Agricultural and Forest Entomology (2016), 18,

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1 Agricultural and Forest Entomology (2016), 18, DOI: /afe Biology of two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), recently invasive in the U.S.A., reared on an ambrosia beetle artificial diet Miriam F. Cooperband, Richard Stouthamer, Daniel Carrillo, Akif Eskalen, Tim Thibault, Allard A. Cossé, Louela A. Castrillo,JohnD.Vandenberg and Paul F. Rugman-Jones Otis Laboratory, USDA-APHIS-PPQ-CPHST, 1398 W. Truck Road, Buzzards Bay, MA 02542, U.S.A., Department of Entomology, 900 University Ave., University of California, Riverside, CA 92521, U.S.A., Tropical Research and Education Center, University of Florida IFAS, SW 280 ST, Homestead, FL 33031, U.S.A., Department of Plant Pathology and Microbiology, 3401 Watkins Ave., University of California, Riverside, CA 92521, U.S.A., Huntington Library, Art Collections, and Botanical Gardens, 1151 Oxford Rd., San Marino, CA 91108, U.S.A., USDA-ARS-NCAUR, 1815 N. University St., Peoria, IL 61604, U.S.A., Department of Entomology, Cornell University, 129 Garden Ave., Ithaca, NY 14853, U.S.A. and USDA, Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, 538 Tower Rd., Ithaca, NY 14853, U.S.A. Abstract 1 Recent molecular studies have found that the ambrosia beetle Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, each carrying a different species of symbiotic fungus, in the genus Fusarium, which they farm within galleries inside woody hosts. Several of these beetle species have become invasive pests around the world for attacking and infecting healthy trees with their phytopathogenic fungal symbionts. 2 Diet and rearing protocols were developed for two members of the E. fornicatus species complex, polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB), using sawdust from host trees, allowing collection of data on beetle biology, phenology and sex ratios. Adults developed within 22 days at 24 C. Single PSHB or TSHB foundresses averaged 32.4 and 24.7 adult female offspring, respectively, and up to 57 and 68 female adults within 6 7 weeks. A strong predictor of the number of offspring in a colony was the number of entry holes. Average sex ratios (% male) for PSHB and TSHB, respectively, were 7.4% and 7.2%. 3 Being haplodiploid, virgin PSHB foundresses were able to produce and mate with male offspring, then subsequently produce female offspring, confirming that they have arrhenotokous reproduction. 4 A cold tolerance study found significant mortality rates among PSHB colonies exposed to 5 or 1 C but not colonies exposed to 0,1 or 5 C. 5 Given Hamilton s local mate competition (LMC) theory, a number of LMC predictions were violated. PSHB sex ratios were not affected by the number of foundresses; approximately 14% of broods did not contain males; males did not usually eclose before females but eclosed around the same time (22 23 days); and PSHB males were found walking outside of their natal galleries on the trunk of a heavily infested tree in the field. Alternatives to LMC are considered, such as early forms of sociality (maternal care, cooperative brood care), local resource enhancement and kin selection. Keywords Artificial rearing, cold tolerance, Fusarium dieback, insect diet, local mate competition, polyphagous shot hole borer, sex ratio, tea shot hole borer. Introduction Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae), the tea shot hole borer (TSHB), was first described from Sri Lanka in 1868 (as Xyleborus fornicatus) (Eichhoff, Correspondence: Miriam F. Cooperband. Tel.: ; fax: ; miriam.f.cooperband@aphis.usda.gov 1868). Long considered the most serious pest of tea in Sri Lanka (Gadd, 1941; Walgama & Pallemulla, 2005), this pest species originates from southeast Asia, occurring in countries from Sri Lanka to Taiwan, and is known for its broad host range and distribution, as well as numerous invasions around the world, such as Hawaii, California, Florida, Israel, Australia, Africa, Vanuatu, Panama, Costa Rica and many more (Danthanarayana, 1968; Published This article is a U.S. Government work and is in the public domain in the USA.

2 224 M. F. Cooperband et al. Rabaglia et al., 2006; Kirkendall & Ødegaard, 2007; Mitchell & Maddox, 2010). However, its status as a single species has recently been questioned based on molecular data. Eskalen et al. (2013) were the first to draw attention to large levels of genetic differences between invasive populations of shot hole borers from Florida and California, both of which matched the morphological description of E. fornicatus, suggesting that they were different species, and coining the common name for the polyphagous shot hole borer (PSHB) in California. O Donnell et al. (2015) subsequently conducted DNA-based phylogenetic analyses on beetles matching the morphological description of TSHB from invasive populations around the world, including beetles from Sri Lanka (where E. fornicatus was first described). Their findings suggested that E. fornicatus was a complex of at least five morphologically indistinguishable species (O Donnell et al., 2015). Invasive populations in both Israel and the greater Los Angeles region of southern California were genetically identical (which they referred to as Euwallacea sp. #1) but differed genetically from the invasive population in Miami-Dade County, Florida (Euwallacea sp. #2), and both differed from a more recently discovered invasive population in San Diego County, southern California (Euwallacea sp. #5). Furthermore, all three of these beetles differed genetically from those in Sri Lanka (Euwallacea sp. #4, potentially the true E. fornicatus) and Australia (Euwallacea sp. #3). Molecular data suggest that Euwallacea sp. #1 (PSHB) most likely originated from Vietnam (R. Stouthamer, unpublished data; Lynch et al., 2016). The genetic differences found between beetle populations were mirrored in the species of Fusarium symbionts that they each carry, and were sufficiently great for the the beetles to be considered as separate species (O Donnell et al., 2015). Fusarium euwallaceae is the principal and obligate symbiont for PSHB (Eskalen et al., 2012, 2013; Freeman