Agricultural and Forest Entomology (2016), 18, 223–237 DOI: 10.1111/afe.12155 Biology of two members of the species complex (Coleoptera: : Scolytinae), recently invasive in the U.S.A., reared on an ambrosia 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 , Riverside, CA 92521, U.S.A., ‡Tropical Research and Education Center, University of Florida–IFAS, 18905 SW 280 ST, Homestead, FL 33031, U.S.A., §Department of 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 Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, each carrying a different species of symbiotic fungus, in the , 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 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 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, diet, local mate competition, polyphagous shot hole borer, sex ratio, tea shot hole borer.

Introduction 1868). Long considered the most serious pest of tea in Sri Lanka (Gadd, 1941; Walgama & Pallemulla, 2005), this pest species Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: originates from southeast Asia, occurring in countries from Sri Scolytinae), the tea shot hole borer (TSHB), was first described from Sri Lanka in 1868 (as fornicatus) (Eichhoff, Lanka to Taiwan, and is known for its broad host range and dis- tribution, as well as numerous invasions around the world, such Correspondence: Miriam F. Cooperband. Tel.: +1 508 563 0934; fax: as , California, Florida, Israel, Australia, Africa, Vanuatu, +1 508 563 0903; e-mail: [email protected] Panama, Costa Rica and many more (Danthanarayana, 1968;

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. 224 M. F. Cooperband et al.

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

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 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 origina

Aceraceae s.s (Sapindaceae s.l.) Acer buergerianum Trident maple As Acer macrophyllum Big leaf mapleb NA Acer negundo Box elderb NA Acer palmatum Japanese maple As Acer paxii Evergreen maple As Aquifoliaceae Ilex cornuta Chinese holly As Betulaceae Alnus rhombifolia White alderb NA Euphorbiaceae Ricinus communis Castor bean Af, Eu, As Acacia sp. Acacia — Albizia julibrissin Mimosa or silk tree As Castanospermum australe Moreton Bay chestnut or blackbean Oe Cercidium floridum ( florida) Blue palo verdeb NA Cercidium x sonorae Brea NA Erythrina corallodendron Coral tree As Parkinsonia aculeata Palo verde SA Prosopis articulata Mesquiteb NA Wisteria floribunda Japanese wisteria As Fagaceae Fagus crenata Japanese beech As Quercus agrifolia Coast live oakb NA Quercus engelmannii Engelmann oakb NA Quercus lobata Valley oakb NA Quercus robur English oak Af, Eu Quercus suber Cork oak Af, Eu Hamamelidaceae s.l. (Altingiaceae s.s.) Liquidambar styraciflua American sweetgum NA 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 sycamoreb NA Platanus x acerifolia London plane tree Eu, As Salicaceae Populus fremontii Fremont cottonwoodb NA Populus trichocarpa Black cottonwoodb NA Salix babylonica Babylon willow or weeping willow or tortuosa As Salix gooddingii Goodding’s black willowb NA Salix laevigata Red willowb NA Sapindaceae Alectryon excelsus Titoki Oe Simaroubaceae Ailanthus altissima Tree of heaven As Theaceae Camellia semiserrata Camellia As aThe 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. bNative 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 Initially Avocado Persea americana (Lauraceae) sawdust successful laboratory mass rearing techniques for two members was unavailable, and so American beech Fagus grandifolia of the E. fornicatus species complex (PSHB and TSHB) and (Fagaceae) and boxelder Acer negundo (Sapindaceae) were report the findings of studies conducted aiming to characterize considered because they were listed as hosts in the litera- basic biological attributes such as the developmental period, ture (Danthanarayana, 1968; Eskalen et al., 2013). As species phenology and sex ratios of both species, as well as the effects became available, PSHB and TSHB were both eventually tested of mating, foundress number and cold tolerance for PSHB. with all three sawdust types (boxelder, beech and avocado) and reared successfully, although the decision was made to continue Materials and methods to rear each species on the same host that it was originally collected from in the field: PSHB on boxelder and TSHB on Diet avocado. A sawdust-based diet was made using methods described in Beech and boxelder trees in Massachusetts and Illinois were Castrillo et al. (2011), with some minor modifications (see felled and peeled, and bolts of wood were allowed to dry at below). Sawdust from three different tree species was tested. room temperature for 2–4 weeks. A miter saw was used to make

