survey for winter moth (lepidoptera: geometridae) in northeastern north america with...

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Survey for Winter Moth (Lepidoptera: Geometridae) inNortheastern North America with Pheromone-BaitedTraps and Hybridization with the Native Bruce Spanworm(Lepidoptera: Geometridae)Author(s): Joseph S. Elkinton, George H. Boettner, Marinko Sremac, RodgerGwiazdowski, Roy R. Hunkins, Julie Callahan, Susan B. Scheufele, Charlene P.Donahue, Adam H. Porter, Ashot Khrimian, Brenda M. Whited, and Nichole K.CampbellSource: Annals of the Entomological Society of America, 103(2):135-145. 2010.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/AN09118URL: http://www.bioone.org/doi/full/10.1603/AN09118

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Survey for Winter Moth (Lepidoptera: Geometridae) in NortheasternNorth America With Pheromone-Baited Traps and Hybridization With

the Native Bruce Spanworm (Lepidoptera: Geometridae)

JOSEPH S. ELKINTON,1,2 GEORGE H. BOETTNER,1 MARINKO SREMAC,1

RODGER GWIAZDOWSKI,1,2 ROY R. HUNKINS,1 JULIE CALLAHAN,3 SUSAN B. SCHEUFELE,1

CHARLENE P. DONAHUE,4 ADAM H. PORTER,1,2 ASHOT KHRIMIAN,5 BRENDA M. WHITED,2

AND NICHOLE K. CAMPBELL6

Ann. Entomol. Soc. Am. 103(2): 135Ð145 (2010); DOI: 10.1603/AN09118

ABSTRACT We used pheromone-baited traps to survey the distribution of winter moth, Operoph-tera brumata (L.) (Lepidoptera: Geometridae), a new invasive defoliator from Europe in eastern NewEngland. The traps also attracted Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera:Geometridae), native to North America. We distinguished between the two species by examining malegenitalia and sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene, the DNAbarcoding region. In 2005, we recovered winter moths at sites stretching from eastern Long Island,southeastern Connecticut, all of Rhode Island, eastern Massachusetts, coastal New Hampshire, andsouthern coastal Maine. At sites further west and north we captured only Bruce spanworm. In 2006,we conÞrmed that both winter moth and Bruce spanworm are present in Nova Scotia and in coastalMaine, but only Bruce spanworm was recovered in coastal New Brunswick, Canada; Pennsylvania;Vermont; or Quebec City, Canada. In 2007, we collected Bruce spanworm, but no winter moths, inNew Brunswick and the interior areas of Maine, New Hampshire, and New York. Winter moth andBruce spanworm differed in the COI sequence by 7.45% of their nucleotides. The prevalence ofintermediate genitalia in the zone of overlap suggested that hybridization between the two speciesmay be occurring. To conÞrm the presence of hybrids, we sequenced the nuclear gene, glucose-6-phosphate dehydrogenase (G6PD). We identiÞed six nucleotides that routinely distinguished wintermoth and Bruce spanworm, of which three were always diagnostic. We showed that eggs producedby hybridizing the two species in the laboratory contained copies of both species at these six sites. Wefound that most of the moths collected in the Þeld with intermediate genitalia had winter moth CO1and G6PD sequences and thus were not hybrids (or at least F1 hybrids). We found three hybrids outof 158 moths with intermediate genitalia in the region where both species were caught. We concludethat hybrids occur in nature, but are not as common as previously reported. Introgression of genesbetween the two species may still be signiÞcant.