et al., 2013a; O Donnell et al., 2015), although, recently, two additional fungal associates, Graphium euwallaceae and Paracremonium pembeum, were identified from PSHB mycangia (Lynch et al., 2016). The fungal associate G. euwallaceae is capable of sustaining offspring development whereas the role of P. pembeum is not known (Sharon et al., 2015). However, infection by a complex of fungal associates may impart an increased risk to the host plant (Lynch et al., 2016). The E. fornicatus species complex is in need of thorough taxonomic revision and, as such, the application of scientific names to the constituent member species would be somewhat preliminary and ambiguous. It also presents a challenge when interpreting the body of literature on the biology of Xyleborus fornicatus (now in genus Euwallacea), which is considered to be a complex of cryptic species rather than a single species. Based on their locality, many of the early studies conducted on the shot hole borer of tea in Sri Lanka (Calnaido, 1965; Danthanarayana, 1968, 1973; Wickremasinghe et al., 1976; Sivapalan & Shivanandarajah, 1977) may have been referring to E. sp. #4 (O Donnell et al., 2015), and E. sp. #1 and E. sp. #2 may be relatively new to science. Among the species invasive in the U.S.A., the common name of PSHB was given to the population in Los Angeles Co., California, aiming to differentiate it from the population in Miami-Dade Co., Florida, which so far has retained the common name of TSHB (Eskalen et al., 2013). The common names PSHB and TSHB are used in the present study, and match with E. sp. #1 and E. sp. #2, respectively, in O Donnell et al. (2015). The TSHB was detected in Florida in 2002 but with minor initial impact (Rabaglia et al., 2008), and was found in avocado groves in south Florida in 2012 where levels started out low, although damage and abundance are increasing (Carrillo et al., 2012, 2015). The PSHB was first discovered in 2003 and 2005 (although it was misidentified as TSHB), in Los Angeles County and Israel, respectively (Rabaglia et al., 2006; Mendel et al., 2012). The Israeli avocado industry sustained major loss as a result of Fusarium dieback, a serious plant disease associated with PSHB (Eskalen et al., 2012; Mendel et al., 2012; Freeman et al., 2013b), and the California avocado industry has started to experience similar losses (Eskalen et al., 2012). Additionally, thousands of other landscape and forest trees have been killed and removed in southern California as a result of Fusarium dieback. Eskalen et al. (2013) reported that, of 335 plant species observed, PSHB attacked 207 species of healthy trees in 58 plant families, and Fusarium was established and recovered in more than half of those species. At the time of their study, Eskalen et al. (2013) reported 19 species as reproductive hosts in which attacks resulted in the establishment of Fusarium and colonies with offspring. Subsequently, the list of attacked tree species has increased to 342 taxa in 63 families (T. Thibault and A. Eskalen, unpublished data). Of these, 39 species in 16 families are known reproductive hosts, 13 of which are species native to California (Table 1) (Eskalen et al., 2013; Eskalen, 2016; Less is known about the full host range of TSHB, although its range is also likely to be extensive because E. fornicatus has been recorded attacking at least 100 different plant species, in 35 families, in India, Java, Malaysia and Sri Lanka, with at least 21 of those being reproductive hosts (Danthanarayana, 1968). The Euwallacea sp. #5 population in San Diego has been found attacking commercial avocado groves and other hosts similar to those of PSHB (Eskalen, 2016; One of the economically important reproductive hosts of all three species in the U.S.A. is avocado, which, in California alone, is an industry worth about $400 million annually (California Avocado Commission, 2015). Mexico is the world s top producer and exporter of avocados, exceeding the avocado production of any other nation by more than four-fold, and is followed by Indonesia, Dominican Republic, U.S.A., Columbia, Peru, Kenya, Chile, Brazil, Rwanda, China, Guatemala, South Africa, Venezuela, Spain and Israel (FAOSTAT, 2012; The list of PSHB hosts that are susceptible to the Fusarium pathogen also includes numerous commonly planted urban and suburban street trees, ornamental trees in public spaces and private yards, 58 native North American tree species, and 19 native Californian tree species. Thus, the monetary and environmental damage potential is already high, and only increasing as beetle populations also increase in southern California. Although laboratory rearing on an artificial substrate in the absence of host material has been conducted to determine symbiont fidelity (Freeman et al., 2013a), to our knowledge, no studies have documented the fecundity and sex ratio of these new species. Timely research is required to (i) improve our understanding of the biology of these species; (ii) develop tools for trapping and detection; and (iii) investigate mitigation options such as biological control. Such research would be greatly expedited by the ability to mass rear

3 Biology and rearing of Euwallacea ambrosia beetles 225 Table 1 An updated list of the 39 plant species in 16 families that are known reproductive hosts for polyphagous shot hole borer (PSHB) and the host plant native origin Family Species Common name Plant origin a Aceraceae s.s (Sapindaceae s.l.) Acer buergerianum Trident maple As Acer macrophyllum Big leaf maple b NA Acer negundo Box elder b NA Acer palmatum Japanese maple As Acer paxii Evergreen maple As Aquifoliaceae Ilex cornuta Chinese holly As Betulaceae Alnus rhombifolia White alder b NA Euphorbiaceae Ricinus communis Castor bean Af, Eu, As Fabaceae Acacia sp. Acacia Albizia julibrissin Mimosa or silk tree As Castanospermum australe Moreton Bay chestnut or blackbean Oe Cercidium floridum (Parkinsonia florida) Blue palo verde b NA Cercidium x sonorae Brea NA Erythrina corallodendron Coral tree As Parkinsonia aculeata Palo verde SA Prosopis articulata Mesquite b NA Wisteria floribunda Japanese wisteria As Fagaceae Fagus crenata Japanese beech As