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 226 M. F. Cooperband et al. multiple cuts into the sapwood of the bolts, avoiding the heart- P526P-13-01673) for further rearing and research within the wood, and the resulting sawdust was collected. Avocado bolts insect containment facility. From these original beetles, the were cut in Florida and allowed to dry. A sander equipped with laboratory stock population that we refer to as PSHB was a vacuum bag was used to make sawdust out of the avocado established. sapwood layer. For all tree species, the heartwood was dis- carded. Large particles were removed from sawdust by passing it through a No. 12 sieve (Hogentogler & Co. Inc., Columbia, TSHB from Florida Maryland). To improve the texture of the boxelder sawdust, a coffee grinder was used to produce a finer sawdust powder. On 5 March 2014, two E. sp. #2 (TSHB) females were dissected Sawdust was packaged and stored in the freezer (−18 ∘C) until out of an infested avocado branch in Miami-Dade County in needed. Florida by Daniel Carrillo. The beetles were surface-sterilized Ingredients were combined in a 600-mL glass beaker: 45 g and placed individually into tubes containing avocado diet. of sawdust (boxelder, beech or avocado), 12 g of agar (Difco Colonies were successfully established and sent to the insect Agar, granulated, Becton, Dickinson and Company, Franklin containment facility at the Otis Laboratory (USDA permit # Lakes, New Jersey), 6 g of sucrose (Bio-Serv, Flemington, New P526P-13-01673) for further rearing and research. From these Jersey), 3 g of casein (Fonterra, Rosemont, Illinois) , 3 g of potato original beetles, the laboratory stock population we refer to as starch (Bio-Serv, Flemington, New Jersey), 3 g of brewer’s yeast TSHB was established. (MP Biomedicals, Santa Ana, California), 0.6 g of Wesson’s salt Rearing tubes containing different beetle species were housed mix (Frontier Agricultural Sciences, Newark, Deleware), 0.21 g in separate containers, which were never opened at the same time of tetracycline (Tetrasol, Med-Pharmex, Pomona, California), to isolate colonies from each other. 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. Verification by DNA Then the oil and ethanol was stirred in, followed by the water. In addition to identification using a key to species for The beaker containing the mixture was covered with foil and E. fornicatus et al. ∘ (Rabaglia , 2006), once laboratory stock autoclaved for 25 min at 121 C and approximately 15 psi. populations of PSHB and TSHB were established in the labora- Tools were also autoclaved, the hood was surface sterilized tory, one female descended from each colony line (eight PSHB with 70% ethanol and lined with aluminum foil, and trays of and two TSHB) was preserved in 95% ethanol and sent to the single-use 50-mL sterile polyethylene centrifuge tubes (Fisher Stouthamer laboratory for genetic confirmation of the cryptic Scientific) were placed in the flow hood and ultraviolet (UV) species. This was achieved by sequencing a section of the surface-sterilized for 45 min. The hot diet was removed from cytochrome c oxidase subunit I (COX I) gene commonly used the autoclave and stirred well to resuspend settled ingredients. in barcoding studies in accordance with protocols described in Working quickly, approximately 15 mL of hot diet was poured Eskalen et al. (2013). All analyzed specimens originating from into each tube. Tubes were loosely capped and tapped to remove the PSHB and TSHB colonies were confirmed as PSHB and air bubbles. Loosely capped tubes of diet were allowed to dry TSHB, respectively, by comparison with GenBank accession in the flow hood and were checked daily for condensation, numbers JX912723–JX912725. 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 Initiation of experimental colonies needed. This volume of diet yielded approximately 25 diet tubes, each containing 15 mL of diet. 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 PSHB from California windows for air exchange, and nested within larger containers Between 10.00 h and 12.00 h on 9 August 2013, beetles were to keep colonies apart, prevent escapes and reduce the risk of collected from a heavily infested boxelder tree in Altadena, contamination by mites or foreign fungi (Rectangular Storage California. Thirteen female and three male PSHB that were Container, Lock & Lock U.S.A., Anaheim, California; Nylon walking on the bark were hand-collected and placed into a 2-mL Mesh 500 μm, 47% open area, Component Supply Co., Fort glass vial with some tissue paper. They were transported to the Meade, Florida). The protocol for handling beetles was as nearby laboratory at the Huntington Library, Art Collections described previously by Castrillo et al. (2011). To allow air and Botanical Gardens in San Marino, California, where each exchange in diet tubes but prevent escapes, a hole was drilled beetle was surface-sterilized by emersion in 70% ethanol for into the lid of each diet tube (diameter 4 mm) and a fine stainless 10 s. Beetles were then dried for 5 s on a piece of filter paper, steel mesh (200 × 200, 33.6% open area) was secured over the and placed, individually or in groups, in 10 rearing tubes hole using a hot glue gun. containing boxelder diet. Surface sterilization was conducted A typical colony was initiated by placing a single mated to prevent microbes, other than the fungi protected inside foundress in a rearing tube with diet. Exceptions to this the mycangia, from colonizing the diet. The newly founded (described below) were required for the investigation of hap- tubes were shipped to the Otis Laboratory (USDA permit # lodiploid reproduction and local mate competition.