KEY WORDS forest defoliator, hybridization, invasive species, pheromone trap survey, DNA bar-coding

In eastern Massachusetts, widespread defoliation bygeometrid larvae in coastal areas north and south ofBoston has been reported since the early 1990s (De-

borah Swanson, personal communication) and wasassumed to be caused by a native species such as thefall cankerworm, Alsophila pometaria (Harris) (Lep-idoptera: Geometridae), which occasionally exhibitsoutbreaks that last several years (Baker 1972). The fallcankerworm is one of several species of geometridmoths that feed in early spring and produce ßightlessadult females that emerge, attract winged males, andlay eggs in November. An examination of larvae andmale moths in 2002 revealed that the outbreak waseither caused by winter moth, Operophtera brumata(L.) (Lepidoptera: Geometridae), which is native toEurope, or its North American congener, the Brucespanworm, Operophtera bruceata (Hulst) (Lepidop-tera: Geometridae). DeÞnitive identiÞcation of thisoutbreak species as winter moth and not Bruce span-

This research may not necessarily express APHISÕ views.1 Corresponding author: Department of Plant, Soil, and Insect Sci-

ences, University of Massachusetts, Amherst, MA 01003 (e-mail:[email protected]).

2 Graduate Program in Organismic and Evolutionary Biology, Uni-versity of Massachusetts, Amherst, MA 01003.

3 Massachusetts Department of Agricultural Resources, Universityof Massachusetts, Amherst, MA 01003.

4 Maine Forest Service, Maine Department of Conservation, Insectand Disease Laboratory, 50 Hospital St., Augusta, ME 04330.

5 USDAÐARS, Bldg. 007, BARC-West, 10300 Baltimore Blvd., Belts-ville, MD 20705.

6 PSS CT/MA/RI USDAÐAPHISÐPPQ, 900 Northrop Rd., Suite C,Wallingford, CT 06492.

0013-8746/10/0135Ð0145$04.00/0 � 2010 Entomological Society of America

worm was provided in December 2003 by David Wag-ner (personal communication) at the University ofConnecticut and Richard Hoebeke (personal commu-nication) at Cornell University.

Invasions of winter moth have occurred at othersites in North America, namely, Nova Scotia, Canada,in the 1930Ð1950s (Embree 1966, 1991) and in thePaciÞc Northwest in the 1970s (Roland 1986). In eachcase, a decade-long outbreak has been successfullyand permanently controlled by the introduction of atachinid parasitoid, Cyzenis albicans (Fallen), fromEurope (Embree 1966, Roland 1986, Roland and Em-bree 1995). This species is highly specialized on wintermoth and efforts are currently underway (by J.S.E.) toget it established in Massachusetts.

Beginning in November 2005, we worked with ateam of cooperators in seven northeastern states(Connecticut, Maine, Massachusetts, New Hamp-shire, New Jersey, New York, and Rhode Island) todeploy a grid of pheromone-baited sticky traps todelineate the extent of the winter moth infestationacross the region. In November 2006, we expanded thesurvey to include Quebec City, Canada; Nova Scotia,Canada; and coastal regions of Maine and New Bruns-wick, Canada. We also trapped in Vermont, south-eastern New Hampshire, and New York (Long Islandand the Hudson Valley), and we sent a small numberof traps to cooperators in Minnesota; New Jersey;Ontario, Canada; Wisconsin; and Michigan. We alsosent traps to cooperators in Great Britain and Austriato obtain winter moths from European populations forDNA comparison. In 2007, we deployed traps in areasof the northeastern United States not surveyed theprevious 2 yr: mainly the interior areas of New York,New Hampshire, New Brunswick, and Maine. Here,we report the combined results of our survey over the3-yr period.

Materials and Methods

Pheromone Synthesis. The pheromone of the win-ter moth, (1,3Z,6Z,9Z)-1,3,6,9-nonadecatetraene (Ro-elofs et al. 1982), was prepared following the approachby Bestmann et al. (1982). In 2005, a 90:10 mixture of3Z and 3E isomers (1,3Z,6Z,9Z-19:H and 1,3E,6Z,9Z-19:H) was prepared from the Þnal Wittig reactionusing (3Z,6Z)-3,6-hexadecadienyltriphenylphos-phonium bromide and butyl lithium as a base. Weincluded the E-isomer because Underhill et al. (1987)indicated it might suppress the trapÕs attractiveness totheBruce spanworm,butnotwintermoth.This turnedout not to be the case, and in 2006Ð2008, a 95:5 mixtureof 1,3Z,6Z,9Z-19:H and 1,3E,6Z,9Z-19:H was obtainedby using sodium bis (trimethylsilyl) amide as a base inthe Wittig reaction.