Quercus agrifolia Coast live oak b NA Quercus engelmannii Engelmann oak b NA Quercus lobata Valley oak b NA Quercus robur English oak Af, Eu Quercus suber Cork oak Af, Eu Hamamelidaceae s.l. (Altingiaceae s.s.) Liquidambar styraciflua American sweetgum NA Lauraceae Persea americana Avocado NA, SA Malvaceae Brachychiton populneus Kurrajong Oe Moraceae Ficus carica Black mission fig As Myrtaceae Corymbia (Eucalyptus) ficifolia Red flowering gum Oe Platanaceae Platanus mexicana Mexican sycamore NA Platanus racemosa California sycamore b NA Platanus x acerifolia London plane tree Eu, As Salicaceae Populus fremontii Fremont cottonwood b NA Populus trichocarpa Black cottonwood b NA Salix babylonica Babylon willow or weeping willow or tortuosa As Salix gooddingii Goodding s black willow b NA Salix laevigata Red willow b NA Sapindaceae Alectryon excelsus Titoki Oe Simaroubaceae Ailanthus altissima Tree of heaven As Theaceae Camellia semiserrata Camellia As a The continent of origin, if known, is indicated: Oe, Oceania; Af, Africa; Eu, Europe; As, Asia; NA, North America including Mexico and above; SA, South and Central America below Mexico. b Native to California. Host records are based on data from the population in and around Los Angeles County (Eskalen et al., 2013; Eskalen, 2016; T. Thibault and A. Eskalen, unpublished data). This list demonstrates the diversity of the hosts both in their origins and taxonomic groupings. these beetles in the laboratory. In the present study, we describe successful laboratory mass rearing techniques for two members of the E. fornicatus species complex (PSHB and TSHB) and report the findings of studies conducted aiming to characterize basic biological attributes such as the developmental period, phenology and sex ratios of both species, as well as the effects of mating, foundress number and cold tolerance for PSHB. Materials and methods Diet A sawdust-based diet was made using methods described in Castrillo et al. (2011), with some minor modifications (see below). Sawdust from three different tree species was tested. Initially Avocado Persea americana (Lauraceae) sawdust was unavailable, and so American beech Fagus grandifolia (Fagaceae) and boxelder Acer negundo (Sapindaceae) were considered because they were listed as hosts in the literature (Danthanarayana, 1968; Eskalen et al., 2013). As species became available, PSHB and TSHB were both eventually tested with all three sawdust types (boxelder, beech and avocado) and reared successfully, although the decision was made to continue to rear each species on the same host that it was originally collected from in the field: PSHB on boxelder and TSHB on avocado. Beech and boxelder trees in Massachusetts and Illinois were felled and peeled, and bolts of wood were allowed to dry at room temperature for 2 4 weeks. A miter saw was used to make

4 226 M. F. Cooperband et al. multiple cuts into the sapwood of the bolts, avoiding the heartwood, and the resulting sawdust was collected. Avocado bolts were cut in Florida and allowed to dry. A sander equipped with a vacuum bag was used to make sawdust out of the avocado sapwood layer. For all tree species, the heartwood was discarded. Large particles were removed from sawdust by passing it through a No. 12 sieve (Hogentogler & Co. Inc., Columbia, Maryland). To improve the texture of the boxelder sawdust, a coffee grinder was used to produce a finer sawdust powder. Sawdust was packaged and stored in the freezer ( 18 C) until needed. Ingredients were combined in a 600-mL glass beaker: 45 g of sawdust (boxelder, beech or avocado), 12 g of agar (Difco Agar, granulated, Becton, Dickinson and Company, Franklin Lakes, New Jersey), 6 g of sucrose (Bio-Serv, Flemington, New Jersey), 3 g of casein (Fonterra, Rosemont, Illinois), 3 g of potato starch (Bio-Serv, Flemington, New Jersey), 3 g of brewer s yeast (MP Biomedicals, Santa Ana, California), 0.6 g of Wesson s salt mix (Frontier Agricultural Sciences, Newark, Deleware), 0.21 g of tetracycline (Tetrasol, Med-Pharmex, Pomona, California), 1.5 ml of wheat germ oil (Bio-Serv), 3 ml 100% of ethanol (Fisher Scientific, Waltham, Massachusetts) and 300 ml of distilled water. Dry ingredients were combined first and stirred. Then the oil and ethanol was stirred in, followed by the water. The beaker containing the mixture was covered with foil and autoclaved for 25 min at 121 C and approximately 15 psi. Tools were also autoclaved, the hood was surface sterilized with 70% ethanol and lined with aluminum foil, and trays of single-use 50-mL sterile polyethylene centrifuge tubes (Fisher Scientific) were placed in the flow hood and ultraviolet (UV) surface-sterilized for 45 min. The hot diet was removed from the autoclave and stirred well to resuspend settled ingredients. Working quickly, approximately 15 ml of hot diet was poured into each tube. Tubes were loosely capped and tapped to remove air bubbles. Loosely capped tubes of diet were allowed to dry in the flow hood and were checked daily for condensation, after which they were UV sterilized for 1 2 h. Once all the condensation had evaporated from inside the tubes (2 8 days), they were capped tightly, labelled and frozen at 20 C until needed. This volume of diet yielded approximately 25 diet tubes, each containing 15 ml of diet. PSHB from California Between h and h on 9 August 2013, beetles were collected from a heavily infested boxelder tree in Altadena, California. Thirteen female and three male PSHB that were walking on the bark were hand-collected and placed into a 2-mL glass vial with some tissue paper. They were transported to the nearby laboratory at the Huntington Library, Art Collections and Botanical Gardens in San Marino, California, where each beetle was surface-sterilized by emersion in 70% ethanol for 10 s. Beetles were then dried for 5 s on a piece of filter paper, and placed, individually or in groups, in 10 rearing tubes containing boxelder diet. Surface sterilization was conducted to prevent microbes, other than the fungi protected inside the mycangia, from colonizing the diet. The newly founded tubes were shipped to the Otis Laboratory (USDA permit # P526P ) for further rearing and