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 Biology and rearing of Euwallacea ambrosia beetles 227

(a) (b) (c)

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 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 As the colonies developed, observations were made to measure brush moistened with 70% ethanol was inserted into the corner of the health of the colony. Colonies were visually evaluated every the bag and used to remove and count the beetles, so as to prevent 1–3 days by examining the sides of the tubes to identify visible any escapes. 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 Haplodiploid reproduction compact cylinder. It was noted whether or not this excavation To confirm the presence of haplodiploid reproduction, andto material was present and, if so, the length of the ejected material determine whether unmated females were capable of initiating was measured using a 5-mm marker for reference (Fig. 1). new colonies by first producing male offspring (from unfertilized The length of excavation material was presumed to be an eggs) and then mating with those ‘sons’ to subsequently produce indication of the extent of gallery formation. The number of eggs, a brood of female offspring, experimental colonies were initiated larvae, pupae and adults on the surface and within any visible using virgin females. To ensure that females were virgins, female galleries was noted. The sex of pupae and adults was recorded pupae were collected from the laboratory stock populations and and, if adult females were found, it was noted whether they placed in an empty rearing tube until adult eclosion. Because were teneral (cuticle was white to brown) or fully sclerotized newly eclosed adults need to acquire their symbionts, diet (cuticle was black). The time and date was recorded for all with Fusarium was taken from an established healthy colony observations. 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, Dissection of colonies individual virgin females were surface-sterilized, transferred to clean diet tubes and allowed to naturally inoculate the sterile diet Colonies were destructively analyzed at various ages to quantify with their symbionts. Tubes were monitored for up to 13 weeks the development of all stages over time. Each tube containing after which they were destructively analyzed and offspring were a beetle colony was systematically dissected under a stereomi- quantified. croscope. 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, Sex ratio and local mate competition and so on. To dissect a tube, all beetle stages that were above the diet surface were removed and counted first. Then the diet Local mate competition (LMC) theory predicts that species with plug was tapped out into a re-sealable zipper storage bag where a high propensity for inbreeding (e.g. shot hole borers) should it was examined under a dissecting microscope. Once all - produce extremely female-biased sex ratios (Hamilton, 1967). tle stages on the surface of the diet plug were removed and As the number of foundresses increases (and the chances of counted, the diet was systematically chipped away in small bits outbreeding increases), sex ratios are expected to approach a starting at the bottom of the plug. As galleries were revealed, more ‘normal’ 1 : 1 ratio (Fisher, 1958; Hamilton, 1967; Nunney they were followed and the diet around them was removed in & Luck, 1988). To test whether PSHB possess this attribute of small increments to allow any beetle stages within to be collected LMC, we evaluated the sex ratio of offspring when one foundress and quantified. This technique was followed until the entire diet or multiple foundresses (two to five) were used to initiate a