The lures were analyzed by gas chromatography(GC) on an 6890N Network GC system (AgilentTechnologies, Santa Clara, CA) equipped with a ßameionization detector and 30- by 0.25-mm (i.d.) DB-210capillary column (Þlm thickness, 0.25 �m) that pro-vided a partial separation of 1,3Z,6Z,9Z-19:H and1,3E,6Z,9Z-19:H geometric isomers (Underhill et al.

1987). The analyses were conducted in a split mode(50:1) by using H2 as a carrier gas at 1 ml/min. Theoven was programmed from 100�C (5 min) to 240�C at10�C/min, the injector temperature was 260�C, andthe detector temperature 270�C.Pheromone Traps and Lures. In 2005, we deployed

Pherocon ICP sticky traps (Zoecon Corp., Palo Alto,CA) baited with a rubber septum impregnated with1,000 �g blend of 90% (Z,Z,Z)-1,3,6,9-nonadecatet-raene (the pheromone) with 10% (E,Z,Z)-1,3,6,9-nonadecatetraene (the Bruce spanworm inhibitor).The trapping extended throughout Connecticut,Rhode Island, eastern and central Massachusetts,southeastern New Hampshire, and coastal Maine in agrid pattern with a spacing of �15 km between traps.Traps were deployed in late November and recoveredin late December or early January. Additional trapswere placed on Long Island and in the Hudson Valleyin New York, and in New Jersey. In 2006 and 2007, weswitched to large capacity Universal Moth Traps(Great Lakes IPM, Inc., Vestaburg, MI) instead ofsticky traps, so that the moths collected would be inbetter condition for subsequent analysis. Traps werebaited with the pheromone only, because we capturedBruce spanworm in all traps in 2005, despite presenceof the inhibitor (except for traps that were quicklysaturated with winter moths at sites within the out-break area). We used the software ARCGIS 9.3 (ESRI,Redlands, CA) to produce maps of the moth capturedata.Identification of Moths and DNA Extraction. Col-

lected specimens were sent to the Elkinton laboratoryat the University of Massachusetts, Amherst, MA. Ini-tial identiÞcation of male winter moths was based ondissection of the male genitalia by G.H.B. (Eidt et al.1966, Troubridge and Fitzpatrick 1993) because wingpatterns and other characteristics are unreliable anddue to the poor condition of moths in sticky traps. In2005, we removed genitalia from 10 moths from eachtrap (�1,500 moths examined) and measured the un-cus (Fig. 1) for three characters: 1) general shape, 2)width of the uncus (in micrometers) near its tip, and3) width of the uncus shaft at its widest point.

We also extracted DNA from individual moths andsequenced the cytochrome oxidase subunit 1 (COI)mitochondrial gene (the barcoding region) to conÞrmour ability to distinguish Bruce spanworm from wintermoth. We used two commonly used methods for DNAextraction. In 2005, we used a standard CTAB protocolfollowed by phenol:chloroform extraction. Beginningin 2006, we used the DNeasy Blood and Tissue kit(catalog no. 69504, QIAGEN GmbH, Hilden, Ger-many) for DNA extraction following manufacturerÕsinstructions. Aliquots of genomic DNA were dilutedeither to 1:10 or 1:20 and stored at �20�C for a sub-sequent use in polymerase chain reaction (PCR) am-pliÞcations.PCR Amplification. For the PCR ampliÞcation of

COI, we used diluted genomic DNA as the templateand commercially synthesized primers (IntegratedDNA Technologies, Inc., Coralville, IA). For the am-pliÞcation of COI gene, the 20-�l reaction mixture

