics Department) for his help with the statistical analysis, Waldemar Klassen and Jorge E. Peña (University of 61

62 Florida) for suggestions to improve the manuscript. This research was in partial fulfillment of an MS degree for Octavio Menocal from the University of Florida. This work was funded in part by FDACS-SCBG grant to DC. 62

63 Table 3-1. Developmental stages and biological parameters of Xyleborus bispinatus on three artificial media based either on avocado or silkbay sawdust inoculated or not inoculated with Raffaelea lauricola. Average no. offspring per tube after 40 days N with Females Adult offspring N with Media* Treatments Brood Male Female in brood mortality (any females N Eggs Larvae Pupae (All stages Adults Adults (%) (%) stage) (%) combined) (%) AM % 6.9% 24 (100%) 24 (100%) 24 Medium 1 Medium 2 AM1 + RL % 6.2% 24 (100%) 19 (79%) 24 SM % 5.0% 24 (100%) 24 (100%) 24 SM1 + RL % 2.7% 24 (100%) 21 (88%) 24 AM % 6.6% 23 (96%) 23 (96%) 24 AM2 + RL % 5.1% 24 (100%) 21 (88%) 24 SM % 2.9% 23 (96%) 22 (92%) 24 SM2 + RL % 3.6% 23 (96%) 22 (92%) 24 AM % 5.2% 22 (92%) 22 (92%) 24 AM3 + RL % 12.9% 24 (100%) 23 (96%) 24 Medium 3 SM % 7.8% 20 (83%) 20 (83%) 24 SM3 + RL % 4.5% 23 (96%) 23 (96%) 24 AM1 = Avocado medium 1; AM1 + RL = Avocado medium 1 inoculated with Raffaelea lauricola; SM1 = Silkbay medium 1; SM1 + RL = Silkbay medium 1 inoculated with Raffaelea lauricola; AM2 = Avocado medium 1; AM2 + RL = Avocado medium 2 inoculated with Raffaelea lauricola; SM2 = Silkbay medium 2; SM2 + RL = Silkbay medium 2 inoculated with Raffaelea lauricola; AM3 = Avocado medium 3; AM3 + RL = Avocado medium 3 inoculated with Raffaelea lauricola; SM3 = Silkbay medium 3; SM3 + RL = Silkbay medium 3 inoculated with Raffaelea lauricola. N = number of batches of the medium. *Note that each medium was evaluated separately, and was considered a separate experiment. 63

64 Table 3-2. Frequency and recovery of Raffaelea lauricola from females of Xyleborus bispinatus reared on artificial media previously inoculated with this fungus. Mean no. of CFUs Mean no. of CFUs Medium Frequency Frequency Host per head & per body lacking type* n/n n/n pronotum head & pronotum Avocado Silkbay Medium / /24 Medium / /24 Medium /24 2 1/24 Medium / /24 Medium /24 2 1/24 Medium 3 6 5/24 2 1/24 n: number of beetles positive for the presence of Raffaelea lauricola. N: Number of beetles tested. CFU: colony-forming units of Raffaelea lauricola. *Note that each medium was evaluated separately, and was considered a separate experiment. 64

