Biology of two members of the Euwallacea fornicatus ......Biology and rearing of Euwallaceaambrosia beetles 225 Table 1 An updated list of the 39 plant species in 16 families that

Agricultural and Forest Entomology (2016), 18, 223–237 DOI: 10.1111/afe.12155

Biology of two members of the Euwallacea fornicatus speciescomplex (Coleoptera: Curculionidae: Scolytinae), recently invasivein 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††, John D. 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, 18905 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 fornicatusEichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, eachcarrying a different species of symbiotic fungus, in the genus Fusarium, which theyfarm within galleries inside woody hosts. Several of these beetle species have becomeinvasive pests around the world for attacking and infecting healthy trees with theirphytopathogenic fungal symbionts.

2 Diet and rearing protocols were developed for two members of the E. fornicatusspecies complex, polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB),using sawdust from host trees, allowing collection of data on beetle biology, phenologyand sex ratios. Adults developed within 22 days at 24 ∘C. Single PSHB or TSHBfoundresses averaged 32.4 and 24.7 adult female offspring, respectively, and up to 57and 68 female adults within 6–7 weeks. A strong predictor of the number of offspringin a colony was the number of entry holes. Average sex ratios (% male) for PSHB andTSHB, respectively, were 7.4% and 7.2%.

3 Being haplodiploid, virgin PSHB foundresses were able to produce and mate withmale offspring, then subsequently produce female offspring, confirming that they havearrhenotokous reproduction.

4 A cold tolerance study found significant mortality rates among PSHB colonies exposedto −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 predictionswere violated. PSHB sex ratios were not affected by the number of foundresses;approximately 14% of broods did not contain males; males did not usually eclosebefore females but eclosed around the same time (22–23 days); and PSHB maleswere found walking outside of their natal galleries on the trunk of a heavily infestedtree 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, localmate 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 describedfrom Sri Lanka in 1868 (as Xyleborus fornicatus) (Eichhoff,

Correspondence: Miriam F. Cooperband. Tel.: +1 508 563 0934; fax:+1 508 563 0903; e-mail: [email protected]

1868). Long considered the most serious pest of tea in Sri Lanka(Gadd, 1941; Walgama & Pallemulla, 2005), this pest speciesoriginates from southeast Asia, occurring in countries from SriLanka to Taiwan, and is known for its broad host range and dis-tribution, as well as numerous invasions around the world, suchas Hawaii, California, Florida, Israel, Australia, Africa, Vanuatu,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& Maddox, 2010). However, its status as a single species hasrecently been questioned based on molecular data. Eskalen et al.(2013) were the first to draw attention to large levels of geneticdifferences between invasive populations of shot hole borersfrom Florida and California, both of which matched the mor-phological description of E. fornicatus, suggesting that theywere different species, and coining the common name for thepolyphagous shot hole borer (PSHB) in California. O’Donnellet al. (2015) subsequently conducted DNA-based phylogeneticanalyses on beetles matching the morphological description ofTSHB from invasive populations around the world, includingbeetles from Sri Lanka (where E. fornicatus was first described).Their findings suggested that E. fornicatus was a complex of atleast five morphologically indistinguishable species (O’Donnellet al., 2015). Invasive populations in both Israel and the greaterLos Angeles region of southern California were genetically iden-tical (which they referred to as Euwallacea sp. #1) but dif-fered genetically from the invasive population in Miami-DadeCounty, Florida (Euwallacea sp. #2), and both differed froma more recently discovered invasive population in San DiegoCounty, southern California (Euwallacea sp. #5). Furthermore,all three of these beetles differed genetically from those in SriLanka (Euwallacea sp. #4, potentially the true E. fornicatus)and Australia (Euwallacea sp. #3). Molecular data suggest thatEuwallacea sp. #1 (PSHB) most likely originated from Viet-nam (R. Stouthamer, unpublished data; Lynch et al., 2016). Thegenetic differences found between beetle populations were mir-rored in the species of Fusarium symbionts that they each carry,and were sufficiently great for the the beetles to be consideredas separate species (O’Donnell et al., 2015). Fusarium euwal-laceae is the principal and obligate symbiont for PSHB (Eskalenet al., 2012, 2013; Freeman et al., 2013a; O’Donnell et al., 2015),although, recently, two additional fungal associates, Graphiumeuwallaceae and Paracremonium pembeum, were identifiedfrom PSHB mycangia (Lynch et al., 2016). The fungal associateG. euwallaceae is capable of sustaining offspring developmentwhereas the role of P. pembeum is not known (Sharon et al.,2015). However, infection by a complex of fungal associates mayimpart an increased risk to the host plant (Lynch et al., 2016).

The E. fornicatus species complex is in need of thorough taxo-nomic revision and, as such, the application of scientific names tothe constituent member species would be somewhat preliminaryand ambiguous. It also presents a challenge when interpreting thebody of literature on the biology of Xyleborus fornicatus (nowin genus Euwallacea), which is considered to be a complex ofcryptic species rather than a single species. Based on their local-ity, many of the early studies conducted on the shot hole borer oftea 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 toscience. Among the species invasive in the U.S.A., the commonname of PSHB was given to the population in Los AngelesCo., California, aiming to differentiate it from the population inMiami-Dade Co., Florida, which so far has retained the commonname of TSHB (Eskalen et al., 2013). The common namesPSHB and TSHB are used in the present study, and match withE. sp. #1 and E. sp. #2, respectively, in O’Donnell et al. (2015).

The TSHB was detected in Florida in 2002 but with minorinitial impact (Rabaglia et al., 2008), and was found in avocadogroves 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 Countyand Israel, respectively (Rabaglia et al., 2006; Mendel et al.,2012). The Israeli avocado industry sustained major loss as aresult of Fusarium dieback, a serious plant disease associatedwith PSHB (Eskalen et al., 2012; Mendel et al., 2012; Freemanet al., 2013b), and the California avocado industry has startedto experience similar losses (Eskalen et al., 2012). Additionally,thousands of other landscape and forest trees have been killedand removed in southern California as a result of Fusariumdieback. Eskalen et al. (2013) reported that, of 335 plant speciesobserved, PSHB attacked 207 species of healthy trees in 58plant families, and Fusarium was established and recovered inmore than half of those species. At the time of their study,Eskalen et al. (2013) reported 19 species as reproductive hostsin which attacks resulted in the establishment of Fusarium andcolonies with offspring. Subsequently, the list of attacked treespecies has increased to 342 taxa in 63 families (T. Thibault andA. Eskalen, unpublished data). Of these, 39 species in 16 familiesare known reproductive hosts, 13 of which are species nativeto California (Table 1) (Eskalen et al., 2013; Eskalen, 2016;http://eskalenlab.ucr.edu/avocado.html). Less is known about thefull host range of TSHB, although its range is also likely tobe extensive because E. fornicatus has been recorded attackingat least 100 different plant species, in 35 families, in India,Java, Malaysia and Sri Lanka, with at least 21 of those beingreproductive hosts (Danthanarayana, 1968). The Euwallacea sp.#5 population in San Diego has been found attacking commercialavocado groves and other hosts similar to those of PSHB(Eskalen, 2016; http://eskalenlab.ucr.edu/avocado.html).