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 228 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 Figure 2 Average excavation material (mm) ejected per rearing tube per week of development by either a mated or virgin polyphagous shot hole The cold-hardiness of PSHB was investigated by exposing borer (PSHB) female in boxelder diet. A total of 80 mated colonies and colonies to different low temperatures. Thirty PSHB colonies 6 virgin colonies, respectively, were monitored several times per week. were initiated by a single foundress and allowed to develop for The number of measurements each week was averaged (e.g. in week 35 days in the environmental chamber at 24 ∘C. At day 35, eleven 1, there were 173 and 21 measurements for mated and virgin colonies, colonies that did not appear to be thriving were discarded and respectively). Bars represent the SE. the remaining 24 colonies were divided into five groups. Four colonies were kept at 24 ∘C (control). The remaining colonies Results were divided into four temperature treatments (n = 5) and placed on a rack. A diet tube without beetles was placed in the centre of Entry holes and excavation the rack with a traceable refrigerator/freezer alarm thermometer The three most prominent signs of a healthy colony were the (Fisher Scientific) inserted into the diet to monitor internal diet growth of the white Fusarium, which ultimately carpeted the temperature. The rack was placed in a freezer at −15 ∘Candan exposed surface of the diet, the presence of one or more fresh alarm sounded when the diet in the monitoring tube reached 5, entry holes, and the presence of excavation material ejected from 1, −1and−5 ∘C, at which time the five tubes in each respective the entry holes (Fig. 1). Entry holes that were not in use would group were removed from the freezer and reunited with the soon become covered over by Fusarium and appear closed or control group at 24 ∘C. An additional temperature probe in the disappear altogether. freezer monitored the temperature outside of the diet. The time Excavation material and entry holes were measured for 80 elapsed between each temperature was recorded. After cold PSHB colonies each founded by a single mated female, and six treatments, the colonies were allowed to recover for 24 h at colonies each founded by a single virgin female, and recorded on 24 ∘C. The next day, all tubes were dissected and the number of average two to three times per week. Colonies founded by mated dead and living larvae, pupae, and adults were quantified. Eggs females initially produced more excavation material than those were excluded because we were unable to visually determine started by virgin females, which lagged by 2–3 weeks (Fig. 2). their health. Dead larvae and pupae were discoloured and Wilcoxon tests revealed significant differences between mated flattened. Adults were considered dead if they lacked movement. and virgin PSHB in the amount of excavation material ejected Mortality rate was calculated as (1 – percentage survival) of in week 2 (𝜒 2 = 4.46, P = 0.0347) and week 5 (𝜒 2 = 15.99, larvae, pupae and adults, and compared for each treatment P < 0.0001). The amount of excavation material peaked for and stage. 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 Statistical analysis second peak was likely suppressed as a result of the diminished Linear regression models were used to predict relationships population capacity and quality of the aging diet. between offspring and entry holes or excavation material (jmp, The average number of active entry holes per colony, founded version 10.0.0; SAS Institute, Cary, North Carolina). Analy- either by a single mated or virgin PSHB, was plotted by week ses comparing sets of data that passed the test for normal- (Fig. 3). In the first 4 weeks, the average number of entry ity were analyzed using analysis of variance (anova) (jmp, holes ranged between 0.6 and 1.0 for both treatments. The first version 10.0.0). For analyses comparing two sets of data that increase in number of entry holes in both mated and virgin were nonparametric, datasets were compared using the Wilcoxon treatments occurred between weeks 4 and 5, corresponding to test (jmp, version 10.0.0). For statistical analyses compar- emerging offspring, and both sexes were observed to partici- ing multiple datasets that failed to satisfy the assumptions of pate in excavation activities. The number of entry holes was anova, data were rank transformed, and then analyzed using similar in the first 5 weeks, after which they started to diverge. anova and Tukey’s mean separation test (sas, version 9.3; Wilcoxon tests found significant differences in the number of SAS Institute Inc., Cary, North Carolina; jmp, version 10.0.0) entry holes between mated and virgin PSHB foundresses in week (Conover, 1980). 3(𝜒 2 = 4.50, P = 0.0338), week 8 (𝜒 2 = 18.806, P < 0.0001),

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 Biology and rearing of Euwallacea ambrosia beetles 229