136 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 103, no. 2

contained the following: 10 �l of HotStarTaq MasterMix (catalog no. 203446, QIAGEN GmbH), 2 �l of 10�M forward primer mtCoI-ptA-F (Folmer et al. 1994)(5�-GGT CAA CAA ATC ATA AAG ATA TTG G-3�),2 �l of 10 �M reverse primer mtCoI-ptA-R. (5�-TAAACT TCA GGG TGA CCA AAA AAT CA-3�), 0.8 �lof 25 mM MgCl2, 3 �l of template DNA, and 2.2 �l ofsterile distilled H2O. The cycling program was: initialactivation for 15 min at 95�C; 30 cycles of denaturationfor 30 s at 94�C, annealing for 30 s at 56�C, extensionfor 1 min at 72�C; and Þnal extension for 10 min at 72�C.

For the ampliÞcation of the nuclear gene glucose-6-phosphate dehydrogenase (G6PD), two sets ofprimers were used, one for a regular PCR ampliÞca-tion and the other for a nested ampliÞcation. The Þrstset of primers was designed by Dr. Baiqing Wang forColias and Papilio butterßies following the methods ofJiggins (2003), based on sequences in GenBank andSoto-Adames et al. (1988). These were, for the for-ward primer (G6PD-F), 5�-TGC CAA AAG TA/ideoxyl/ ATG AAA GTT CGG-3�, and for the reverseprimer (G6PD-R), 5�-TTT CCC AAG TAA TGA TCGATT CTG-3�. The nested ampliÞcation, designed forthis work, used forward primer (G6PD-nested-F) 5�-ACT TAT CAC GCT GGT GCC TAC GAT-3� andreverse primer (G6PD-nested-R) 5�-ACT TCG CTGAGC TTA CAT CAT CCC-3�. The PCR reaction mix-tures contained the same reagents as for COI, with theannealing time of 45 s at 50�C for 35 cycles for theinitial G6PD ampliÞcation, and 45 s at 59�C for 30cycles for the nested ampliÞcation.

The ampliÞed DNA products were resolved elec-trophoretically on 1.5 or 2.0% agarose gels and visu-alized by ethidium bromide staining. Samples with thecorrect fragment size were sent out for sequencing toLaragen, Inc. (Los Angeles, CA), and the DNA se-quences were edited and aligned using Sequencher 4.2(Gene Codes Corporation, Ann Arbor, MI).Detection of Hybrids. Previous trapping studies of

winter moth and Bruce spanworm in western Canada(Pivnick et al. 1988) detected hybrids between the

two species based on intermediate male genitalia. Inprevious laboratory studies, Smith and Ring (unpub-lished, cited in Underhill et al. 1987) bred winter mothfemales with Bruce spanworm males in cages andproduced viable progeny to the F2 generation. Brucespanworm females did not mate with male wintermoths, so any hybrids would have had winter mothmitochondrial genes. To determine whether wintermoth was hybridizing with Bruce spanworm in oursurvey, we Þrst determined whether any specimenshad intermediate genitalia, then sequenced the nu-clear gene G6PD for these and a sample of morpho-logically unambiguous moths, including winter mothsfrom Europe. We compared the sequences to identifynucleotides in the G6PD gene that uniquely and re-liably distinguished the two species. Hybrids (at leastF1 hybrids) would be expected to have both. WeconÞrmed this in 2007 by extracting DNA from eggsproduced by mating female winter moths collected inBritish Columbia with male Bruce spanworm col-lected with pheromone traps in western Massachu-setts. Individual mating pairs were held at 16�C and aphotoperiod of 12:12 [L:D] h, and females were al-lowed to oviposit on paper strips. Given the small sizeof the eggs, we pooled all eggs from each female beforeDNA extraction. Hybrid families exhibit a heterozy-gous genotypic pattern, i.e., overlapping peaks in thechromatogram, at the species-speciÞc sequence sitesin these pooled samples.