65 Table 3-3. Fungal species isolated from 12 X. bispinatus beetles and their galleries collected either from avocado or silkbay based media. Fungi were isolated from the head and pronotum or from the body lacking the head and pronotum Medium containing avocado sawdust Medium containing silkbay sawdust Treatments Isolate ID Head and pronotum Body 1 Gallery Head and pronotum Body 1 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 Candida multigemmis 9/ /12 0 0/12 9/ /12 0 0/12 Media inoculated with R. lauricola Phaeoacremonium inflatipes 0/12 0 0/12 0 4/12 0/12 0 0/12 0 2/12 Raffaelea arxii 10/ /12 0 0/12 7/ /12 0 0/12 Raffaelea lauricola 3/ /12 0 8/12 6/ / /12 Raffaelea subalba 11/ /12 0 0/12 11/ / /12 Raffaelea subfusca 0/12 0 0/12 0 9/12 0/12 0 0/12 0 0/12 Media non-inoculated with R. lauricola Alloascoidea africana 0/12 0 0/12 0 6/12 0/12 0 0/12 0 0/12 Candida multigemmis 10/ /12 0 0/12 10/ /12 0 0/12 Phaeoacremonium inflatipes 0/12 0 0/12 0 0/12 0/12 0 0/12 0 4/12 Raffaelea arxii 9/ /12 0 0/12 3/ /12 0 0/12 Raffaelea lauricola 0/12 0 0/12 0 0/12 0/12 0 0/12 0 0/12 Raffaelea subalba 11/ /12 0 0/12 12/ /12 0 0/12 Raffaelea subfusca 12/ / /12 0/12 0 0/ /12 1 Body separated from the head and the pronotum. Freq. = Frequency of fungal species detected; Avg. = Average; n = Number of beetle body parts or the number of galleries that were positive for a fungal species; N = Number of specimens examined, or number of galleries assayed for the presence of various species of fungi. 65

66 Figure 3-1. Number of Xyleborus bispinatus females and total brood produced by one female reared in one of three artificial media (A = medium 1; B = medium 2 and C = medium 3) prepared either of avocado or silkbay sawdust each inoculated or not inoculated with Raffaelea lauricola*. Bars represent the mean (± SE) number of females (black) and brood produced (gray) per female. Columns with the same letters are not significantly different (P < 0.05) 66

67 *AM1 = Avocado medium 1; AM1 + RL = Avocado medium 1 inoculated with Raffaelea lauricola; SM1 = Silkbay medium 1; SM1 + RL = Silkbay medium 1 inoculated with Raffaelea lauricola; AM2 = Avocado medium 1; AM2 + RL = Avocado medium 2 inoculated with Raffaelea lauricola; SM2 = Silkbay medium 2; SM2 + RL = Silkbay medium 2 inoculated with Raffaelea lauricola; AM3 = Avocado medium 3; AM3 + RL = Avocado medium 3 inoculated with Raffaelea lauricola; SM3 = Silkbay medium 3; SM3 + RL = Silkbay medium 3 inoculated with Raffaelea lauricola. 67

68 CHAPTER 4 VERTICAL DISTRIBUTION OF IN-FLIGHT AMBROSIA BEETLES ASSOCIATED WITH LAUREL WILT AFFECTED IN AVOCADO ORCHARDS Abstract In recent years ambrosia beetles have emerged as significant pests of avocado (Persea americana Mill.; Lauraceae) because of their association with fungal pathogens, in particular, the causal agent of laurel wilt disease, Raffaelea lauricola. Knowing the height at which ambrosia beetles fly could provide insights into the beetle s interaction with host avocado trees, and guide the development of strategies to protect against beetle attacks. The objective of this study was to document the flight height of ambrosia beetle species in three avocado orchards affected by laurel wilt. Ladder like traps with unbaited sticky pannels arranged at three height levels (i.e., low: 0 2 m; middle: 2 4 m; high: 4 6 m) were placed in close proximity of laurel wilt affected trees. During a three-month period, a total of 1,306 individuals of 12 species of ambrosia beetles were captured. The primary vector of R. lauricola, Xyleborus glabratus, was not captured in this study. Six species accounted for ~95% of the captures: Xyleborus volvulus (31.5%), Xyleborinus saxesenii (18.2%), Euplatypus parallelus (17.0%), Xyleborus bispinatus (13.4%), Xyleborus affinis (7.9%), and Hypothenemus spp. (6.6%). All species were captured at the three height levels tested, and only X. volvulus showed a preference for flight at the low-level. Xyleborus bispinatus showed no preference between the low and middle levels, but the low-level traps captured significantly more beetles than the high-level traps. The results suggest that these two ambrosia beetles interact with the main trunk and major scaffold limbs but that the other captured species appeared to have no clear flight preference. 68