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 genera- tion 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 off- Figure 3 Average number of fresh entry holes observed per colony per spring from both single and multiple mated foundresses was not week in colonies founded by a single mated (solid) or virgin (dotted) female polyphagous shot hole borer (PSHB) foundress in boxelder diet significantly affected by the age grouping (one generation versus over a period of 11 or 13 weeks, respectively. A total of 80 mated colonies two generations), although the limitations of the diet may have and 6 virgin colonies, respectively, were monitored several times per been reached. Conversely, single TSHB foundresses had fewer week. The number of observations for each week was averaged (e.g. first-generation offspring than PSHB (Wilcoxon test, 𝜒 2 = 4.93, in week 1, there were 188 and 21 measurements for mated and virgin P = 0.0264) and generation had a significant effect on the num- colonies, respectively). Bars represent the SE. ber of female offspring (Wilcoxon test, 𝜒 2 = 5.10, P = 0.0239) (Tables 3 and 4). Generation also had a significant effect on the week 9 (𝜒 2 = 13.572, P = 0.0002) and week 10 (𝜒 2 = 15.972, number of female offspring of virgin PSHB colonies (Wilcoxon 𝜒 2 P < 0.0001). test, = 4.51, P = 0.0337) because the first generation of off- For each colony, the maximum number of active entry holes spring contained no females. was compared with the total number of male and female off- spring harvested. Because the sex of pupae could also be determined, they were included in the tally. Entry hole num- Sex ratio and haplodiploidy ber was found to be a significant predictor of the number Virgin foundresses did not initially produce female offspring; of PSHB offspring in a colony (Fig. 4) (linear regression, r2 = 0.6148, P < 0.0001 for females; r2 = 0.1153, P = 0.0033 for rather, they produced sons with which they mated and subse- males; n = 73). Excavation material was also found to be signif- quently produced daughters. Although there appeared to be a icantly correlated with the number of PSHB offspring, although trend for older PSHB colonies to have higher sex ratios (% it was not as strong a predictor as entry holes (linear regression, males), there was no significant difference in sex ratio between r2 = 0.4748, P < 0.0001 for females; r2 = 0.0787, P = 0.0162 for colonies of different ages, species or foundress number (Tables 3 males; n = 73). Similarly, the linear regression model for TSHB and 4). The only factor that affected sex ratio was the mating sta- offspring to the number of entry holes is presented in Fig. 5 tus of the foundress, in which colonies initiated by virgin females (r2 = 0.6916, P < 0.0001 for females; r2 = 0.3405, P < 0.0001 were 100% male in 5–8-week-old colonies, and remained pre- for males; n = 46). As with PSHB, the relationship between the dominantly male in older colonies. On average, when harvested number of TSHB offspring and excavation material was weaker between 5 and 8 weeks, typical mated single-foundress PSHB than with entry holes (r2 = 0.4145, P < 0.0001 for females; and TSHB colonies had average sex ratios of 7.4% and 7.2%, r2 = 0.4670, P < 0.0001 for males; n = 46). 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. Offspring development Whether these foundresses failed to produce males or their male The first day that each stage was observed either on the diet offspring died prematurely and were concealed by fungus prior surface or by examining galleries along the rearing tube walls to dissection is not known, and so these colonies are reported of any colony founded by a mated or virgin female PSHB separately (Table 3). is presented in Table 2. This observational approach was a In colonies dissected between 14 and 28 days, no adults were nondestructive way of evaluating the timing of the different found prior to day 22 (Fig. 7). The colonies in which only the first stages. With mated foundresses, eggs and larvae were first adult had emerged were aged 22–24 days. Unexpectedly, the first observed 3–4 days earlier than with virgin foundresses, although adult to emerge was not always a male as predicted by LMC. Of the first male was observed 2 days later. The first appearance of six PSHB rearing tubes aged 22–24 days old, two had a female daughters in the virgin treatment was 18–19 days later than the emerge first on day 22 and one had a male emerge first onday first appearance of daughters in the mated treatment, presumably 24. Of six TSHB rearing tubes aged 22–24 days old, two had a after a haploid son matured and mated with the foundress. female emerge first, one on day 22 and the other on day 23.The Colonies were dissected from each week of development, and remaining tubes examined had either no adult emergence yet, or the numbers of eggs, larvae, male pupae, female pupae, male both sexes had already emerged.