research within the insect containment facility. From these original beetles, the laboratory stock population that we refer to as PSHB was established. TSHB from Florida On 5 March 2014, two E. sp. #2 (TSHB) females were dissected out of an infested avocado branch in Miami-Dade County in Florida by Daniel Carrillo. The beetles were surface-sterilized and placed individually into tubes containing avocado diet. Colonies were successfully established and sent to the insect containment facility at the Otis Laboratory (USDA permit # P526P ) for further rearing and research. From these original beetles, the laboratory stock population we refer to as TSHB was established. Rearing tubes containing different beetle species were housed in separate containers, which were never opened at the same time to isolate colonies from each other. Verification by DNA In addition to identification using a key to species for E. fornicatus (Rabaglia et al., 2006), once laboratory stock populations of PSHB and TSHB were established in the laboratory, one female descended from each colony line (eight PSHB and two TSHB) was preserved in 95% ethanol and sent to the Stouthamer laboratory for genetic confirmation of the cryptic species. This was achieved by sequencing a section of the cytochrome c oxidase subunit I (COX I) gene commonly used in barcoding studies in accordance with protocols described in Eskalen et al. (2013). All analyzed specimens originating from the PSHB and TSHB colonies were confirmed as PSHB and TSHB, respectively, by comparison with GenBank accession numbers JX JX Initiation of experimental colonies Beetles were reared in a walk-in environmental chamber under an LD 16 : 8 h photocycle at 24 C in the insect containment facility at the Otis Laboratory. Rearing tubes were enclosed within locking food storage containers that were modified with mesh windows for air exchange, and nested within larger containers to keep colonies apart, prevent escapes and reduce the risk of contamination by mites or foreign fungi (Rectangular Storage Container, Lock & Lock U.S.A., Anaheim, California; Nylon Mesh 500μm, 47% open area, Component Supply Co., Fort Meade, Florida). The protocol for handling beetles was as described previously by Castrillo et al. (2011). To allow air exchange in diet tubes but prevent escapes, a hole was drilled into the lid of each diet tube (diameter 4 mm) and a fine stainless steel mesh ( , 33.6% open area) was secured over the hole using a hot glue gun. A typical colony was initiated by placing a single mated foundress in a rearing tube with diet. Exceptions to this (described below) were required for the investigation of haplodiploid reproduction and local mate competition.

5 (a) (b) (c) Biology and rearing of Euwallacea ambrosia beetles 227 Figure 1 A vial containing a polyphagous shot hole borer (PSHB) colony is depicted from the side showing galleries against the wall of the vial (a); looking down at the surface of a 2-week-old colony, Fusarium and excavation material (arrow) can be seen coming from a single entry hole (b); and, in the same vial, 1 month later, several entry holes made by offspring are observed (arrows) (c). Colony development As the colonies developed, observations were made to measure the health of the colony. Colonies were visually evaluated every 1 3 days by examining the sides of the tubes to identify visible galleries, and by removing the lid and examining the surface of the diet. The number of active entry holes (not covered over by fungus) was quantified. These beetles push excavated material and frass out through the entry hole in the form of a compact cylinder. It was noted whether or not this excavation material was present and, if so, the length of the ejected material was measured using a 5-mm marker for reference (Fig. 1). The length of excavation material was presumed to be an indication of the extent of gallery formation. The number of eggs, larvae, pupae and adults on the surface and within any visible galleries was noted. The sex of pupae and adults was recorded and, if adult females were found, it was noted whether they were teneral (cuticle was white to brown) or fully sclerotized (cuticle was black). The time and date was recorded for all observations. Dissection of colonies Colonies were destructively analyzed at various ages to quantify the development of all stages over time. Each tube containing a beetle colony was systematically dissected under a stereomicroscope. All eggs, larvae, pupae and adults were quantified. Week 1 included the day of colony initiation (day 0) until day 7, week 2 included days 8 14, week 3 included days 15 21, and so on. To dissect a tube, all beetle stages that were above the diet surface were removed and counted first. Then the diet plug was tapped out into a re-sealable zipper storage bag where it was examined under a dissecting microscope. Once all beetle stages on the surface of the diet plug were removed and counted, the diet was systematically chipped away in small bits starting at the bottom of the plug. As galleries were revealed, they were followed and the diet around them was removed in small increments to allow any beetle stages within to be collected and quantified. This technique was followed until the entire diet plug had been dissected. If at any time a large number of beetles emerged from the diet at once, the bag was sealed and a paint brush moistened with 70% ethanol was inserted into the corner of the bag and used to remove and count the beetles, so as to prevent any escapes. Haplodiploid reproduction To confirm the presence of haplodiploid reproduction, and to determine whether unmated females were capable of initiating new colonies by first producing male offspring (from unfertilized eggs) and then mating with those sons to subsequently produce a brood of female offspring, experimental colonies were initiated using virgin females. To ensure that females were virgins, female pupae were collected from the laboratory stock populations and placed in an empty rearing tube until adult eclosion. Because newly eclosed adults need to acquire their symbionts, diet with Fusarium was taken from an established healthy colony