69 Introduction Flight is critical to the biology and ecological success of insects. Flight assists insects in various ways, including location of mates, evasion of predators, utilization of food resources, and colonization of new habitats (Sane 2003). In the case of ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) dispersal flight is used to locate new host trees suitable for cultivation of their symbiotic fungi, the primary nutritional source required to sustain new beetle colonies (Farrell 2001). Most ambrosia beetles are not considered pests as their symbiotic fungi are not pathogens; however, the recent incursion of exotic beetles and their pathogenic fungal symbionts has raised the status of some ambrosia beetles to important forest and agricultural pests. Over the past five years, avocado trees in south Florida have been affected by the ambrosia beetle transmitted pathogen, Raffaelea lauricola T.C. Harr. Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the causal agent of laurel wilt (LW) disease. The redbay ambrosia beetle (Xyleborus glabratus Eichhoff) is the primary vector of laurel wilt in natural hammocks but is rare in commercial avocado groves. Despite this, LW has become established in commercial avocado orchards and now is spread most likely by other ambrosia beetles species documented to carry R. lauricola (Carrillo et al. 2014, Ploetz et al. 2017). Brar et al. (2012) reported that X. glabratus flight occurs between 1600 and 1800 h EDST in North Florida, and mainly at very low heights (0.35 to 1 m above the ground). Kendra et al. (2012) documented that X. glabratus initiates flight at 1600 h, with peak flight occurring between 1800 to 1900 h, followed by a sharp decline near dusk. Except Euwallacea nr. fornicatus, which has peak flight in the early afternoon (Kendra et al. unpublished), little is known about the flight behavior of other ambrosia beetles associated with LW in avocado. 69

70 The timing of dispersal flight in ambrosia beetles is influenced by an interaction of abiotic (i.e., temperature, light intensity, and winds) and biotic (resource availability and beetle s energy reserves) factors (Rudinsky 1962, Martikainen 2000, Reding et al. 2010, Evenden et al. 2014, Kirkendall et al. 2015). Like all insects, ambrosia beetles are poikilothermic; their body temperature depends on the ambient environmental temperature. All aspects of their biology are influenced by temperature including the generation length, the rate of development, mating activity, and initiation of dispersal flight (Gaylord et al. 2008). Several studies report that some ambrosia beetles exhibit a crepuscular flight habit (i.e., light intensity resembling twilight) (Saunders and Knoke 1966; Kendra et al. 2012, Brar et al. 2012, Chen and Seybold 2014). In addition, ambrosia beetles, like most small insects are slow fliers and with strong winds can simply be "blown off course" (Farrow 1986). Flying is critical for female dispersal and location of new reproductive substrates, but this activity exposes them to harsh environmental conditions. Mortality in bark beetles during dispersal flight is approximately 50% (Garraway and Freeman 1981) whereas mortality for ambrosia beetles is estimated to be between 70-80% (Milne and Giese 1970). During long dispersal periods, mortality increases primarily due to depletion of energy reserves, exposure to adverse weather conditions, and susceptibility to predation (Kirkendall et al. 2015). Resource availability or the quantity and quality of hosts are other important factors affecting the flight and dispersal behaviors of scolytine species (Ulyshen and Hanula 2007). Knowing the height at which pest ambrosia beetle species fly could provide insight into the beetle s interaction with different structures of avocado trees, key 70