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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 Discussion The time that it took to drop to target temperatures resulted Biology and rearing ∘ in rates of temperature change of 0.08–0.25 C/min for each The two newly invasive species of the E. fornicatus species treatment. Dissections of colonies exposed to different tem- complex found in Los Angeles County, California (PSHB), and peratures revealed that one tube from each of the 5, −1and Miami-Dade County, Florida (TSHB), were similar with respect −5 ∘C treatments contained no offspring, reducing the num- to their biology and rearing capabilities. The first successful ber for those treatments. There were significant differences in rearing attempt on artificial diet of scolytine ambrosia beetles mortality between temperature exposures for larvae (anova of was described by Saunders and Knoke (1967) using Xyleborus ranked data: F = 3.22, P = 0.0354), pupae (F = 4.34, P = 0.0117) ferrugineus (Fabricius) and its Fusarium symbiont. By omission and adults (F = 4.44, P = 0.0106) (Fig. 8). Mortality increased of key ingredients, they that found cellulose powder or fresh host significantly with a lower temperature and longer cold exposure, sawdust was essential, and the importance of wood vitamins was for all stages. Larvae and pupae appeared to be more sensitive further demonstrated by Norris and Baker (1968). Diet variations have similarly been tested for rearing a member of the E. for- to cold than adults, as indicated by their higher mortality rates at nicatus species complex (Sivapalan & Shivanandarajah, 1977), the lowest temperatures. After exposure to −5 ∘C, 100% of lar- showing that cellulose powder, salt mixture and yeast extract vae, 95.7% of pupae and 69.2% of adults died. Of those surviving were important for members of this group. Similar diets have also adults, many were on their backs, moving their legs only slightly been developed for other species of ambrosia beetles (Mizuno & ∘ after the 24-h recovery period. Exposure to −1 C produced sim- Kajimura, 2002, 2009; Peer & Taborsky, 2004; Castrillo et al., ilarly high levels of mortality, whereas most individuals survived 2011; Maner et al., 2013). Based on a diet for rearing Xylosan- the 0 and 5 ∘C treatments, and those treatments did not differ drus germanus (Blandford) (Peer & Taborsky, 2004; Castrillo from controls. et al., 2011), the diets in the present study were developed using

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

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Table 2 Day (and week) of the earliest observed life stages of PSHB those that were not (Formby et al., 2013), and may also vary offspring, either from a virgin or a mated foundress, and the difference in with the exposure time or rate of cooling (Kostal et al., 2011). developmental time between the two The slow rate of cooling used in the present study may have allowed for a rapid cold-hardening response (Lee, 1989; Kelty Day (week) of earliest Difference in & Lee, 1999). Our empirical study of mortality rates for var- surface observation development times (days) between ious stages inside galleries exposed to different low tempera- Mated Virgin mated and virgin tures and exposure times found that PSHB was somewhat tol- Offspring stage foundress foundress foundress erant to brief exposures of near freezing temperatures. When ∘ Egga 17 (3) 21 (3) 4 temperatures were reduced to 5 and 0 C, although some mor- Larva 13 (2) 16 (3) 3 tality was observed, this was not significantly different from Male pupa 24 (4) 22 (4) −2 the control group. However, when diet temperatures dropped Male adult 27 (4) 25 (4) −2 below freezing and for longer time periods, mortality was sig- Female pupa 20 (3) 38 (6) 18 nificantly affected. PSHB survival at and above0 ∘C in the lab- Female adult 24 (4) 43 (7) 19 oratory suggests they can survive similar temperatures inside Number of colonies 269 45 trees. observed Temperatures inside overwintering trees, particularly in the aDoes not include concealed offspring inside galleries, and so outer xylem and the centres of overwintering trees, may be much the first observation of eggs did not define the first presence of higher than winter temperatures outside of trees, and tempera- eggs. tures in the sapwood, where these beetles mostly reside, can be Observations were conducted nondestructively for 8 weeks by examining much higher than temperatures at the centres of trees (Sakai, the surface of the diet and inside any galleries that were visible through 1966). When negative air temperatures occur, temperatures in the sides of the rearing tubes. the sapwood may remain well above freezing and be able to safely harbour these beetles. Internal tree temperatures fluctuate and 7.2% males, respectively. Colony sex ratio was not affected greatly and are influenced by solar radiation, the face of the tree by the number of foundresses. being measured, the diameter of the tree, bark colour and thick- Freeman et al. (2013a) studied groups of PSHB placed on a ness, and tree species. Therefore, PSHB may be able to survive lawn of F. euwallaceae on potato dextrose agar (PDA) in Petri in regions with harsher winters than their current distribution in dishes and observed development times to the first observation southern California if internal tree temperatures can safely har- of each developmental stage, including three larval instars, bour them. Although phloem temperatures in pine forests have incubated at 25 ∘C. They recorded a slightly longer overall been investigated to predict overwintering capabilities of bark development time than that observed in the present study at beetles (Tran et al., 2007), more information on the internal over- 24 ∘C. They found that time to oviposit was greatly affected wintering temperatures of preferred host trees of PSHB, such as by natal host experience, depending on whether the foundress boxelder, under various growing conditions would improve our had been reared on PDA, in which case oviposition was greatly ability to estimate the potential range of these beetles and similar delayed, or in avocado branches. species.