In 2007, we ran an eastÐwest transect along Route 2in northern Massachusetts, with traps spaced everythree km to further delineate the boundary of thewinter moth infestations. Near the boundary of thewinter moth infestation that we had mapped in 2005,we decreased the spacing between traps to 1.5 km toprovide a more exact delineation of the boundarybetween the populations. We speculated that in thisregion we would be most likely to collect hybrids,because in areas further east where winter moth wasin outbreak phase, the number of winter moths wouldvastly outnumber Bruce spanworm, which has re-

Fig. 1. Uncus from male genitalia of (A) winter moth and (B) Bruce spanworm. Horizontal lines indicate where widthmeasurements were made.

March 2010 ELKINTON ET AL.: WINTER MOTH SURVEY IN NORTHEASTER NORTH AMERICA 137

mained at low density everywhere in New Englandthroughout our study.Ground Survey. In May and June 2005, we con-

ducted a ground survey of towns in eastern Massa-chusetts by car, where we recorded presence andabsence of observable defoliation by winter moth. WeconÞrmed that defoliation was caused by winter mothby noting the presence of caterpillars. After the cat-erpillars had pupated in June, we also surveyed defo-liation by examining the damage (holes in leaves)caused by the very early feeding of winter moth onunexpanded buds. This winter moth damage is uniquefrom that of other common defoliators such as gypsymoth, Lymantria dispar L., or forest tent caterpillars(Malacosoma disstria Hubner), which were alsopresent in the region.

Results

Winter moths were trapped at sites that stretchedfrom eastern Long Island, the southeastern corner ofConnecticut, all of Rhode Island, eastern Massachu-setts, coastal New Hampshire, and southern coastal

Maine in 2005. They were initially identiÞed based onmale genitalia (Fig. 2). In contrast, defoliation bywinter moth was observed only in Massachusettswithin a region surrounding Boston and on Cape Cod(Fig. 2). We caught winter moths in areas that wereat least 50 km from any areas known to be defoliatedby winter moths. Traps further west and north caughtexclusively Bruce spanworm. These include othertraps in New Jersey and the Hudson Valley in NewYork that are not shown on the map.

In 2006, we conÞrmed that both winter moth andBruce spanworm were present in Nova Scotia and incoastal Maine, but only Bruce spanworm was recov-ered in coastal New Brunswick, Pennsylvania, Ver-mont, and Quebec City (Fig. 3A). We also recoveredBruce spanworm but not winter moths in Minnesota;Wisconsin; Michigan; and Sault Ste. Marie, ON, Can-ada. In 2007, there were no winter moths collectedanywhere in New Brunswick or the interior areas ofMaine, New Hampshire, or New York, whereas trapsin all these locations captured Bruce spanworm (Fig.3A). The resulting distribution of winter moths in theNortheast has a striking correspondence to the winter

Fig. 2. Distribution of winter moth, Bruce spanworm, and intermediate phenotypes captured in pheromone-baited trapsin New England in 2005. IdentiÞcation is based on male genitalia only. Each circle represents a pie chart of 10 dissected mothsampled from all moths in a trap. In the few cases when �10 moths were caught, we dissected all moths in the trap. Hatchedmarkings show towns where we detected defoliation by winter moth.


hardiness zones (Cathey 1990; Fig. 3B). Winter mothswere recovered in areas corresponding to hardinesszone 5b (minimum temperature, �26 to �23�C) butnot 5a (�29 to �26�C).

Analysis of COI gene sequences demonstrated veryclear differences between the winter moth and Brucespanworm (Table 1). We sequenced 124 winter mothsand106Bruce spanwormsand foundthat theydifferedby 7.4% of the 679 bp in our sequences. The entire 679bp sequence (GenBank accessions GQ424954 andGQ424955) was identical for all winter moths we an-alyzed, whereas there was some variation among theBruce spanworm (Table 1). There was also some vari-ation in the six winter moths we analyzed from Austriaand the United Kingdom (Table 1).