71 information for effective protection of trees against beetle attacks. Typically, ambrosia beetles fly near the ground (Chapman and Kinghorn 1958, Roling and Kearby et al. 1975, Ulyshen and Hanula 2007, Ranger et al. 2010; Hanula et al. 2011, Brar et al. 2012). Based on these reports, the standard control tactic to manage ambrosia beetles in nurseries and agricultural areas is to apply preventive pesticide treatments to the trunk of the trees (Reding et al. 2010). Knowing the flight height of ambrosia beetles associated with LW in avocado orchards could be used to optimize trapping and monitoring techniques, to detect the beginning of ambrosia beetle emergence, and to determine where and when to apply preventive treatments. The objective of this study was to evaluate the flight height of ambrosia beetles species that comprise the Scolytinae community in avocado orchards affected by laurel wilt in south Florida. Materials and Methods Study Sites The vertical distribution of ambrosia beetles in three avocado orchards with laurel wilt-diagnosed trees was investigated from August 2016 to December 2016 in Homestead, FL. The first orchard (N W ) had approximately 4 ha of avocado trees (cv. Booth 7 and Monroe ). Since July 2013 laurel wilt has killed approximately 80% of the trees. During the experiment, this grove had numerous standing laurel-wilt affected trees with large ambrosia beetle infestations. The second orchard composed of Nadir, Tower II, Simmonds, and a Tower II/Nestbitt hybrid (N W ) trees was 18.5 ha and LW was detected in August Approximately 40% of the trees had been affected by LW and harbored large ambrosia beetle infestations. The first and second orchards were not 71

72 being actively managed. The third orchard (N W ) had approximately 81 ha of avocado trees of different cultivars ( Lula, Miguel, and Monroe ) Laurel wilt was first detected in September 2013, and since then, 5% avocado trees had been removed and chipped to avoid spreading of this disease. Management practices in this orchard include irrigation, fertilization, pest management, pruning, and harvesting. The avocado trees were year-old in the three orchards. Row orientation was East-West in the first two orchards and North-South in the third orchard. Beetle Trapping Ten ladder-like traps were set 5 m away from laurel wilt symptomatic avocado trees in each orchard. Each trap was assembled with two half-inch electrical conduits (Home Depot, Model #101543) attached by a fitting made out of a three-quarter electrical conduit (Home Depot, Model #101550). Six transparent Plexiglas panels (25 30 cm) were attached to the conduit with plastic tie straps. The panels were located at 1, 2, 3, 4, 5, and 6 m above the ground. Two white sticky traps (23 28 cm, Sentry wing trap bottoms; Great Lakes IPM, Vestaburg, MI, USA) were attached back-to-back onto each Plexiglas panel with small binder clips (3 cm, Office Depot). Each ladder-like trap was divided into three levels. Sticky panels located between 0 to 2 m were considered the low-level corresponding to the main trunk; 2 to 4 m the middle-level, corresponding to main scaffold branches; and 4 to 6 m the high-level, corresponding to branches and stems. No lures were used in order to capture ambrosia beetles passively without altering their natural flight behavior. Each ladder-like trap was placed onto a concrete reinforcement steel bar nailed two feet below ground acting as anchorage. In addition, 72

73 for stability, the ladder traps were guyed with ropes to three to four adjacent avocado trees. Traps were collected after three months, and the number of ambrosia beetles trapped at each height level was recorded. Ambrosia beetles were morphologically identified according to Rabaglia et al. (2006) at the University of Florida-Tropical Fruit Entomology Laboratory. The number of beetles trapped per orchard was small but the data were similar from all three sites (Table 4-1). Therefore, data from three orchards were combined for the statistical analysis. Statistical Analysis The number of beetles of each species captured per height level was analyzed using the Kruskal-Wallis non-parametric statistical test. A multiple comparison analysis (DSCF) was performed to identify differences among height levels using the SAS package (SAS Institute 2010, v. 9.3). Results A total of 1,306 ambrosia beetles were captured during this experiment. Six species accounted for ~95% of all recorded ambrosia beetles: Xyleborus volvulus Eichhoff (31.5%), Xyleborinus saxesenii Ratzeburg (18.2 %), Euplatypus parallelus (F.) (17.0%), Xyleborus bispinatus Eichhoff (13.4%), Xyleborus affinis Eichhoff (7.9%), and Hypothenemus spp. (6.6%). Other ambrosia beetle species captured in small numbers (5.43%) included Xylosandrus crassisculus (Motschulsky), Xyleborus ferrugineus (F.), Xylosandrus compactus (Eichhoff), Ambrosiodmus devexulus (Wood), Euwallacea fornicatus Eichhoff, and Xyleborinus andrewesi Blandford (Table 4-1). 73