One of the economically important reproductive hosts of allthree species in the U.S.A. is avocado, which, in Californiaalone, is an industry worth about $400 million annually(California Avocado Commission, 2015). Mexico is theworld’s top producer and exporter of avocados, exceedingthe avocado production of any other nation by more thanfour-fold, and is followed by Indonesia, Dominican Repub-lic, U.S.A., Columbia, Peru, Kenya, Chile, Brazil, Rwanda,China, Guatemala, South Africa, Venezuela, Spain and Israel(FAOSTAT, 2012; http://faostat.fao.org). The list of PSHB hoststhat are susceptible to the Fusarium pathogen also includesnumerous commonly planted urban and suburban street trees,ornamental trees in public spaces and private yards, 58 nativeNorth American tree species, and 19 native Californian treespecies. Thus, the monetary and environmental damage poten-tial is already high, and only increasing as beetle populationsalso increase in southern California. Although laboratory rear-ing on an artificial substrate in the absence of host material hasbeen conducted to determine symbiont fidelity (Freeman et al.,2013a), to our knowledge, no studies have documented thefecundity and sex ratio of these new species. Timely research isrequired to (i) improve our understanding of the biology of thesespecies; (ii) develop tools for trapping and detection; and (iii)investigate mitigation options such as biological control. Suchresearch 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 hostplant native origin

Family Species Common name Plant origina

Aceraceae s.s (Sapindaceae s.l.) Acer buergerianum Trident maple AsAcer macrophyllum Big leaf mapleb NAAcer negundo Box elderb NAAcer palmatum Japanese maple AsAcer paxii Evergreen maple As

Aquifoliaceae Ilex cornuta Chinese holly AsBetulaceae Alnus rhombifolia White alderb NAEuphorbiaceae Ricinus communis Castor bean Af, Eu, AsFabaceae Acacia sp. Acacia —

Albizia julibrissin Mimosa or silk tree AsCastanospermum australe Moreton Bay chestnut or blackbean OeCercidium floridum (Parkinsonia florida) Blue palo verdeb NACercidium x sonorae Brea NAErythrina corallodendron Coral tree AsParkinsonia aculeata Palo verde SAProsopis articulata Mesquiteb NAWisteria floribunda Japanese wisteria As

Fagaceae Fagus crenata Japanese beech AsQuercus agrifolia Coast live oakb NAQuercus engelmannii Engelmann oakb NAQuercus lobata Valley oakb NAQuercus robur English oak Af, EuQuercus suber Cork oak Af, Eu

Hamamelidaceae s.l. (Altingiaceae s.s.) Liquidambar styraciflua American sweetgum NALauraceae Persea americana Avocado NA, SAMalvaceae Brachychiton populneus Kurrajong OeMoraceae Ficus carica Black mission fig AsMyrtaceae Corymbia (Eucalyptus) ficifolia Red flowering gum OePlatanaceae Platanus mexicana Mexican sycamore NA

Platanus racemosa California sycamoreb NAPlatanus x acerifolia London plane tree Eu, As

Salicaceae Populus fremontii Fremont cottonwoodb NAPopulus trichocarpa Black cottonwoodb NASalix babylonica Babylon willow or weeping willow or tortuosa AsSalix gooddingii Goodding’s black willowb NASalix laevigata Red willowb NA

Sapindaceae Alectryon excelsus Titoki OeSimaroubaceae Ailanthus altissima Tree of heaven AsTheaceae 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, Southand 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 describesuccessful laboratory mass rearing techniques for two membersof the E. fornicatus species complex (PSHB and TSHB) andreport the findings of studies conducted aiming to characterizebasic biological attributes such as the developmental period,phenology and sex ratios of both species, as well as the effectsof mating, foundress number and cold tolerance for PSHB.

Materials and methods

Diet

A sawdust-based diet was made using methods described inCastrillo et al. (2011), with some minor modifications (seebelow). Sawdust from three different tree species was tested.

Initially Avocado Persea americana (Lauraceae) sawdustwas unavailable, and so American beech Fagus grandifolia(Fagaceae) and boxelder Acer negundo (Sapindaceae) wereconsidered because they were listed as hosts in the litera-ture (Danthanarayana, 1968; Eskalen et al., 2013). As speciesbecame available, PSHB and TSHB were both eventually testedwith all three sawdust types (boxelder, beech and avocado) andreared successfully, although the decision was made to continueto rear each species on the same host that it was originallycollected from in the field: PSHB on boxelder and TSHB onavocado.

Beech and boxelder trees in Massachusetts and Illinois werefelled and peeled, and bolts of wood were allowed to dry atroom temperature for 2–4 weeks. A miter saw was used to make



multiple cuts into the sapwood of the bolts, avoiding the heart-wood, and the resulting sawdust was collected. Avocado boltswere cut in Florida and allowed to dry. A sander equipped witha vacuum bag was used to make sawdust out of the avocadosapwood layer. For all tree species, the heartwood was dis-carded. Large particles were removed from sawdust by passingit through a No. 12 sieve (Hogentogler & Co. Inc., Columbia,Maryland). To improve the texture of the boxelder sawdust, acoffee grinder was used to produce a finer sawdust powder.Sawdust was packaged and stored in the freezer (−18 ∘C) untilneeded.

Ingredients were combined in a 600-mL glass beaker: 45 gof sawdust (boxelder, beech or avocado), 12 g of agar (DifcoAgar, granulated, Becton, Dickinson and Company, FranklinLakes, New Jersey), 6 g of sucrose (Bio-Serv, Flemington, NewJersey), 3 g of casein (Fonterra, Rosemont, Illinois) , 3 g of potatostarch (Bio-Serv, Flemington, New Jersey), 3 g of brewer’s yeast(MP Biomedicals, Santa Ana, California), 0.6 g of Wesson’s saltmix (Frontier Agricultural Sciences, Newark, Deleware), 0.21 gof 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 ofdistilled 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 andautoclaved for 25 min at 121 ∘C and approximately 15 psi.Tools were also autoclaved, the hood was surface sterilizedwith 70% ethanol and lined with aluminum foil, and trays ofsingle-use 50-mL sterile polyethylene centrifuge tubes (FisherScientific) were placed in the flow hood and ultraviolet (UV)surface-sterilized for 45 min. The hot diet was removed fromthe autoclave and stirred well to resuspend settled ingredients.Working quickly, approximately 15 mL of hot diet was pouredinto each tube. Tubes were loosely capped and tapped to removeair bubbles. Loosely capped tubes of diet were allowed to dryin the flow hood and were checked daily for condensation,after which they were UV sterilized for 1–2 h. Once all thecondensation had evaporated from inside the tubes (2–8 days),they were capped tightly, labelled and frozen at −20 ∘C untilneeded. This volume of diet yielded approximately 25 diet tubes,each containing 15 mL of diet.

PSHB from California

Between 10.00 h and 12.00 h on 9 August 2013, beetles werecollected from a heavily infested boxelder tree in Altadena,California. Thirteen female and three male PSHB that werewalking on the bark were hand-collected and placed into a 2-mLglass vial with some tissue paper. They were transported to thenearby laboratory at the Huntington Library, Art Collectionsand Botanical Gardens in San Marino, California, where eachbeetle was surface-sterilized by emersion in 70% ethanol for10 s. Beetles were then dried for 5 s on a piece of filter paper,and placed, individually or in groups, in 10 rearing tubescontaining boxelder diet. Surface sterilization was conductedto prevent microbes, other than the fungi protected insidethe mycangia, from colonizing the diet. The newly foundedtubes were shipped to the Otis Laboratory (USDA permit #

P526P-13-01673) for further rearing and research within theinsect containment facility. From these original beetles, thelaboratory stock population that we refer to as PSHB wasestablished.