and provided to the newly eclosed virgin females for feeding, allowing beetles to acquire their symbionts in their mycangia. After being allowed the opportunity to feed for at least 1 day, individual virgin females were surface-sterilized, transferred to clean diet tubes and allowed to naturally inoculate the sterile diet with their symbionts. Tubes were monitored for up to 13 weeks after which they were destructively analyzed and offspring were quantified. Sex ratio and local mate competition Local mate competition (LMC) theory predicts that species with a high propensity for inbreeding (e.g. shot hole borers) should produce extremely female-biased sex ratios (Hamilton, 1967). As the number of foundresses increases (and the chances of outbreeding increases), sex ratios are expected to approach a more normal 1 : 1 ratio (Fisher, 1958; Hamilton, 1967; Nunney & Luck, 1988). To test whether PSHB possess this attribute of LMC, we evaluated the sex ratio of offspring when one foundress or multiple foundresses (two to five) were used to initiate a

6 228 M. F. Cooperband et al. colony. The number of male and female offspring were quantified and sex ratios were calculated and compared for colonies allowed to develop 5 8 weeks (one generation) (n = 54 and 10 for single and multiple foundresses, respectively) and 9 13 weeks (two generations) (n = 13 and 5 for single and multiple foundresses, respectively). For TSHB, the sex ratios were only examined for colonies initiated by a single foundress. Another prediction of LMC is that males in a brood emerge before their sisters, and so PSHB and TSHB were studied for the presence of this attribute as well. PSHB and TSHB colonies were dissected between 14 and 28 days to determine the time of the first adult eclosion and also which sex reaches adulthood first. Cold tolerance The cold-hardiness of PSHB was investigated by exposing colonies to different low temperatures. Thirty PSHB colonies were initiated by a single foundress and allowed to develop for 35 days in the environmental chamber at 24 C. At day 35, eleven colonies that did not appear to be thriving were discarded and the remaining 24 colonies were divided into five groups. Four colonies were kept at 24 C (control). The remaining colonies were divided into four temperature treatments (n = 5) and placed on a rack. A diet tube without beetles was placed in the centre of the rack with a traceable refrigerator/freezer alarm thermometer (Fisher Scientific) inserted into the diet to monitor internal diet temperature. The rack was placed in a freezer at 15 Candan alarm sounded when the diet in the monitoring tube reached 5, 1, 1 and 5 C, at which time the five tubes in each respective group were removed from the freezer and reunited with the control group at 24 C. An additional temperature probe in the freezer monitored the temperature outside of the diet. The time elapsed between each temperature was recorded. After cold treatments, the colonies were allowed to recover for 24 h at 24 C. The next day, all tubes were dissected and the number of dead and living larvae, pupae, and adults were quantified. Eggs were excluded because we were unable to visually determine their health. Dead larvae and pupae were discoloured and flattened. Adults were considered dead if they lacked movement. Mortality rate was calculated as (1 percentage survival) of larvae, pupae and adults, and compared for each treatment and stage. Statistical analysis Linear regression models were used to predict relationships between offspring and entry holes or excavation material (jmp, version ; SAS Institute, Cary, North Carolina). Analyses comparing sets of data that passed the test for normality were analyzed using analysis of variance (anova) (jmp, version ). For analyses comparing two sets of data that were nonparametric, datasets were compared using the Wilcoxon test (jmp, version ). For statistical analyses comparing multiple datasets that failed to satisfy the assumptions of anova, data were rank transformed, and then analyzed using anova and Tukey s mean separation test (sas, version 9.3; SAS Institute Inc., Cary, North Carolina; jmp, version ) (Conover, 1980). Figure 2 Average excavation material (mm) ejected per rearing tube per week of development by either a mated or virgin polyphagous shot hole borer (PSHB) female in boxelder diet. A total of 80 mated colonies and 6 virgin colonies, respectively, were monitored several times per week. The number of measurements each week was averaged (e.g. in week 1, there were 173 and 21 measurements for mated and virgin colonies, respectively). Bars represent the SE. Results Entry holes and excavation The three most prominent signs of a healthy colony were the growth of the white Fusarium, which ultimately carpeted the exposed surface of the diet, the presence of one or more fresh entry holes, and the presence of excavation material ejected from the entry holes (Fig. 1). Entry holes that were not in use would soon become covered over by Fusarium and appear closed or disappear altogether. Excavation material and entry holes were measured for 80 PSHB colonies each founded by a single mated female, and six colonies each founded by a single virgin female, and recorded on average two to three times per week. Colonies founded by mated females initially produced more excavation material than those started by virgin females, which lagged by 2 3 weeks (Fig. 2). Wilcoxon tests revealed significant differences between mated and virgin PSHB in the amount of excavation material ejected in week 2 (χ 2 = 4.46, P = ) and week 5 (χ 2 = 15.99, P < ). The amount of excavation material peaked for mated foundresses at weeks 6 and 9, and for virgin foundresses at weeks 7 and 11, most likely corresponding to the peak maturation of the first and second generations of female offspring. The second peak was likely suppressed as a result of the diminished population capacity and quality of the aging diet. The average number of active entry holes per colony, founded either by a single mated or virgin PSHB, was plotted by week (Fig. 3). In the first 4 weeks, the average number of entry holes ranged between 0.6 and 1.0 for both treatments. The first increase in number of entry holes in both mated and virgin treatments occurred between weeks 4 and 5, corresponding to emerging offspring, and both sexes were observed to participate in excavation activities. The number of entry holes was similar in the first 5 weeks, after which they started to diverge. Wilcoxon tests found significant differences in the number of entry holes between mated and virgin PSHB foundresses in week 3(χ 2 = 4.50, P = ), week 8 (χ 2 = , P < ),