74 Low-level traps caught significantly more X. volvulus than mid and high-level traps [X 2 (2) = , P = ] (Figure. 4-1; Table 4-1). Traps at mid and highlevels captured similar numbers of X. volvulus. Trap height significantly affected X. bispinatus captures [X 2 (2) = , P = ]. There was no difference in the number of X. bispinatus caught at the low and middle-levels (P = ). However, high traps captured significantly fewer X. bispinatus than low traps (P = ) (Figure. 4-1; Table 4-1). The number of X. saxesenii caught at any level was not significantly different [X 2 (2) = , P = ] (Figure. 4-1). Similarly, the number of X. affinis caught at low, middle and high-levels were not statistically different [X 2 (2) = , P = ]. Traps located at mid-level caught slightly more Hypothenemus spp. than low and high-levels, although these differences were not statistically different [X 2 (2) = , P = ]. Euplatypus parallelus was trapped more frequently at mid-levels than low and high levels, although these difference were not statistically significant [X 2 (2) = , P = ]. Other ambrosia beetles were also trapped at different levels; however, their numbers were relatively low for valid statistical analysis (Table 4-1). Discussion Ambrosia beetles, like other insects, only engage in flight when suitable conditions for flight are present. Typically, abiotic factors such as temperature (Six and Bracewell 2015), light intensity (Chen and Seybold 2014), and wind speed (Aluja et al. 1993) influence the flight behavior of insects. In addition, biotic factors such as energy reserves and host availability affect insect flight. These factors have repercussions on the flight behavior and biology of ambrosia beetles. 74

75 Temperature is a critical abiotic factor that influences many biological aspects of ambrosia beetle, especially the ability to engage in flight (Six and Bracewell 2015). At low temperatures, the metabolic reactions that influence flight may be compromised, and the beetles may be unable to operate flight muscles properly (Pasek 1988). Flight activity increases gradually with temperature until a threshold; above this, further temperature increases causes a fall in activity (Taylor 1963). The lower and upper flight thresholds for the mountain pine beetle (Dendroctonus ponderosae Hopkins) are 19 and 41 C, respectively, with optimum flight conditions between C (McCambridge 1971). However, great differences exist in suitable temperatures for flight among ambrosia and bark beetles (Rudinsky 1962). For instance, Ipis pini (Say) initiates flight between 15.6 and 21.1 C (Livingston 1979); Dendroctonus brevicomis (LeConte) at 15.6 C (Miller and Keen 1960), and Ips typographus (L.) at 18 C (Rudinsky 1962). By contrast, other beetles initiate flight at lower temperatures such as Dendroctonus frontalis (Zimm.) at 6.7 C (Thompson and Moser 1986) and Myelophilus piniperda L. at 9 C (Rudinsky 1962). Although daily average low and high temperatures were not recorded in each orchard, temperatures recorded at the University of Florida, Tropical Research and Education Center, <5 km from these orchards ranged from 8.7 to 34.7 C during this investigation (FAWN 2017). Light intensity is another important abiotic factor that influences ambrosia beetle dispersal. For instance, Chen and Seybold (2014) found an inverse relationship between light intensity and flight activity of Pityophthorus juglandis Blackman. In addition, more beetles were caught at dusk (76.4%) when light intensity decreases. In a different study in Ecuador, X. ferrugineus emerged and engaged in flight between 18:00 75