TSHB from Florida

On 5 March 2014, two E. sp. #2 (TSHB) females were dissectedout of an infested avocado branch in Miami-Dade County inFlorida by Daniel Carrillo. The beetles were surface-sterilizedand placed individually into tubes containing avocado diet.Colonies were successfully established and sent to the insectcontainment facility at the Otis Laboratory (USDA permit #P526P-13-01673) for further rearing and research. From theseoriginal beetles, the laboratory stock population we refer to asTSHB was established.

Rearing tubes containing different beetle species were housedin separate containers, which were never opened at the same timeto isolate colonies from each other.

Verification by DNA

In addition to identification using a key to species forE. fornicatus (Rabaglia et al., 2006), once laboratory stockpopulations of PSHB and TSHB were established in the labora-tory, one female descended from each colony line (eight PSHBand two TSHB) was preserved in 95% ethanol and sent to theStouthamer laboratory for genetic confirmation of the crypticspecies. This was achieved by sequencing a section of thecytochrome c oxidase subunit I (COX I) gene commonly usedin barcoding studies in accordance with protocols described inEskalen et al. (2013). All analyzed specimens originating fromthe PSHB and TSHB colonies were confirmed as PSHB andTSHB, respectively, by comparison with GenBank accessionnumbers JX912723–JX912725.

Initiation of experimental colonies

Beetles were reared in a walk-in environmental chamber under anLD 16 : 8 h photocycle at 24 ∘C in the insect containment facilityat the Otis Laboratory. Rearing tubes were enclosed withinlocking food storage containers that were modified with meshwindows for air exchange, and nested within larger containersto keep colonies apart, prevent escapes and reduce the risk ofcontamination by mites or foreign fungi (Rectangular StorageContainer, Lock & Lock U.S.A., Anaheim, California; NylonMesh 500 μm, 47% open area, Component Supply Co., FortMeade, Florida). The protocol for handling beetles was asdescribed previously by Castrillo et al. (2011). To allow airexchange in diet tubes but prevent escapes, a hole was drilledinto the lid of each diet tube (diameter 4 mm) and a fine stainlesssteel mesh (200× 200, 33.6% open area) was secured over thehole using a hot glue gun.

A typical colony was initiated by placing a single matedfoundress in a rearing tube with diet. Exceptions to this(described below) were required for the investigation of hap-lodiploid reproduction and local mate competition.



(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, inthe 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 measurethe health of the colony. Colonies were visually evaluated every1–3 days by examining the sides of the tubes to identify visiblegalleries, and by removing the lid and examining the surfaceof the diet. The number of active entry holes (not coveredover by fungus) was quantified. These beetles push excavatedmaterial and frass out through the entry hole in the form of acompact cylinder. It was noted whether or not this excavationmaterial was present and, if so, the length of the ejected materialwas measured using a 5-mm marker for reference (Fig. 1).The length of excavation material was presumed to be anindication of the extent of gallery formation. The number of eggs,larvae, pupae and adults on the surface and within any visiblegalleries was noted. The sex of pupae and adults was recordedand, if adult females were found, it was noted whether theywere teneral (cuticle was white to brown) or fully sclerotized(cuticle was black). The time and date was recorded for allobservations.

Dissection of colonies

Colonies were destructively analyzed at various ages to quantifythe development of all stages over time. Each tube containinga beetle colony was systematically dissected under a stereomi-croscope. All eggs, larvae, pupae and adults were quantified.Week 1 included the day of colony initiation (day 0) until day7, week 2 included days 8–14, week 3 included days 15–21,and so on. To dissect a tube, all beetle stages that were abovethe diet surface were removed and counted first. Then the dietplug was tapped out into a re-sealable zipper storage bag whereit was examined under a dissecting microscope. Once all bee-tle stages on the surface of the diet plug were removed andcounted, the diet was systematically chipped away in small bitsstarting at the bottom of the plug. As galleries were revealed,they were followed and the diet around them was removed insmall increments to allow any beetle stages within to be collectedand quantified. This technique was followed until the entire diet

plug had been dissected. If at any time a large number of beetlesemerged from the diet at once, the bag was sealed and a paintbrush moistened with 70% ethanol was inserted into the corner ofthe bag and used to remove and count the beetles, so as to preventany escapes.

Haplodiploid reproduction

To confirm the presence of haplodiploid reproduction, and todetermine whether unmated females were capable of initiatingnew colonies by first producing male offspring (from unfertilizedeggs) and then mating with those ‘sons’ to subsequently producea brood of female offspring, experimental colonies were initiatedusing virgin females. To ensure that females were virgins, femalepupae were collected from the laboratory stock populations andplaced in an empty rearing tube until adult eclosion. Becausenewly eclosed adults need to acquire their symbionts, dietwith Fusarium was taken from an established healthy colonyand 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 toclean diet tubes and allowed to naturally inoculate the sterile dietwith their symbionts. Tubes were monitored for up to 13 weeksafter which they were destructively analyzed and offspring werequantified.

Sex ratio and local mate competition

Local mate competition (LMC) theory predicts that species witha high propensity for inbreeding (e.g. shot hole borers) shouldproduce extremely female-biased sex ratios (Hamilton, 1967).As the number of foundresses increases (and the chances ofoutbreeding increases), sex ratios are expected to approach amore ‘normal’ 1 : 1 ratio (Fisher, 1958; Hamilton, 1967; Nunney& Luck, 1988). To test whether PSHB possess this attribute ofLMC, we evaluated the sex ratio of offspring when one foundressor multiple foundresses (two to five) were used to initiate a



colony. The number of male and female offspring were quantifiedand sex ratios were calculated and compared for colonies allowedto develop 5–8 weeks (one generation) (n= 54 and 10 for singleand multiple foundresses, respectively) and 9–13 weeks (twogenerations) (n= 13 and 5 for single and multiple foundresses,respectively). For TSHB, the sex ratios were only examined forcolonies initiated by a single foundress. Another prediction ofLMC is that males in a brood emerge before their sisters, and soPSHB and TSHB were studied for the presence of this attributeas well. PSHB and TSHB colonies were dissected between 14and 28 days to determine the time of the first adult eclosion andalso which sex reaches adulthood first.

Cold tolerance

The cold-hardiness of PSHB was investigated by exposingcolonies to different low temperatures. Thirty PSHB colonieswere initiated by a single foundress and allowed to develop for35 days in the environmental chamber at 24 ∘C. At day 35, elevencolonies that did not appear to be thriving were discarded andthe remaining 24 colonies were divided into five groups. Fourcolonies were kept at 24 ∘C (control). The remaining colonieswere divided into four temperature treatments (n= 5) and placedon a rack. A diet tube without beetles was placed in the centre ofthe rack with a traceable refrigerator/freezer alarm thermometer(Fisher Scientific) inserted into the diet to monitor internal diettemperature. The rack was placed in a freezer at −15 ∘C and analarm sounded when the diet in the monitoring tube reached 5,1, −1 and −5 ∘C, at which time the five tubes in each respectivegroup were removed from the freezer and reunited with thecontrol group at 24 ∘C. An additional temperature probe in thefreezer monitored the temperature outside of the diet. The timeelapsed between each temperature was recorded. After coldtreatments, the colonies were allowed to recover for 24 h at24 ∘C. The next day, all tubes were dissected and the number ofdead and living larvae, pupae, and adults were quantified. Eggswere excluded because we were unable to visually determinetheir health. Dead larvae and pupae were discoloured andflattened. Adults were considered dead if they lacked movement.Mortality rate was calculated as (1 – percentage survival) oflarvae, pupae and adults, and compared for each treatmentand stage.