Measurements of male genitalia were found to beintermediate in the region where both species werecaptured, suggestive of possible hybrids. AlthoughBruce spanworm unci were generally longer and thin-ner (Fig. 1); we identiÞed specimens as intermediatesif they fell within the region where measurementsoverlapped (Fig. 4). We found that 24% of moths inthe band where the two populations came together in

Massachusetts and Rhode Island had intermediategenitalia, suggesting that they might be hybrids (Fig.2). When we sequenced G6PD (Table 2), we foundthat only 3/158 (1.9%) were unmistakable hybrids.These three moths all came from traps near the centerof the 2007 eastÐwest transect in Massachusetts indi-cated by the closely spaced line of traps (Fig. 3A,inset). Two of these hybrids had winter moth mothersas indicated by their CO1 sequences and one had aBruce spanworm mother. As with our analysis of CO1,all winter moths that we analyzed had identical G6PDsequences (Table 2), whereas Bruce spanwormshowed heterozygosity at some sites. We found sixnucleotides that routinely distinguished winter mothfrom Bruce spanworm (Table 2), but only the Þrst twoand the sixth (sequence numbers 21, 23, 141, Table 2)were invariably homozygous in Bruce spanworm. OurDNA analyses revealed that most of moths with in-termediate genitalia had winter moth rather thanmixed ancestry genotypes. If these intermediates didindeed have mixed ancestry, then they would haveoriginated from backcrosses extending one or more

Fig. 3. (A) Distribution of winter moth and Bruce spanworm in pheromone-baited traps in northeastern North Americain 2005Ð2007. IdentiÞcation of moths is based on male genitalia and the DNA sequence of the COI mitochondrial gene, forwhich there are no intermediates. Inset, enlarged view of captures in southern New England.


generations into the winter-moth genetic background,to restore homozygosity at G6PD.

When we plotted our measurements of uncus shape(uncus tip width/shaft width, see Fig. 1) versus uncuswidth (Fig. 4), we discovered a broad range of overlapbetween the two species, indicating that uncus shapewas an imperfect tool for distinguishing the two spe-cies. The range of overlap included moths from Eu-rope, where there has been no possibility of hybrid-ization between winter moth and Bruce spanworm.

Discussion

The COI sequences show that DNA analysis is adeÞnitive method to distinguish these two moth spe-cies. It also conÞrms that we have recovered wintermoths from near Worcester, MA, �50 km west of anyknown winter moth infestation. We also caught wintermoths at various places along the coast of Maine (Figs.2 and 3A). Thus, we cannot rule out the possibility thatwinter moth spread along the coast from Nova Scotiato Massachusetts. However, to our knowledge, therehas never been an outbreak of winter moth in Maineor New Brunswick. Review of the Canadian ForestInsect Survey for New Brunswick for 1970Ð1990 (Nat-ural Resources Canada 2008) conÞrms that winter

moth has not been recovered in interior New Bruns-wick, but it is sometimes recovered along the coast. Itis possible that male moths in coastal Maine and NewBrunswick may have blown across the Bay of Fundyfrom Nova Scotia. Furthermore, in New England wehave not recovered either of the two parasitoids[Cyzenis albicans (Fallen) and Agrypon flaveolatum(Gravenhorst); J.S.E., unpublished data] that controlwinter moth in Nova Scotia, from the collection andrearing of several thousand mature winter moth larvaefrom various sites. These facts may suggest a separateintroduction of winter moth to New England insteadof spread along the coast, although the source of thepopulations might still have been Nova Scotia.

We found that the COI sequences were identical forall winter moths that we analyzed in North Americaincluding moths from Nova Scotia, British Columbia,andNewEngland.The lackofpolymorphism inwintermoth, in contrast to Bruce spanworm, suggests a pos-sible founder effect from an initial invasion of one orvery few individuals and the spread of winter mothfrom Nova Scotia to New England and to British Co-lumbia. We are currently collecting more wintermoths from Europe in hopes of identifying the sourcepopulation of North American winter moths.