76 and 19:00 h, coinciding with the evening crepuscule (Saunders and Knoke 1966). Kendra et al. (2012) examined the daily flight periodicity of three species of ambrosia beetles. Xyleborus glabratus flew as early as 16:00 to 20:30 h with a peak flight at 18:00 and 19:00 h, these results where similar to Brar et al. (2012). In contrast, X. affinis and X. ferrugineus were observed flying at 18:30 and 19:00 h respectively; the highest number of these two beetles was caught just after sunset (Kendra et al. 2012), coinciding once again with the evening crepuscule. In contrast, flight in females of Hypothenemus spp. occurred at 11:00 h with a peak flight occurring at 15:00 h. (Johnson et al. 2016). Chen and Seybold (2014) stated that temperature followed by light intensity are the main abiotic factors affecting ambrosia beetle flight activity. However, wind speed is an important factor that has been largely overlooked. Wind speed and wind direction are critical for ambrosia beetle dispersal. Typically, insects fly against wind currents to detect host-emitted volatiles (Millar et al. 1986, Miller and Rabaglia 2009). Ambrosia beetles, like most small insects, are slow fliers and can simply be "blown off course" by strong or even moderate wind currents (Farrow 1986). Wind speed increases with altitude from the surface to the atmosphere; this is called wind speed gradient. In biological terms, the atmosphere can be divided into layers. Taylor (1974) defined the insect boundary layer (i.e., the lowest part of the atmosphere that is in contact with a planetary surface) as the air layer in which an insect can control its movement relative to the ground because its flight speed may surpass the wind speed. This layer is just a few meters deep during the day, and sometimes the vertical motion of air may exceed 1-2 m/s, although it might get deeper at dusk and during the night when the surface airs become calm (Farrow 1986). Small insects such 76

77 as bark and ambrosia beetles generally fly at a speed less than 1 m/s (Kinn et al. 1994, Williams and Robertson 2008, Evenden et al. 2014). Thus, the vertical distribution and displacement of bark and ambrosia beetles are controlled largely by atmospheric processes at this layer (Pasek 1988). The second layer is called the planetary layer, and in this case, the wind flow is reduced by the earth's friction. This layer could be extend 1000 m or more from the surface of the earth during the day (Farrow 1986). This layer is hard to reach by ambrosia beetles during the day due to updrafts and downdrafts that make flight difficult to control, the turbulences represent an obvious flight challenge for weakly (i.e., ambrosia beetles) and strongly (i.e., moths) flying insects. However, by dusk, the planetary boundary layer starts to collapse near sunset in response to changing surface heat fluxes (Farrow 1986). Under cloudless conditions, this system develops rapidly, and the air is slowed by the friction effect of the surface. Average wind speeds are less and the planetary layer becomes more stable with relatively calm winds (Farrow 1986). This window could represent for ambrosia beetles an opportunity for increased flight activity, higher flight, and possibly long-range migration due to the effect of wind shear. Overall, these studies suggest that vertical distribution of ambrosia beetles is determined by meteorological factors that are beyond the immediate control of the insect. In this study most of X. volvulus were caught in panels located at low levels (0 2 m) than those at middle and high levels (2 6 m). Similar results were found in other studies. Brar et al. (2012) found that X. glabratus typically fly at m above ground and the number of beetles decreased with increasing height, however, X. 77

78 glabratus was caught up to 3.45 m above ground. In a similar study, Hanula et al. (2011) evaluated different trap heights for capturing X. glabratus in forests. Using sticky traps, 171 of 202 X. glabratus were captured between 1.5 and 2 m above ground. Similarly, Reding et al. (2010) found that X. germanus was mostly caught at 0.5 m above the ground. It could be hypothesized that ambrosia beetles fly at low levels to avoid high wind speeds and turbulences. For instance, when wind speed increased from 0.3 to 1.1 m/s, P. juglandis flight decreased (Chen and Seybold 2014). Another beetle closely related to P. juglandis, Trypodendron lineatum (Oliver) dropped linearly as wind speed increased from 0 to 0.9 m/s (Salom and McLean 1991). Other studies have shown that wind speed is an important factor that always affects flight activity in other insects. Aluja et al. (1993) reported that under laboratory conditions, flies (Rhagoletis pomonella Walsh) were most active under windless conditions and Juillet (1964) documented a reduction in flight activity of ichneumonids and braconids during periods of high wind speed. Flight speed is relatively constant in bark and ambrosia beetles (Evenden et al. 2014). Dendroctonus ponderosae ranged between 0.43 and 0.54 m/s (Evenden et al. 2014). It could be inferred that ambrosia beetles fly at low levels because their flight velocity is greater than the wind speed in the insect boundary layer allowing greater control of their flight. In avocado orchards windless conditions are rare, but when present, ambrosia beetles are quite active (O. Menocal, personal observation). It has to be noted that wind conditions in avocado orchards are quite variable and could be described as follows. First, windless conditions during the day are uncommon; second, light winds of varying speeds within orchards are modified by the tree height, canopy density, row orientation 78