Statistical analysis

Linear regression models were used to predict relationshipsbetween offspring and entry holes or excavation material (jmp,version 10.0.0; SAS Institute, Cary, North Carolina). Analy-ses comparing sets of data that passed the test for normal-ity were analyzed using analysis of variance (anova) (jmp,version 10.0.0). For analyses comparing two sets of data thatwere nonparametric, datasets were compared using the Wilcoxontest (jmp, version 10.0.0). For statistical analyses compar-ing multiple datasets that failed to satisfy the assumptions ofanova, data were rank transformed, and then analyzed usinganova and Tukey’s mean separation test (sas, version 9.3;SAS Institute Inc., Cary, North Carolina; jmp, version 10.0.0)(Conover, 1980).

Figure 2 Average excavation material (mm) ejected per rearing tube perweek of development by either a mated or virgin polyphagous shot holeborer (PSHB) female in boxelder diet. A total of 80 mated colonies and6 virgin colonies, respectively, were monitored several times per week.The number of measurements each week was averaged (e.g. in week1, 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 thegrowth of the white Fusarium, which ultimately carpeted theexposed surface of the diet, the presence of one or more freshentry holes, and the presence of excavation material ejected fromthe entry holes (Fig. 1). Entry holes that were not in use wouldsoon become covered over by Fusarium and appear closed ordisappear altogether.

Excavation material and entry holes were measured for 80PSHB colonies each founded by a single mated female, and sixcolonies each founded by a single virgin female, and recorded onaverage two to three times per week. Colonies founded by matedfemales initially produced more excavation material than thosestarted by virgin females, which lagged by 2–3 weeks (Fig. 2).Wilcoxon tests revealed significant differences between matedand virgin PSHB in the amount of excavation material ejectedin week 2 (𝜒2 = 4.46, P= 0.0347) and week 5 (𝜒2 = 15.99,P< 0.0001). The amount of excavation material peaked formated foundresses at weeks 6 and 9, and for virgin foundresses atweeks 7 and 11, most likely corresponding to the peak maturationof the first and second generations of female offspring. Thesecond peak was likely suppressed as a result of the diminishedpopulation capacity and quality of the aging diet.

The average number of active entry holes per colony, foundedeither by a single mated or virgin PSHB, was plotted by week(Fig. 3). In the first 4 weeks, the average number of entryholes ranged between 0.6 and 1.0 for both treatments. The firstincrease in number of entry holes in both mated and virgintreatments occurred between weeks 4 and 5, corresponding toemerging offspring, and both sexes were observed to partici-pate in excavation activities. The number of entry holes wassimilar in the first 5 weeks, after which they started to diverge.Wilcoxon tests found significant differences in the number ofentry holes between mated and virgin PSHB foundresses in week3 (𝜒2 = 4.50, P= 0.0338), week 8 (𝜒2 = 18.806, P< 0.0001),



Figure 3 Average number of fresh entry holes observed per colony perweek in colonies founded by a single mated (solid) or virgin (dotted)female polyphagous shot hole borer (PSHB) foundress in boxelder dietover a period of 11 or 13 weeks, respectively. A total of 80 mated coloniesand 6 virgin colonies, respectively, were monitored several times perweek. The number of observations for each week was averaged (e.g.in week 1, there were 188 and 21 measurements for mated and virgincolonies, respectively). Bars represent the SE.

week 9 (𝜒2 = 13.572, P= 0.0002) and week 10 (𝜒2 = 15.972,P< 0.0001).

For each colony, the maximum number of active entry holeswas compared with the total number of male and female off-spring harvested. Because the sex of pupae could also bedetermined, they were included in the tally. Entry hole num-ber was found to be a significant predictor of the numberof PSHB offspring in a colony (Fig. 4) (linear regression,r2 = 0.6148, P< 0.0001 for females; r2 = 0.1153, P= 0.0033 formales; n= 73). Excavation material was also found to be signif-icantly correlated with the number of PSHB offspring, althoughit was not as strong a predictor as entry holes (linear regression,r2 = 0.4748, P< 0.0001 for females; r2 = 0.0787, P= 0.0162 formales; n= 73). Similarly, the linear regression model for TSHBoffspring to the number of entry holes is presented in Fig. 5(r2 = 0.6916, P< 0.0001 for females; r2 = 0.3405, P< 0.0001for males; n= 46). As with PSHB, the relationship between thenumber of TSHB offspring and excavation material was weakerthan with entry holes (r2 = 0.4145, P< 0.0001 for females;r2 = 0.4670, P< 0.0001 for males; n= 46).

Offspring development

The first day that each stage was observed either on the dietsurface or by examining galleries along the rearing tube wallsof any colony founded by a mated or virgin female PSHBis presented in Table 2. This observational approach was anondestructive way of evaluating the timing of the differentstages. With mated foundresses, eggs and larvae were firstobserved 3–4 days earlier than with virgin foundresses, althoughthe first male was observed 2 days later. The first appearance ofdaughters in the virgin treatment was 18–19 days later than thefirst appearance of daughters in the mated treatment, presumablyafter a haploid son matured and mated with the foundress.

Colonies were dissected from each week of development, andthe numbers of eggs, larvae, male pupae, female pupae, male

adults, teneral female adults and mature female adults, and alsothe number of dissected colonies, are presented in Fig. 6 for bothPSHB and TSHB colonies founded by a single mated female.There were no significant differences between PSHB and TSHBin the number of each stage of offspring harvested (Fig. 6). Adultoffspring were not observed in colonies prior to 22 days. Thegreatest number of adult females produced by a single PSHB was57 (in week 7), and that produced by a TSHB foundress was 68(in week 6).

The number of offspring was not significantly influenced byfoundress number, although colony age grouping (first genera-tion at 5–8 weeks versus second generation at 9–13 weeks) andmating status had a significant effect on the number of femaleoffspring (Tables 3 and 4). For PSHB, the number of female off-spring from both single and multiple mated foundresses was notsignificantly affected by the age grouping (one generation versustwo generations), although the limitations of the diet may havebeen reached. Conversely, single TSHB foundresses had fewerfirst-generation offspring than PSHB (Wilcoxon test, 𝜒 2 = 4.93,P= 0.0264) and generation had a significant effect on the num-ber of female offspring (Wilcoxon test, 𝜒 2 = 5.10, P= 0.0239)(Tables 3 and 4). Generation also had a significant effect on thenumber of female offspring of virgin PSHB colonies (Wilcoxontest, 𝜒 2 = 4.51, P= 0.0337) because the first generation of off-spring contained no females.

Sex ratio and haplodiploidy

Virgin foundresses did not initially produce female offspring;rather, they produced sons with which they mated and subse-quently produced daughters. Although there appeared to be atrend for older PSHB colonies to have higher sex ratios (%males), there was no significant difference in sex ratio betweencolonies of different ages, species or foundress number (Tables 3and 4). The only factor that affected sex ratio was the mating sta-tus of the foundress, in which colonies initiated by virgin femaleswere 100% male in 5–8-week-old colonies, and remained pre-dominantly male in older colonies. On average, when harvestedbetween 5 and 8 weeks, typical mated single-foundress PSHBand TSHB colonies had average sex ratios of 7.4% and 7.2%,respectively. Unexpectedly, of 95 PSHB colonies with matedfoundresses that were examined, only female offspring werefound in 13 (13.6%) of them. A similar percentage of colonieswith mated TSHB foundresses also resulted in no male offspring.Whether these foundresses failed to produce males or their maleoffspring died prematurely and were concealed by fungus priorto dissection is not known, and so these colonies are reportedseparately (Table 3).