Cold tolerance LMC Cold tolerance plays a crucial role in the population dynamics of Ambrosia beetles in the typically have arrhenotok- the scolitine Dendroctonus ponderosae (Régnière & ous (haplodiploid) sex determination, in which fertilized eggs Bentz, 2007) and may be equally important for ambrosia bee- develop into diploid females and unfertilized eggs develop tles despite the paucity of cold tolerance studies on them. To into haploid males (Kirkendall, 1993; Peer & Taborsky, 2005), our knowledge, the present study is the first aiming to quan- and also have extreme sex ratios as characteristically found tify survival at low temperatures in an ambrosia beetle. Another in LMC, in which inbreeding is the rule (Hamilton, 1967). invasive xyleborine ambrosia beetle, ,was Brothers generally mate with their sisters within the natal found to have temperature-dependent development (Brar et al., galleries prior to female dispersal, and males are flightless 2015), and it is the only ambrosia beetle for which the supercool- and unlikely to disperse. Initially, it would appear that these ing point (SCP) was determined (Formby et al., 2013). The SCP species exhibit LMC. Hamilton (1967) described eight biofacies has also been explored in a number of bark beetles (Lombardero defining LMC as having: (i) a female-biased sex ratio; (ii) et al., 2000; Régnière & Bentz, 2007; Kostal et al., 2011; Lester arrehnotokous reproduction; (iii) at least one male in every & Irwin, 2012). However, because SCP is the temperature at batch of offspring; (iv) gregarious development; (v) adult males which the body fluids change phase from liquid to solid, mortal- eclosing first and mating many times; (vi) mating occurring ity typically occurs at temperatures above the SCP of an insect, before, during or immediately after adult female eclosion; and SCP is not always a good predictor of intrinsic cold toler- (vii) males not dispersing; and (viii) females storing sperm ance (Lombardero et al., 2000; Renault et al., 2002). Cold tol- from one insemination to fertilize her entire egg production. erance can be highly dynamic within a single species (Rég- These Euwallacea species adhered to some but not all of the nière & Bentz, 2007); for example, a lower SCP may occur criteria strictly. Of the listed criteria, they do have extremely when beetles were previously cold-hardened compared with female-biased sex ratios (i); arrhenotokous reproduction (ii);

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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 clear whether females mated immediately upon eclosion (vi) an entire brood of sisters, with sperm stored by a mated female because we discovered that, in six cases, mature PSHB females capable of inseminating an entire clutch of eggs (viii). Although removed from their natal colony produced only male offspring, males were capable of multiple matings, we found they were suggesting they were still virgins. In another xyleborine not strictly the first to eclose in either species (v). In addition, X. germanus, females were found to adjust the sex ratio of their approximately 14% of clutches of both PSHB and TSHB did broods as predicted by LMC when there are more foundresses not contain a male (iii). The phenomenon of male-less broods present (Peer & Taborsky, 2004). However, the sex ratio of PSHB has been reported in other LMC xyleborines, and at similar was not significantly affected by the number of foundresses, frequencies of approximatey 14% (Entwhistle, 1964; Peer & Taborsky, 2004; Biedermann, 2010). This, together with the and alternative explanations for this species should be consid- fact that we collected PSHB males walking on the bark of ered. One possibility is that multiple females in a rearing tube trees (despite being wingless) represents potential evidence of maintained separate galleries, thus avoiding mixing of offspring male migration and outbreeding, another violation (vii). Male (Kirkendall, 1993). However, this does not explain the other migratory behaviour in E. fornicatus sensu lato was previ- violations, and thus LMC may not suitably describe the traits of ously documented in Sri Lanka by Calnaido (1965). It is not these species.

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Table 3 Average numbers of eggs, larvae, adults, and males and females (adults and pupae), and the sex ratio (percentage males), in colonies initiated either by a single foundress or multiple (two to five) foundresses, either mated or virgin

Average number of offspring per colony Initial mating status Number of Sex of foundress Speciesa foundresses Generationsb Eggs Larvae nc Pupae Adults Femalesd Malesd ratiod (%) ne