79 and tree spacing; third, generally, wind speeds are less in mature orchards with full canopies (J. Crane, personal communication) than young orchards or orchards recently pruned; fourth, short gusts of slight wind mixed with calm periods may be common inside orchards and; fifth, moderate to strong winds are more common during cold fronts and tropical storms. We hypothesize that ambrosia beetles could be found flying in greater numbers under the third and fourth conditions. This interpretation is based mostly on observation and could not be inferred from the data collected. Ambrosia beetle flight at low heights could also be related to kairomones. During flight, most ambrosia beetles are attracted to ethanol (Hanula and Sullivan 2008, Miller and Rabaglia 2009) which is a clear sign of stressed trees (Millar et al. 1986). Different tree structures could release ethanol at different concentrations. For instance, X. glabratus is attracted to natural volatiles emitted from Lauraceae trees (Kendra et al. 2011a). Niogret at al. (2013) and Kendra et al. (2014) documented four sesquiterpenes related with attraction: α-cubebene, α-copaene, α-humulene, and calamenene. In avocado, α-cubebene and α-copaene are found at higher concentrations in the trunk (the dominant site of beetle attack) followed by the branches and then leaves (Niogret at al. 2013). Therefore, the flight height of ambrosia beetles may be influenced by chemical cues emitted at different concentrations by different tree structures. Another possible reason for flights at low levels of ambrosia beetles could be related to biotic factors such as the amount of energy that a female requires to engage in flight (Kirkendall et al. 2015). The main sources of flight fuel in mosquitoes are glycogen and sugar (Bargielowski et al. 2012). Glycogen and triglyceride are known to be the energy reserves in animal cells (Arrese and Soulages 2010). According to Arrese 79

80 and Soulages (2010), the amount of nutrients accumulated in the fat body regulates several important aspects of insect's life. These fat reserves are used by insects to provide energy for developing embryo (Ziegler and Van Antwerpen 2006) and to fuel prolonged periods of flight (Beenakkers et al. 1984). In a different experiment, Evenden et al. (2014) found that D. ponderosae beetles that flew had a median of 7% of their mass attributed to lipids compared to flightless control beetles contained a median of 15% of their body mass as lipid. In a similar study, Thompson and Bennett (1971) found that after 5 h of flight, Dendroctonus pseudotsugae Hopkins fat content decreased from to 5.21%. It is likely that not all but only vigorous ambrosia beetle females that are already fertilized engage in flight. It is reasonable to think that females will fly at low levels and short distances not to deplete their fat reserves because they will use those reserves to produce offspring. Further research is warranted to identify specific fuels used by ambrosia beetles for flying activities. In this study, ambrosia beetles were found flying at heights of 0 6 m and possibly interacting with all structures (i.e., trunk, scaffold limbs, stems, etc.) of avocado trees. An explanation for these differences could be related to the use of visual cues and resource availability. As suggested by Byers et al. (1989) and Mayfield and Brownie (2013) some insects can target visual silhouettes (i.e., tree trunk and branches). Ulyshen and Hanula (2007) recorded significantly more Pityophthorus in the canopy than near the ground. Individuals that belong to this genus typically attack twigs and small branches in the canopy (Wood 1982). In a different study, Maner et al. (2014) hypothesized that X. glabratus is not likely to attack trees with small DBH (diameter at breast height), probably because small trees cannot support a high number of progeny. 80