In colonies dissected between 14 and 28 days, no adults werefound prior to day 22 (Fig. 7). The colonies in which only the firstadult had emerged were aged 22–24 days. Unexpectedly, the firstadult to emerge was not always a male as predicted by LMC. Ofsix PSHB rearing tubes aged 22–24 days old, two had a femaleemerge first on day 22 and one had a male emerge first on day24. Of six TSHB rearing tubes aged 22–24 days old, two had afemale emerge first, one on day 22 and the other on day 23. Theremaining tubes examined had either no adult emergence yet, orboth sexes had already emerged.



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 acolony.

Cold tolerance

The time that it took to drop to target temperatures resultedin rates of temperature change of 0.08–0.25 ∘C/min for eachtreatment. Dissections of colonies exposed to different tem-peratures revealed that one tube from each of the 5, −1 and−5 ∘C treatments contained no offspring, reducing the num-ber for those treatments. There were significant differences inmortality between temperature exposures for larvae (anova ofranked data: F = 3.22, P= 0.0354), pupae (F = 4.34, P= 0.0117)and adults (F = 4.44, P= 0.0106) (Fig. 8). Mortality increasedsignificantly with a lower temperature and longer cold exposure,for all stages. Larvae and pupae appeared to be more sensitiveto cold than adults, as indicated by their higher mortality rates atthe lowest temperatures. After exposure to −5 ∘C, 100% of lar-vae, 95.7% of pupae and 69.2% of adults died. Of those survivingadults, many were on their backs, moving their legs only slightlyafter the 24-h recovery period. Exposure to −1 ∘C produced sim-ilarly high levels of mortality, whereas most individuals survivedthe 0 and 5 ∘C treatments, and those treatments did not differfrom controls.

Discussion

Biology and rearing

The two newly invasive species of the E. fornicatus speciescomplex found in Los Angeles County, California (PSHB), andMiami-Dade County, Florida (TSHB), were similar with respectto their biology and rearing capabilities. The first successfulrearing attempt on artificial diet of scolytine ambrosia beetleswas described by Saunders and Knoke (1967) using Xyleborusferrugineus (Fabricius) and its Fusarium symbiont. By omissionof key ingredients, they that found cellulose powder or fresh hostsawdust was essential, and the importance of wood vitamins wasfurther demonstrated by Norris and Baker (1968). Diet variationshave similarly been tested for rearing a member of the E. for-nicatus species complex (Sivapalan & Shivanandarajah, 1977),showing that cellulose powder, salt mixture and yeast extractwere important for members of this group. Similar diets have alsobeen 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 Xylosan-drus germanus (Blandford) (Peer & Taborsky, 2004; Castrilloet al., 2011), the diets in the present study were developed using



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 andpupae). The linear regression models are shown in each plot.

the host tree species from which our beetles were captured, anda mass rearing protocol was successfully established.

Brood production has been described in a number of otherspecies of ambrosia beetles (Ngoan et al., 1976; Minuzo &Kajimura, 2002; Peer & Taborsky, 2005), although this is thefirst comprehensive study quantifying a brood at increments incolony age for these two members of the E. fornicatus speciescomplex. Because offspring stages overlap and are not discrete,the number of brood in each stage varies by the age of the colonyat the time it is dissected. Foundresses typically began excavatingas soon as they were placed on the diet, introducing Fusariuminto their galleries. Excavations were monitored by measuringthe amount of material ejected from the entry hole and, althoughin Xylosandrus pfeili (Ratzeburg) (Minuzo & Kajimura, 2002),gallery length was found to be strongly correlated with numberof offspring, we found that the number of entry holes in the dietwas a stronger predictor of the number of offspring produced in aparticular tube than the amount of excavation material. Becauseeach entry hole is presumably formed by a single foundresswhen she initiates a new colony, this parameter can also be usedin estimating the population size, both in the rearing tube and

in the field. The timing of their appearance can also be usedwhen describing their phenology. Adult female offspring wereobserved as early as 24 days after the initiation of a colony bya mated foundress, and an average PSHB and TSHB foundressproduced 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 astheir own offspring. Generations clearly overlapped, although theappearance of mature adult F1 daughters began at week 5, peakedat week 6 or 7, and began to decline by week 8, after which theF2 daughters started to emerge.

Approximately 14% of PSHB and TSHB colonies producedonly 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 (possiblysp. #4) in Sri Lanka (Calnaido, 1965) wandering outside of theirgalleries on the surfaces of infested host trees during morninghours, suggests that there may be a built-in mechanism foroutbreeding to occur in a portion of the population. Both PSHBand TSHB had similarly female-biased sex ratios, with only 7.4%



Table 2 Day (and week) of the earliest observed life stages of PSHBoffspring, either from a virgin or a mated foundress, and the difference indevelopmental time between the two

Day (week) of earliestsurface observation

Offspring stageMatedfoundress

Virginfoundress

Difference indevelopment times(days) betweenmated and virginfoundress

Egga 17 (3) 21 (3) 4Larva 13 (2) 16 (3) 3Male pupa 24 (4) 22 (4) −2Male adult 27 (4) 25 (4) −2Female pupa 20 (3) 38 (6) 18Female adult 24 (4) 43 (7) 19Number of colonies

observed269 45

aDoes not include concealed offspring inside galleries, and sothe first observation of eggs did not define the first presence ofeggs.Observations were conducted nondestructively for 8 weeks by examiningthe surface of the diet and inside any galleries that were visible throughthe sides of the rearing tubes.

and 7.2% males, respectively. Colony sex ratio was not affectedby the number of foundresses.

Freeman et al. (2013a) studied groups of PSHB placed on alawn of F. euwallaceae on potato dextrose agar (PDA) in Petridishes and observed development times to the first observationof each developmental stage, including three larval instars,incubated at 25 ∘C. They recorded a slightly longer overalldevelopment time than that observed in the present study at24 ∘C. They found that time to oviposit was greatly affectedby natal host experience, depending on whether the foundresshad been reared on PDA, in which case oviposition was greatlydelayed, or in avocado branches.