7 Biology and rearing of Euwallacea ambrosia beetles 229 Figure 3 Average number of fresh entry holes observed per colony per week in colonies founded by a single mated (solid) or virgin (dotted) female polyphagous shot hole borer (PSHB) foundress in boxelder diet over a period of 11 or 13 weeks, respectively. A total of 80 mated colonies and 6 virgin colonies, respectively, were monitored several times per week. The number of observations for each week was averaged (e.g. in week 1, there were 188 and 21 measurements for mated and virgin colonies, respectively). Bars represent the SE. week 9 (χ 2 = , P = ) and week 10 (χ 2 = , P < ). For each colony, the maximum number of active entry holes was compared with the total number of male and female offspring harvested. Because the sex of pupae could also be determined, they were included in the tally. Entry hole number was found to be a significant predictor of the number of PSHB offspring in a colony (Fig. 4) (linear regression, r 2 = , P < for females; r 2 = , P = for males; n = 73). Excavation material was also found to be significantly correlated with the number of PSHB offspring, although it was not as strong a predictor as entry holes (linear regression, r 2 = , P < for females; r 2 = , P = for males; n = 73). Similarly, the linear regression model for TSHB offspring to the number of entry holes is presented in Fig. 5 (r 2 = , P < for females; r 2 = , P < for males; n = 46). As with PSHB, the relationship between the number of TSHB offspring and excavation material was weaker than with entry holes (r 2 = , P < for females; r 2 = , P < for males; n = 46). Offspring development The first day that each stage was observed either on the diet surface or by examining galleries along the rearing tube walls of any colony founded by a mated or virgin female PSHB is presented in Table 2. This observational approach was a nondestructive way of evaluating the timing of the different stages. With mated foundresses, eggs and larvae were first observed 3 4 days earlier than with virgin foundresses, although the first male was observed 2 days later. The first appearance of daughters in the virgin treatment was days later than the first appearance of daughters in the mated treatment, presumably after a haploid son matured and mated with the foundress. Colonies were dissected from each week of development, and the numbers of eggs, larvae, male pupae, female pupae, male adults, teneral female adults and mature female adults, and also the number of dissected colonies, are presented in Fig. 6 for both PSHB and TSHB colonies founded by a single mated female. There were no significant differences between PSHB and TSHB in the number of each stage of offspring harvested (Fig. 6). Adult offspring were not observed in colonies prior to 22 days. The greatest number of adult females produced by a single PSHB was 57 (in week 7), and that produced by a TSHB foundress was 68 (in week 6). The number of offspring was not significantly influenced by foundress number, although colony age grouping (first generation at 5 8 weeks versus second generation at 9 13 weeks) and mating status had a significant effect on the number of female offspring (Tables 3 and 4). For PSHB, the number of female offspring from both single and multiple mated foundresses was not significantly affected by the age grouping (one generation versus two generations), although the limitations of the diet may have been reached. Conversely, single TSHB foundresses had fewer first-generation offspring than PSHB (Wilcoxon test, χ 2 = 4.93, P = ) and generation had a significant effect on the number of female offspring (Wilcoxon test, χ 2 = 5.10, P = ) (Tables 3 and 4). Generation also had a significant effect on the number of female offspring of virgin PSHB colonies (Wilcoxon test, χ 2 = 4.51, P = ) because the first generation of offspring contained no females. Sex ratio and haplodiploidy Virgin foundresses did not initially produce female offspring; rather, they produced sons with which they mated and subsequently produced daughters. Although there appeared to be a trend for older PSHB colonies to have higher sex ratios (% males), there was no significant difference in sex ratio between colonies of different ages, species or foundress number (Tables 3 and 4). The only factor that affected sex ratio was the mating status of the foundress, in which colonies initiated by virgin females were 100% male in 5 8-week-old colonies, and remained predominantly male in older colonies. On average, when harvested between 5 and 8 weeks, typical mated single-foundress PSHB and TSHB colonies had average sex ratios of 7.4% and 7.2%, respectively. Unexpectedly, of 95 PSHB colonies with mated foundresses that were examined, only female offspring were found in 13 (13.6%) of them. A similar percentage of colonies with mated TSHB foundresses also resulted in no male offspring. Whether these foundresses failed to produce males or their male offspring died prematurely and were concealed by fungus prior to dissection is not known, and so these colonies are reported separately (Table 3). In colonies dissected between 14 and 28 days, no adults were found prior to day 22 (Fig. 7). The colonies in which only the first adult had emerged were aged days. Unexpectedly, the first adult to emerge was not always a male as predicted by LMC. Of six PSHB rearing tubes aged days old, two had a female emerge first on day 22 and one had a male emerge first on day 24. Of six TSHB rearing tubes aged days old, two had a female emerge first, one on day 22 and the other on day 23. The remaining tubes examined had either no adult emergence yet, or both sexes had already emerged.