Mated, produced PSHB 1 First 31.8 4.3 53 7.6 34.6 32.4 2.1 7.4 54 male offspring 1 Second 22.3 7.8 8 4.5 30.8 27.1 3.8 12.6 13 2–5 First 33.2 4.6 9 2.6 34.6 32.2 2.4 7.8 10 2–5 Second 41.7 0 3 0 33.0 29.6 3.4 13.9 5 TSHB 1 First 27.5 4.3 21 7.8 26.3 24.7 1.6 7.2 26 1 Second 19.8 0 4 3.5 50.5 47.3 3.3 6.8 4 Mated, only female PSHB 1 First 11.8 3.7 6 3.0 7.9 7.9 0 0 9 offspring found 1 Second 12.0 0 1 4.5 15.5 15.5 0 0 2 5 First 18.0 0 1 0 19.0 19.0 0 0 1 5 Second 29.0 0 1 0 34.0 34.0 0 0 1 TSHB 1 First 7.8 3.0 4 1.6 9.2 9.2 0 0 5 Virgin PSHB 1 First 9.0 15.7 3 2.3 9.0 0 9.0 100 3 1 Second 17.3 1.0 3 3.1 19.4 7.7 11.7 64.5 7 2 Second 14.7 0.7 3 8.3 23.7 5.0 18.7 80.5 3 aPSHB, polyphagous shot hole borer; TSHB, tea shot hole borer. bGeneration of offspring is defined by the colony age at the time of dissection: first = 5–8 weeks old, second = 9–13 weeks old. cNumber of colony vials used to quantify eggs and larvae when the colony was dissected. dPupae and adults combined. eNumber of colony vials used to quantify pupae and adults during dissection of each colony including some that were removed prior to dissection. Colonies harvested at 5–8 weeks likely included only one generation, whereas colonies harvested at 9–13 weeks could have included offspring from a second generation. Colonies that produced female but no male offspring were analyzed separately.

Table 4 The number of female offspring (Table 3) was significantly different depending on the combination of variables and the factor being tested

Independent variables Significant differences Comparison Status Species Foundress number Generation 𝜒2 P

Species: PSHB versus TSHB Mated — Single First 4.93 0.0264 Species: PSHB versus TSHB Mated — Single Second - - Generation: first versus second Mated PSHB Single — - - Generation: first versus second Mated PSHB Multiple — - - Generation: first versus second Mated TSHB Single — 5.10 0.0239 Foundress number: single versus multiple Mated PSHB — First - - Foundress number: single versus multiple Mated PSHB — Second - - Generation: first versus second Virgin PSHB Single — 4.51 0.0337 Foundress number: single versus multiple Virgin PSHB — Second - - Status: virgin versus mated — PSHB Single First 8.39 0.0038 Status: virgin versus mated — PSHB Single Second 7.72 0.0055 Status: virgin versus mated — PSHB Multiple Second - -

Test statistics for the significantly different factors are specified (Wilcoxon test). -, the factors were not significantly different.

A possible alternative to LMC could be that interactions ratio (Tang et al., 2014). Similar to findings reported for Xyle- between nest mates are mutually beneficial and represent borinus saxeseni (Peer & Taborsky, 2007), we saw no increase quasi-social behaviours including forms of parental care, as well in oviposition after the appearance of adult offspring, supporting as care of siblings (Kirkendall et al., 1997; Peer & Taborsky, the notion that cooperative breeding may also take place in these 2007). Forms of maternal care or cooperative brood care species. have been found previously in xyleborine ambrosia beetles, Considering the number of LMC violations, it appears that and suggest a level of sociality. Such behaviours include this group does not conform well with LMC theory, and may post-ovipositional blocking of the gallery entrance, fungus be better explained by such alternative theories relating to farming, cropping symbiotic fungus to avoid overgrowth of early forms of sociality. Unlike most ambrosia beetles, PSHB galleries, rolling of eggs and larvae to patches of food inside are found attacking healthy trees rather than dead or weak- galleries, feeding, extending galleries, and clearing the galleries ened trees, and so they may rely more on each other for of larval waste. If such social behaviours extend to the roles of mass attack to overcome their host (Kirkendall et al., 1997). adult daughters caring for their sisters prior to dispersal, then Because individual success may be enhanced by the success the reproductive value of daughters would increase and local of siblings or the larger group on a resource patch, addi- resource enhancement might help explain the female-biased sex tional considerations to explain the female-biased sex ratio

Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237 Biology and rearing of Euwallacea ambrosia beetles 235

are grateful to Kerry O’Donnell and Tracy Sink for genetic confirmation of Fusarium euwallaceae. Mention of a com- mercial product does not constitute its endorsement by the USDA.

References

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Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Agricultural and Forest Entomology, 18, 223–237