Cold tolerance

Cold tolerance plays a crucial role in the population dynamics ofthe scolitine bark beetle Dendroctonus ponderosae (Régnière &Bentz, 2007) and may be equally important for ambrosia bee-tles despite the paucity of cold tolerance studies on them. Toour knowledge, the present study is the first aiming to quan-tify survival at low temperatures in an ambrosia beetle. Anotherinvasive xyleborine ambrosia beetle, Xyleborus glabratus, wasfound to have temperature-dependent development (Brar et al.,2015), and it is the only ambrosia beetle for which the supercool-ing point (SCP) was determined (Formby et al., 2013). The SCPhas also been explored in a number of bark beetles (Lombarderoet al., 2000; Régnière & Bentz, 2007; Kostal et al., 2011; Lester& Irwin, 2012). However, because SCP is the temperature atwhich the body fluids change phase from liquid to solid, mortal-ity typically occurs at temperatures above the SCP of an insect,and SCP is not always a good predictor of intrinsic cold toler-ance (Lombardero et al., 2000; Renault et al., 2002). Cold tol-erance can be highly dynamic within a single species (Rég-nière & Bentz, 2007); for example, a lower SCP may occurwhen beetles were previously cold-hardened compared with

those that were not (Formby et al., 2013), and may also varywith the exposure time or rate of cooling (Kostal et al., 2011).The slow rate of cooling used in the present study may haveallowed for a rapid cold-hardening response (Lee, 1989; Kelty& Lee, 1999). Our empirical study of mortality rates for var-ious stages inside galleries exposed to different low tempera-tures and exposure times found that PSHB was somewhat tol-erant to brief exposures of near freezing temperatures. Whentemperatures were reduced to 5 and 0 ∘C, although some mor-tality was observed, this was not significantly different fromthe control group. However, when diet temperatures droppedbelow freezing and for longer time periods, mortality was sig-nificantly affected. PSHB survival at and above 0 ∘C in the lab-oratory suggests they can survive similar temperatures insidetrees.

Temperatures inside overwintering trees, particularly in theouter xylem and the centres of overwintering trees, may be muchhigher than winter temperatures outside of trees, and tempera-tures in the sapwood, where these beetles mostly reside, can bemuch higher than temperatures at the centres of trees (Sakai,1966). When negative air temperatures occur, temperatures inthe sapwood may remain well above freezing and be able tosafely harbour these beetles. Internal tree temperatures fluctuategreatly and are influenced by solar radiation, the face of the treebeing measured, the diameter of the tree, bark colour and thick-ness, and tree species. Therefore, PSHB may be able to survivein regions with harsher winters than their current distribution insouthern California if internal tree temperatures can safely har-bour them. Although phloem temperatures in pine forests havebeen investigated to predict overwintering capabilities of barkbeetles (Tran et al., 2007), more information on the internal over-wintering temperatures of preferred host trees of PSHB, such asboxelder, under various growing conditions would improve ourability to estimate the potential range of these beetles and similarspecies.

LMC

Ambrosia beetles in the Xyleborini typically have arrhenotok-ous (haplodiploid) sex determination, in which fertilized eggsdevelop into diploid females and unfertilized eggs developinto haploid males (Kirkendall, 1993; Peer & Taborsky, 2005),and also have extreme sex ratios as characteristically foundin LMC, in which inbreeding is the rule (Hamilton, 1967).Brothers generally mate with their sisters within the natalgalleries prior to female dispersal, and males are flightlessand unlikely to disperse. Initially, it would appear that thesespecies exhibit LMC. Hamilton (1967) described eight biofaciesdefining LMC as having: (i) a female-biased sex ratio; (ii)arrehnotokous reproduction; (iii) at least one male in everybatch of offspring; (iv) gregarious development; (v) adult maleseclosing first and mating many times; (vi) mating occurringbefore, during or immediately after adult female eclosion;(vii) males not dispersing; and (viii) females storing spermfrom one insemination to fertilize her entire egg production.These Euwallacea species adhered to some but not all of thecriteria strictly. Of the listed criteria, they do have extremelyfemale-biased sex ratios (i); arrhenotokous reproduction (ii);



Figure 6 The development of two Euwallacea spp. [polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB)] showing average number ofeach stage per colony, harvested at different ages. The number of colonies used for each age and species are provided (bottom left). Note differencesin the scale of the y-axes across the different stages.

gregarious development (iv); and males capable of inseminatingan entire brood of sisters, with sperm stored by a mated femalecapable of inseminating an entire clutch of eggs (viii). Althoughmales were capable of multiple matings, we found they werenot strictly the first to eclose in either species (v). In addition,approximately 14% of clutches of both PSHB and TSHB didnot contain a male (iii). The phenomenon of male-less broodshas been reported in other LMC xyleborines, and at similarfrequencies of approximatey 14% (Entwhistle, 1964; Peer &Taborsky, 2004; Biedermann, 2010). This, together with thefact that we collected PSHB males walking on the bark oftrees (despite being wingless) represents potential evidence ofmale migration and outbreeding, another violation (vii). Malemigratory behaviour in E. fornicatus sensu lato was previ-ously 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 femalesremoved from their natal colony produced only male offspring,suggesting they were still virgins. In another xyleborineX. germanus, females were found to adjust the sex ratio of theirbroods as predicted by LMC when there are more foundressespresent (Peer & Taborsky, 2004). However, the sex ratio of PSHBwas not significantly affected by the number of foundresses,and alternative explanations for this species should be consid-ered. One possibility is that multiple females in a rearing tubemaintained separate galleries, thus avoiding mixing of offspring(Kirkendall, 1993). However, this does not explain the otherviolations, and thus LMC may not suitably describe the traits ofthese species.



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

Average number of offspring per colonyInitial mating statusof foundress Speciesa

Number offoundresses Generationsb Eggs Larvae nc Pupae Adults Femalesd Malesd

Sexratiod (%) ne

Mated, producedmale offspring

PSHB 1 First 31.8 4.3 53 7.6 34.6 32.4 2.1 7.4 541 Second 22.3 7.8 8 4.5 30.8 27.1 3.8 12.6 132–5 First 33.2 4.6 9 2.6 34.6 32.2 2.4 7.8 102–5 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 7.2 261 Second 19.8 0 4 3.5 50.5 47.3 3.3 6.8 4

Mated, only femaleoffspring found

PSHB 1 First 11.8 3.7 6 3.0 7.9 7.9 0 0 91 Second 12.0 0 1 4.5 15.5 15.5 0 0 25 First 18.0 0 1 0 19.0 19.0 0 0 15 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 5Virgin 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 72 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 asecond 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 0.0264Species: 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 0.0239Foundress number: single versus multiple Mated PSHB — First - -Foundress number: single versus multiple Mated PSHB — Second - -Generation: first versus second Virgin PSHB Single — 4.51 0.0337Foundress number: single versus multiple Virgin PSHB — Second - -Status: virgin versus mated — PSHB Single First 8.39 0.0038Status: virgin versus mated — PSHB Single Second 7.72 0.0055Status: 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 interactionsbetween nest mates are mutually beneficial and representquasi-social behaviours including forms of parental care, as wellas care of siblings (Kirkendall et al., 1997; Peer & Taborsky,2007). Forms of maternal care or cooperative brood carehave been found previously in xyleborine ambrosia beetles,and suggest a level of sociality. Such behaviours includepost-ovipositional blocking of the gallery entrance, fungusfarming, cropping symbiotic fungus to avoid overgrowth ofgalleries, rolling of eggs and larvae to patches of food insidegalleries, feeding, extending galleries, and clearing the galleriesof larval waste. If such social behaviours extend to the roles ofadult daughters caring for their sisters prior to dispersal, thenthe reproductive value of daughters would increase and localresource enhancement might help explain the female-biased sex

ratio (Tang et al., 2014). Similar to findings reported for Xyle-borinus saxeseni (Peer & Taborsky, 2007), we saw no increasein oviposition after the appearance of adult offspring, supportingthe notion that cooperative breeding may also take place in thesespecies.