8 230 M. F. Cooperband et al. Figure 4 Data from 73 colonies, aged 5 13 weeks, each founded by a single mated polyphagous shot hole borer (PSHB) female, were examined. The number of fresh entry holes (left) or the amount of excavation material (right) was plotted against the total number of female (top) or male (bottom) offspring (adults and pupae). Linear regression models show that the number of entry holes was a superior predictor of the number of offspring in a colony. Cold tolerance The time that it took to drop to target temperatures resulted in rates of temperature change of C/min for each treatment. Dissections of colonies exposed to different temperatures revealed that one tube from each of the 5, 1 and 5 C treatments contained no offspring, reducing the number for those treatments. There were significant differences in mortality between temperature exposures for larvae (anova of ranked data: F = 3.22, P = ), pupae (F = 4.34, P = ) and adults (F = 4.44, P = ) (Fig. 8). Mortality increased significantly with a lower temperature and longer cold exposure, for all stages. Larvae and pupae appeared to be more sensitive to cold than adults, as indicated by their higher mortality rates at the lowest temperatures. After exposure to 5 C, 100% of larvae, 95.7% of pupae and 69.2% of adults died. Of those surviving adults, many were on their backs, moving their legs only slightly after the 24-h recovery period. Exposure to 1 C produced similarly high levels of mortality, whereas most individuals survived the 0 and 5 C treatments, and those treatments did not differ from controls. Discussion Biology and rearing The two newly invasive species of the E. fornicatus species complex found in Los Angeles County, California (PSHB), and Miami-Dade County, Florida (TSHB), were similar with respect to their biology and rearing capabilities. The first successful rearing attempt on artificial diet of scolytine ambrosia beetles was described by Saunders and Knoke (1967) using Xyleborus ferrugineus (Fabricius) and its Fusariumsymbiont. By omission of key ingredients, they that found cellulose powder or fresh host sawdust was essential, and the importance of wood vitamins was further demonstrated by Norris and Baker (1968). Diet variations have similarly been tested for rearing a member of the E. fornicatus species complex (Sivapalan & Shivanandarajah, 1977), showing that cellulose powder, salt mixture and yeast extract were important for members of this group. Similar diets have also been developed for other species of ambrosia beetles (Mizuno & Kajimura, 2002, 2009; Peer & Taborsky, 2004; Castrillo et al., 2011; Maner et al., 2013). Based on a diet for rearing Xylosandrus germanus (Blandford) (Peer & Taborsky, 2004; Castrillo et al., 2011), the diets in the present study were developed using

9 Biology and rearing of Euwallacea ambrosia beetles 231 Figure 5 Data were examined from 46 colonies, aged 5 9 weeks, each founded by a single mated tea shot hole borer (TSHB) female in avocado diet. The number of fresh entry holes and the amount of excavation material was plotted against the total number of female and male offspring (adults and pupae). The linear regression models are shown in each plot. the host tree species from which our beetles were captured, and a mass rearing protocol was successfully established. Brood production has been described in a number of other species of ambrosia beetles (Ngoan et al., 1976; Minuzo & Kajimura, 2002; Peer & Taborsky, 2005), although this is the first comprehensive study quantifying a brood at increments in colony age for these two members of the E. fornicatus species complex. Because offspring stages overlap and are not discrete, the number of brood in each stage varies by the age of the colony at the time it is dissected. Foundresses typically began excavating as soon as they were placed on the diet, introducing Fusarium into their galleries. Excavations were monitored by measuring the amount of material ejected from the entry hole and, although in Xylosandrus pfeili (Ratzeburg) (Minuzo & Kajimura, 2002), gallery length was found to be strongly correlated with number of offspring, we found that the number of entry holes in the diet was a stronger predictor of the number of offspring produced in a particular tube than the amount of excavation material. Because each entry hole is presumably formed by a single foundress when she initiates a new colony, this parameter can also be used in estimating the population size, both in the rearing tube and in the field. The timing of their appearance can also be used when describing their phenology. Adult female offspring were observed as early as 24 days after the initiation of a colony by a mated foundress, and an average PSHB and TSHB foundress produced 32.4 and 24.7 adult female offspring, respectively, which was significantly different (Table 4). If left in tubes longer, those offspring started to produce their own galleries, as well as their own offspring. Generations clearly overlapped, although the appearance of mature adult F 1 daughters began at week 5, peaked at week 6 or 7, and began to decline by week 8, after which the F 2 daughters started to emerge. Approximately 14% of PSHB and TSHB colonies produced only female offspring, and no male offspring were found. This, along with observations of males of PSHB in California (M. F. Cooperband, personal observations) and E. fornicatus (possibly sp. #4) in Sri Lanka (Calnaido, 1965) wandering outside of their galleries on the surfaces of infested host trees during morning hours, suggests that there may be a built-in mechanism for outbreeding to occur in a portion of the population. Both PSHB and TSHB had similarly female-biased sex ratios, with only 7.4%

10 232 M. F. Cooperband et al. Table 2 Day (and week) of the earliest observed life stages of PSHB offspring, either from a virgin or a mated foundress, and the difference in developmental time between the two Offspring stage Day (week) of earliest surface observation Mated foundress Virgin foundress Egg a 17 (3) 21 (3) 4 Larva 13 (2) 16 (3) 3 Male pupa 24 (4) 22 (4) 2 Male adult 27 (4) 25 (4) 2 Female pupa 20 (3) 38 (6) 18 Female adult 24 (4) 43 (7) 19 Number of colonies observed Difference in development times (days) between mated and virgin foundress a Does not include concealed offspring inside galleries, and so the first observation of eggs did not define the first presence of eggs. Observations were conducted nondestructively for 8 weeks by examining the surface of the diet and inside any galleries that were visible through the sides of the rearing tubes. and 7.2% males, respectively. Colony sex ratio was not affected by the number of foundresses. Freeman et al. (2013a) studied groups of PSHB placed on a lawn of F. euwallaceae on potato dextrose agar (PDA) in Petri dishes and observed development times to the first observation of each developmental stage, including three larval instars, incubated at 25 