Fig. 3. (B) Plant cold hardiness zone map for northeastern North America (adapted from Cathey 1990). Zones are basedon average absolute minimum winter temperature.


Table 1. Nucleotide sequences for the barcoding fragment of the mitochondrial gene Cytochrome oxidase subunit 1 (CO1) for allNorth American winter moths (first line), six winter moths from Europe, and Bruce spanworm from various locations in the northeasternUnited States

Spa Locb Hapc Nd 1,515e

WM-NAmer 1 31 AAGATATTGG AACTTTATAC TTTATTTTTG GAATTTGAGC CGGTATAATT GGAACTTCACWM-UK 1 2 .......... .......... .......... .......... .......... ..........WM-UK 2 2 .......... .......... .......... .......... .........C ..........WM-UK 1 .......... .......... .......... .......... .........C ..........WM-AUS 1 .......... .......... .......... .......... .......... ..........BS NAmer 1 16 .......... .........T .....C.... .......... T.....G... ........TTBS NAmer 2 9 .......... .........T .....C.... .......... T.....G... ........TTBS NH 1 .......... .........T .....C.... .......... T.....G... ........TTBS MA 1 .......... .........T .....C.... .......... T.....G... ........TTBS VT 1 .......... .........T .....C.... .......... T.....G... ........TT1,576TAAGTTTATT AATTCGAGCT GAATTAGGTA ACCCTGGTTC TTTAATTGGG GATGACCAAA TTTACAACAC.......... .......... .......... .......... .......... .......... .................... .......... .......... .......C.. .......... .......... .................... .......... .......... .......C.. .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... ........A. .T..A..... .......... .......... .................... .......... ........A. .T..A..... .........A .......... .................... .......... ........A. .T..A..... .......... .......... .................... .......... ........A. .T..A..... .......... .......... .................... .......... ........A. .T..A..... .......... .......... ..........1,646TATTGTTACA GCACATGCTT TTATTATAAT TTTTTTTATA GTTATACCAA TTATAATTGG AGGATTTGGT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... ........C. .......... .........G .......... .......... .................... ........C. .......... .........G .......... .......... .................... ........C. .......... .........G .......... .......... .................... ........C. .......... .........G .......... .......... .................... ........C. .......... .........G .......... .......... ..........1,716AATTGATTAG TACCTTTAAT ACTTGGAGCT CCTGATATAG CTTTCCCCCG AATAAATAAT ATAAGATTTT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... ..................G. .....C.T.. ......G... .......... .......... T......... .....T............G. .....C.T.. ......G... .......... .......... T......... .....T............G. .....C.T.. ......G... .......... .......... T......... .....T............G. .....C.T.. ......G... .......... .......... T......... .....T............G. .....C.T.. ......G... .......... .......... T......... .....T....1,786GATTATTACC TCCCTCTATT ACTCTTTTAA TTTCTAGAAG AATTGTAGAA AATGGGGCAG GAACTGGATG.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .....A.... .................... .......... .......... .......... .......... .....A.... .................... .......... .......... .......... .......... .......... .................... ...G...... ..A..A.... .......... .......... .......... .................... ...G...... ..A..A.... .......... .......... .......... .................... ...G...... ..A..A.... .......... .......... .......... .................... ...G...... ..A..A.... .......... .......... .......... .................... ...A...... ..A..A.... .......... .......... .......... ..........1,856AACTGTTTAC CCCCCTTTAT CTTCTAATAT TGCCCATGGA GGAAGATCCG TAGATCTAGC TATCTTTTCT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... ........A. .................... .......... .......... .......... .......... .......... ...................T ..G..CC.G. .......... C........G ........T. .......... ...T.....C.........T ..G..CC.G. .......... C........G ........T. .......... ...T.....C.........T ..G..CC.G. .......... C........G ........T. .....T.... ...T.....C.........T ..G..CC.A. .......... C........G ........T. .......... ...T.....C.........T ..G..CC.A. .......... C........G ........T. .......... ...T.....C1,926CTTCATTTAG CTGGTATTTC CTCAATTTTA GGTGCAATTA ACTTTATTAC CACTATTATC AATATACGAT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... ..........