81 Interestingly, Fraedrich et al. (2008) documented that 1 of 222 redbay trees with DBH < 2.5 cm died from laurel wilt two years after X. glabratus arrived at the study site, and redbay trees with 10.3 cm DBH or greater were dead in less than a year. Therefore, we hypothesize that some ambrosia beetles are likely to fly near ground guided by visual cues looking for larger diameter trees that will represent a suitable host for their reproduction. In addition, ambrosia beetles are dependent on certain fungi, and many fungal species are sensitive to water content (Jonsell et al. 2005). Maner et al. (2014) suggested that lower portions of the host trunk may retain more moisture for ambrosia beetle fungal growth over a longer period than upper portions (i.e., branches) because they tend to dry out faster. The results of this investigation provide more information on the biology of ambrosia beetles regarding their flight activity. The results show that most ambrosia beetles species were captured at all the height levels tested but that X. bispinatus and X. volvulus showed a preference for the low- and middle height levels. The results suggest that the ambrosia beetles associated with LW affected trees may interact with the main trunk, scaffold branches, and stems. Knowledge of these distribution patterns should be taken into consideration when planning pest management control strategies. This study only investigated the vertical flight distribution; however other factors such as wind speed should be measured to have a better interpretation of what factors affect ambrosia beetle flight in avocado orchards in south Florida. Acknowledgments Thanks to, Jose Alegría, Manuela Angel, Julio Mantilla, and Teresa Narváez for experiment set-up and data collection. Thanks to James Colee (UF-IFAS-Statistics 81

82 Department) for his help with the statistical analysis. This research was in partial fulfillment of an MS degree for Octavio Menocal from the University of Florida. This work was funded in part by FDACS-SCBG grant to DC. 82

83 Table 4-1. Mean number ± SE of ambrosia beetles caught at three different height levels in three avocado orchards in Homestead, Florida. Beetle Field 1 Field 2 Field 3 Total z Species Low Middle High Low Middle High Low Middle High Low Middle High Xyleborus volvulus 8.50 ± ± ± ± ± ± ± ± ± ± 1.12a 3.37 ± 1.27b 4.30 ± 1.69b Xyleborinus saxesenii 4.90 ± ± ± ± ± ± ± ± ± ± 0.86a 2.87 ± 0.81a 2.03 ± 0.74a Euplatypus parallelus 4.10 ± ± ± ± ± ± ± ± ± ± 0.70a 2.80 ± 1.21a 2.33 ± 1.01a Xyleborus bispinatus 5.60 ± ± ± ± ± ± ± ± ± ± 0.56a 1.70 ± 0.41ab 0.90 ± 0.30b Xyleborus affinis 3.30 ± ± ± ± ± ± ± ± ± ± 0.47a 0.93 ± 0.34a 0.80 ± 0.38a Hypothenemus sp ± ± ± ± ± ± ± ± ± ± 0.20a 1.27 ± 0.31a 0.83 ± 0.21a Xylosandrus crassiusculus 0.20 ± ± ± ± ± ± ± ± ± ± ± ± 0.08 Xyleborus ferrugineus 0.40 ± ± ± ± ± ± ± ± ± ± ± ± 0.09 Xylosandrus compactus 0.00 ± ± ± ± ± ± ± ± ± ± ± ± 0.00 Ambrosiodmus develuxus 0.00 ± ± ± ± ± ± ± ± ± ± ± ± 0.03 Euwallacea fornicatus 0.00 ± ± ± ± ± ± ± ± ± ± ± ± 0.10 Xyleborinus andrewesii 0.00 ± ± ± ± ± ± ± ± ± ± ± ± 0.03 Low level: 0 2 m. Middle level: 2 4 m. High level: 4 6 m. Z Those columns not followed by a letter were not statistically different. 83

84 Figure 4-1. Effect of three height levels on numbers of ambrosia beetles trapped. (Low = 0 2 m; Middle = 2 4 m; Top = 4 6 m). Bars represent the mean ± SE of ambrosia beetles caught per height level. Bars with the same letters in the same column are not significantly different (P < 0.05) 84