Considering the number of LMC violations, it appears thatthis group does not conform well with LMC theory, and maybe better explained by such alternative theories relating toearly forms of sociality. Unlike most ambrosia beetles, PSHBare found attacking healthy trees rather than dead or weak-ened trees, and so they may rely more on each other formass attack to overcome their host (Kirkendall et al., 1997).Because individual success may be enhanced by the successof siblings or the larger group on a resource patch, addi-tional considerations to explain the female-biased sex ratio



Figure 7 The first adults to emerge, by sex, in polyphagous shothole borer (PSHB) and tea shot hole borer (TSHB) colonies, dissectedbetween 14 and 28 days. The first adults were observed on days 23 and22 for PSHB and TSHB (n=21 and 18, respectively).

Figure 8 Percentage mortality (mean±SE) of 5-week-old polyphagousshot hole borer (PSHB) colonies exposed to different minimum diettemperatures. Bars with the same lowercase letters above them are notsignificantly different (analysis of variance and Tukey’s mean separationon ranked data, P=0.05). The minimum diet temperature, amount oftime the diet was below freezing and the time that vials were inside thefreezer is indicated on the x-axis.

might include kin selection (Queller & Strassmann, 1998), aswell as group selection or the haystack model (Bulmer, 1986;Frank, 1986).

Acknowledgements

We thank Tanya Dockray and Katie Cleary for their assis-tance with diet preparation and colony maintenance. We thankthe Huntington Library, Art Collection and Botanical Gar-dens for the use of their field and laboratory facilities, aswell as Dan Berry for his contribution to this project. We

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

References

Biedermann, P.H.W. (2010) Observations on sex ratio and behavior ofmales in Xyleborinus saxesenii Ratzeburg (Scolytinae, Coleoptera).ZooKeys, 56, 253–267.

Brar, G.S., Capinera, J.L., Kendra, P.E., Smith, J.A. & Peña, J.E.(2015) Temperature-dependent development of Xyleborus glabratus(Coleoptera: Curculionidae: Scolytinae). Florida Entomologist, 98,856–864.

Bulmer, M. (1986) Sex ratios in geographically structured populations.Trends in Ecology and Evolution, 1, 35–38.

California Avocado Commission (2015) Industry Statistical Data[WWW document]. URL http://www.californiaavocadogrowers.com/industry/industry-statistical-data [accessed on 5 January 2015].

Calnaido, D. (1965) The flight and dispersal of shot-hole borer of tea(Xyleborus fornicatus Eichh., Coleoptera: Scolytidae). EntomologiaExperimentalis et Applicata, 8, 249–262.

Carrillo, D., Duncan, R.E. & Peña, J.E. (2012) Ambrosia beetles(Coleoptera: Curculionidae: Scolytinae) that breed in avocado woodin Florida. Florida Entomologist, 95, 573–579.

Carrillo, D., Narvaez, T., Cossé, A.A., Stouthamer, R. & Cooperband,M.F. (2015) Attraction of Euwallacea nr. fornicatus to lures containingquercivorol. Florida Entomologist, 98, 780–782.

Castrillo, L.A., Griggs, M.H., Ranger, C.M., Reding, M.E. & Van-denberg, J.D. (2011) Virulence of commercial strains of Beauve-ria bassiana and Metarhizium brunneum (Ascomycota: Hypocreales)against adult Xylosandrus germanus (Coleoptera: Curculionidae)andimpact on brood. Biological Control, 58, 121–126.

Conover, W.J. (1980) Practical Nonparametric Statistics, 2nd edn. JohnWiley & Sons, Inc., New York, New York.

Danthanarayana, W. (1968) The distribution and host-range of theshot-hole borer (Xyleborus fornicatus Eichh.) of tea. Tea Quarterly,39, 61–69.

Danthanarayana, W. (1973) Host-plant relationships of the shot-holeborer of tea (Xyleborus fornicatus) (Coleoptera: Scolytidae). Ento-mologia Experimentalis et Applicata, 16, 305–312.

Eichhoff, W.J. (1868) New american bark beetle genera and species.Berliner Entomologische Zeitschrift, 12, 145–152.

Entwhistle, P.F. (1964) Inbreeding and arrhenotoky in the ambrosia bee-tle Xyleborus compactus (Eichh.) (Coleoptera: Scolytidae). Proceed-ings of the Royal Entomological Society of London Series A GeneralEntomology, 39, 83–88.

Eskalen, A. (2016) Eskalen Lab: Fusarium Dieback/PolyphagousShot Hole Borer [WWW document]. URL http://eskalenlab.ucr.edu/avocado.html [accessed on 16 February 2016].

Eskalen, A., Gonzalez, A., Wang, D.H., Twizeyimana, M. & Mayorquin,J.S. (2012) First report of a Fusarium sp. and its vector tea shot holeborer (Euwallacea fornicatus) causing Fusarium dieback on avocadoin California. Plant Disease, 96, 1070.

Eskalen, A., Stouthamer, R., Lynch, S.C., Rugman-Jones, P.F., Twizey-imana, M., Gonzalez, A. & Thibault, T. (2013) Host range of Fusar-ium dieback and its ambrosia beetle (Coleoptera: Scolytinae) vector insouthern California. Plant Disease, 97, 938–951.

FAOSTAT (2012) Top Production – Avocados – 2012. United Nations,FAO, Rome, Italy. [WWW document]. URL http://faostat.fao.org[accessed on 16 February 2016].

Fisher, R.A. (1958) The nature of inheritance. The genetical theory ofnatural selection, pp. 1–21. Dover Publications, Inc., New York, NewYork.



Formby, J.P., Krishnan, N. & Riggins, J.J. (2013) Supercooling inthe redbay ambrosia beetle (Coleoptera: Curculionidae). FloridaEntomologist, 96, 1530–1540.

Frank, S.A. (1986) Hierarchical selection theory and sex ratios. I. Generalsolutions for structured populations. Theoretical Population Biology,29, 312–342.

Freeman, S., Protasov, A., Sharon, M. et al. (2013a) Obligate feedrequirement of Fusarium sp. nov., an avocado wilting agent, bythe ambrosia beetle Euwallacea aff. Fornicata Symbiosis, 58,245–251.

Freeman, S., Sharon, M., Maymon, M. et al. (2013b) Fusarium euwal-laceae sp. nov – a symbiotic fungus of Euwallacea sp., an inva-sive ambrosia beetle in Israel and California. Mycologia, 105,1595–1606.

Gadd, C.H. (1941) The life-history of the shot-hole borer of tea. TeaQuarterly, 14, 5–22.

Hamilton, W.D. (1967) Extraordinary sex ratios. Science, 156, 477–488.Kelty, J.D. & Lee, R.E. Jr. (1999) Induction of rapid cold hardening

by cooling at ecologically relevant rates in Drosophila melanogaster.Journal of Insect Physiology, 45, 719–726.

Kirkendall, L.R. (1993) Ecology and Evolution of Biased Sex Ratiosin Bark and Ambrosia Beetles: Evolution and Diversity of Sex Ratioin Insects and Mites (ed. by D. L. Wrensch and M. A. Ebbert), pp.235–345. Chapman and Hall, New York, New York.

Kirkendall, L.R. & Ødegaard, F. (2007) Ongoing invasions of old-growthtropical forests: establishment of three incestuous beetle species insouthern Central America (Curculionidae: Scolytinae). Zootaxa, 1588,53–62.

Kirkendall, L.R., Kent, D.S. & Raffa, K.F. (1997) Interactions amongmales, females and offspring in bark and ambrosia beetles: thesignificance of living in tunnels for the evolution of social behavior.The Evolution of Social Behavior in Insects and Arachnids (ed. byJ. C. Choe and B. J. Crespi), pp. 181–215. Cambridge UniversityPress, U.K.