C. They recorded a slightly longer overall development time than that observed in the present study at 24 C. They found that time to oviposit was greatly affected by natal host experience, depending on whether the foundress had been reared on PDA, in which case oviposition was greatly delayed, or in avocado branches. those that were not (Formby et al., 2013), and may also vary with the exposure time or rate of cooling (Kostal et al., 2011). The slow rate of cooling used in the present study may have allowed for a rapid cold-hardening response (Lee, 1989; Kelty & Lee, 1999). Our empirical study of mortality rates for various stages inside galleries exposed to different low temperatures and exposure times found that PSHB was somewhat tolerant to brief exposures of near freezing temperatures. When temperatures were reduced to 5 and 0 C, although some mortality was observed, this was not significantly different from the control group. However, when diet temperatures dropped below freezing and for longer time periods, mortality was significantly affected. PSHB survival at and above 0 C in the laboratory suggests they can survive similar temperatures inside trees. Temperatures inside overwintering trees, particularly in the outer xylem and the centres of overwintering trees, may be much higher than winter temperatures outside of trees, and temperatures in the sapwood, where these beetles mostly reside, can be much higher than temperatures at the centres of trees (Sakai, 1966). When negative air temperatures occur, temperatures in the sapwood may remain well above freezing and be able to safely harbour these beetles. Internal tree temperatures fluctuate greatly and are influenced by solar radiation, the face of the tree being measured, the diameter of the tree, bark colour and thickness, and tree species. Therefore, PSHB may be able to survive in regions with harsher winters than their current distribution in southern California if internal tree temperatures can safely harbour them. Although phloem temperatures in pine forests have been investigated to predict overwintering capabilities of bark beetles (Tran et al., 2007), more information on the internal overwintering temperatures of preferred host trees of PSHB, such as boxelder, under various growing conditions would improve our ability to estimate the potential range of these beetles and similar species. Cold tolerance Cold tolerance plays a crucial role in the population dynamics of the scolitine bark beetle Dendroctonus ponderosae (Régnière & Bentz, 2007) and may be equally important for ambrosia beetles despite the paucity of cold tolerance studies on them. To our knowledge, the present study is the first aiming to quantify survival at low temperatures in an ambrosia beetle. Another invasive xyleborine ambrosia beetle, Xyleborus glabratus, was found to have temperature-dependent development (Brar et al., 2015), and it is the only ambrosia beetle for which the supercooling point (SCP) was determined (Formby et al., 2013). The SCP has also been explored in a number of bark beetles (Lombardero et al., 2000; Régnière & Bentz, 2007; Kostal et al., 2011; Lester & Irwin, 2012). However, because SCP is the temperature at which the body fluids change phase from liquid to solid, mortality typically occurs at temperatures above the SCP of an insect, and SCP is not always a good predictor of intrinsic cold tolerance (Lombardero et al., 2000; Renault et al., 2002). Cold tolerance can be highly dynamic within a single species (Régnière & Bentz, 2007); for example, a lower SCP may occur when beetles were previously cold-hardened compared with LMC Ambrosia beetles in the Xyleborini typically have arrhenotokous (haplodiploid) sex determination, in which fertilized eggs develop into diploid females and unfertilized eggs develop into haploid males (Kirkendall, 1993; Peer & Taborsky, 2005), and also have extreme sex ratios as characteristically found in LMC, in which inbreeding is the rule (Hamilton, 1967). Brothers generally mate with their sisters within the natal galleries prior to female dispersal, and males are flightless and unlikely to disperse. Initially, it would appear that these species exhibit LMC. Hamilton (1967) described eight biofacies defining LMC as having: (i) a female-biased sex ratio; (ii) arrehnotokous reproduction; (iii) at least one male in every batch of offspring; (iv) gregarious development; (v) adult males eclosing first and mating many times; (vi) mating occurring before, during or immediately after adult female eclosion; (vii) males not dispersing; and (viii) females storing sperm from one insemination to fertilize her entire egg production. These Euwallacea species adhered to some but not all of the criteria strictly. Of the listed criteria, they do have extremely female-biased sex ratios (i); arrhenotokous reproduction (ii);

11 Biology and rearing of Euwallacea ambrosia beetles 233 Figure 6 The development of two Euwallacea spp. [polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB)] showing average number of each stage per colony, harvested at different ages. The number of colonies used for each age and species are provided (bottom left). Note differences in the scale of the y-axes across the different stages. gregarious development (iv); and males capable of inseminating an entire brood of sisters, with sperm stored by a mated female capable of inseminating an entire clutch of eggs (viii). Although males were capable of multiple matings, we found they were not strictly the first to eclose in either species (v). In addition, approximately 14% of clutches of both PSHB and TSHB did not contain a male (iii). The phenomenon of male-less broods has been reported in other LMC xyleborines, and at similar frequencies of approximatey 14% (Entwhistle, 1964; Peer & Taborsky, 2004; Biedermann, 2010). This, together with the fact that we collected PSHB males walking on the bark of trees (despite being wingless) represents potential evidence of male migration and outbreeding, another violation (vii). Male migratory behaviour in E. fornicatus sensu lato was previously documented in Sri Lanka by Calnaido (1965). It is not clear whether females mated immediately upon eclosion (vi) because we discovered that, in six cases, mature PSHB females removed from their natal colony produced only male offspring, suggesting they were still virgins. In another xyleborine X. germanus, females were found to adjust the sex ratio of their broods as predicted by LMC when there are more foundresses present (Peer & Taborsky, 2004). However, the sex ratio of PSHB was not significantly affected by the number of foundresses, and alternative explanations for this species should be considered. One possibility is that multiple females in a rearing tube maintained separate galleries, thus avoiding mixing of offspring (Kirkendall, 1993). However, this does not explain the other violations, and thus LMC may not suitably describe the traits of these species.