Kostal, V., Dolezal, P., Rozsypal, J., Moravcova, M., Zahradnickova, H.& Simek, P. (2011) Physiological and biochemical analysis of over-wintering and cold tolerance in two central European populations ofthe spruce bark beetle, Ips typographus. Journal of Insect Physiology,57, 1136–1146.

Lee, R.E. Jr. (1989) Insect cold-hardiness: to freeze or not to freeze howinsects survive low temperatures. Bioscience, 39, 308–313.

Lester, J.D. & Irwin, J.T. (2012) Metabolism and cold tolerance of over-wintering adult mountain pine beetles (Dendroctonus ponderosae):evidence of facultative diapause? Journal of Insect Physiology, 58,808–815.

Lombardero, M.J., Ayres, M.P., Ayres, B.D. & Reeve, J.D. (2000) Coldtolerance of four species of bark beetle (Coleoptera: Scolytidae) inNorth America. Environmental Entomology, 29, 421–432.

Lynch, S.C., Twizeyimana, M., Mayorquin, J.S. et al. (2016) Identi-fication, pathogenicity, and abundance of Paracremonium pembiumsp. nov. and Graphium euwallaceae sp. nov. – two newly discoveredmycangial associates of the polyphagous shot hole borer (Euwallaceasp.) in California. Mycologia, 108, 313–329. DOI: 10.3852/15-063.

Maner, M.L., Hanula, J.L. & Braman, S.K. (2013) Rearing red-bay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculion-idae: Scolytinae), on semi-artificial media. Florida Entomologist, 96,1042–1051.

Mendel, Z., Protasov, A., Sharon, M. et al. (2012) An Asian ambrosiabeetle Euwallacea fornicatus and its novel symbiotic fungus Fusariumsp. pose a serious threat to the Israeli avocado industry. Phytoparasit-ica, 40, 235–238.

Mitchell, A. & Maddox, C. (2010) Bark beetles (Coleoptera: Curculion-idae: Scolytinae) of importance to the Australian macadamia industry:an integrative taxonomic approach to species diagnostics. AustralianJournal of Entomology, 49, 104–113.

Mizuno, T. & Kajimura, H. (2002) Reproduction of the ambrosia beetle,Xyloborus pfeili (Ratzeburg) (Col., Scolytidae), on semi-artificial diet.Journal of Applied Entomology, 126, 455–462.

Mizuno, T. & Kajimura, H. (2009) Effects of ingredients and structureof semi-artificial diet on the reproduction of an ambrosia beetle,Xyleborus pfeili (Ratzeburg) (Coleoptera: Curculionidae: Scolytinae).Applied Entomology and Zoology, 44, 363–370.

Ngoan, N.D., Wilkinson, R.C., Short, D.E., Moses, C.S. & Mangold, J.R.(1976) Biology of an introduced ambrosia beetle, Xylosandrus com-pactus, in Florida. Annals of the Entomological Society of America,69, 872–876.

Norris, D.M. & Baker, J.M. (1968) A minimal nutritional substraterequired by Fusarium solani to fulfill its mutualistic relationshipwith Xyleborus ferrugineus. Annals of the Entomological Society ofAmerica, 61, 1473–1475.

Nunney, L. & Luck, R.F. (1988) Factors influencing the optimum sexratio in a structured population. Theoretical Population Biology, 33,1–30.

O’Donnell, K., Sink, S., Libeskind-Hadas, R. et al. (2015) Dis-cordant phylogenies suggest repeated host shifts in the Fusar-ium – Euwallacea ambrosia beetle mutualism. Fungal Genetics andBiology, 82, 277–290.

Peer, K. & Taborsky, M. (2004) Female ambrosia beetles adjust theiroffspring sex ratio according to outbreeding opportunities for theirsons. Journal of Evolutionary Biology, 17, 257–264.

Peer, K. & Taborsky, M. (2005) Outbreeding depression, but no inbreed-ing depression in haplodiploid ambrosia beetles with regular siblingmating. Evolution, 59, 317–323.

Peer, K. & Taborsky, M. (2007) Delayed dispersal as a potential routeto cooperative breeding in ambrosia beetles. Behavioral Ecology andSociobiology, 61, 729–739.

Queller, D.C. & Strassmann, J.E. (1998) Kin selection and social insects.Bioscience, 48, 165–175.

Rabaglia, R.J., Dole, S.A. & Cognato, A.I. (2006) Review of AmericanXyloborina (Coleoptera: Curculionidae: Scolytinae) occurring northof Mexico, with an illustrated key. Annals of the Entomological Societyof America, 99, 1034–1056.

Rabaglia, R., Duerr, D., Acciavatti, R. & Ragenovich, I. (2008) EarlyDetection and Rapid Response for Non-Native Bark and AmbrosiaBeetles, pp. 1–12. USDA Forest Service, Forest Health Protection,Washington, District of Columbia.

Régnière, J. & Bentz, B. (2007) Modeling cold tolerance in the mountainpine beetle, Dendroctonus ponderosae. Journal of Insect Physiology,53, 559–572.

Renault, D., Salin, C., Vannier, G. & Vernon, P. (2002) Survival at lowtemperatures in insects: what is the ecological significance of thesupercooling point? Cryo Letters, 23, 217–228.

Sakai, A. (1966) Temperature fluctuation in wintering trees. PhysiologiaPlantarum, 19, 105–114.

Saunders, J.L. & Knoke, J.K. (1967) Diets for rearing the ambrosiabeetle Xyleborus ferrugineus (Fabricius) in vitro. Science, 157,460–463.

Sharon, M., Maymon, M., Protasov, A. (2015) Dissemination of the fungiFusarium euwallaceae, Graphium sp. and Acremonium sp., in sym-biosis with the ambrosia beetle Euwallacea nr. fornicatus. Abstractsof Presentations at the 36th Congress of the Israeli PhytopathologicalSociety, Phytoparasitica, Vol. 43, p. 378. Israel.

Sivapalan, P. & Shivanandarajah, V. (1977) Diets for rearing the ambrosiabeetle of tea, Xyleborus fornicatus (Coleoptera: Scolytidae), in vitro.Entomologia Experimentalis et Applicata, 21, 1–8.

Tang, X., Meng, L., Kapranas, A., Xu, F., Hardy, I.C.W. & Li,B. (2014) Mutually beneficial host exploitation and ultra-biasedsex ratios in quasisocial parasitoids. Nature Communications, 5,4942.



Tran, J.K., Ylioja, T., Billings, R.F., Regniere, J. & Ayres, M.P.(2007) Impact of minimum winter temperatures on the populationdynamics of Dendroctonus frontalis. Ecological Applications, 17,882–899.

Walgama, R.S. & Pallemulla, R.M.D.T. (2005) The distribution ofshot-hole borer, Xyleborus fornicatus Eichh. (Coleoptera: Scolytidae),across tea-growing areas in Sri Lanka: a reassessment. Sri LankaJournal of Tea Science, 70, 105–120.

Wickremasinghe, R.L., Perera, B.P.M. & Perera, K.P.W.C. (1976)𝛼-Spinasterol, tempmerature and moisture content as determining fac-tors in the infestation of Camelia sinensis by Xyleborus fornicatus.Biochemical Systematics and Ecology, 4, 103–110.

Accepted 19 February 2016First published online 21 March 2016


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