Continued on following page


The close correspondence of the distribution ofwinter moth to the cold hardiness zones based onaverage annual minimum winter temperatures

(Cathey 1990) provides a possible explanation as towhy winter moth became common in Nova Scotia inthe 1930s (Embree 1965) but did not spread beyondthat region until recently. The spread may have beenblocked by unfavorable winter conditions in NewBrunswick. Macphee (1967) and Tenow and Nilssen(1990) have shown that winter moth eggs are killed bytemperatures below �36�C, but winter temperaturesrarely get that low in many parts of Maine and NewBrunswick where winter moths are absent (Fig. 3B).A similar pattern exists in the birch forests of northernScandinavia (Jepson et al. 2008), where the distribu-tion of winter moth is seemingly limited by cold wintertemperatures in areas that never get as low as �36�C.Winter moth has a life cycle that depends on adultßight and oviposition in late fall and early winter. It ispossible that temperatures during this period in NewBrunswick and interior Maine may be inimical to thisbehavior. It is also possible that temperatures in the

Table 1. Continued

.......... .......... .......... .......... .......... .......... ..........

.......... .......... .......... .......... .......... .......... ..........

.......... .......... T......... ..A....... .T........ T........T ..........

.......... .......... T......... ..A....... .T........ T........T ..........

.......... .......... T......... ..A....... .T........ T........T ..........

.......... .......... T......... ..A....... .T........ T........T ..........

.......... .......... T......... ..A....... .T........ T........T ..........1996TAAATAATAT ATTTTTTGAC CAATTACCAT TATTTGTTTG AGCTGTAGGA ATCACAGCAT TTTTACTTTT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .........T .......... .......... .......... .......... .................... .........T .......... .......... ...A...... ..T....... .....T.A............ .........T .......... .......... ...A...... ..T....... .....T.A............ .........T .......... .......... ...A...... ..T....... .....T.A............ .........T .......... .......... ...A...... ..T....... .....T.A............ .........T .......... .......... ...A...... ..T....... .....T.A..2,066ATTGTCATTA CCAGTATTAG CGGGAGCTAT TACTATATTA TTAACAGATC GAAATTTAAA TACATCATTT.......... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... .................... .......... .......... .......... .......... .......... ...........C.A...... .......... .T..G..... .......... .......... .......... ...........C.A...... .......... .T..G..... .......... .......... .......... ...........C.A...... .......... .T..G..... .......... .......... .......... ...........C.A...... .......... .T..G..... .......... .......... .......... ...........C.A...... .......... .T..G..... .......... .......... .......... ..........2,136 2194TTCGATCCTG CTGGGGGGGG AGATCCTATT CTTTATCAAC ACTTATTTTG ATTTTTTGG.......... .......... .......... .......... .......... ................ ....A..... .......... .......... .......... ................ ....A.............. ...........T..C.... ....A..... G......... .......... .......... ...........T..C.... ....A..... G......... .......... .......... ...........T..C.... ....A..... G......... .......... .......... ...........T..C.... ....A..... G......... .......... .......... ...........T..C.... ....A..... G......... .......... .......... .........

a Sp, species; WM, winter moth; BS, Bruce spanworm.b Location code: NAmer, North America; UK, United Kingdom; AUS, Austria; NH, New Hampshire; ME, Maine; MA, Massachusetts; PA,

Pennsylvania; VT, Vermont.cHap, haplotype.dN, number of moths in category out of 37 winter moths and 43 Bruce spanworm sequenced. Five of the winter moths came from the United

Kingdom., one from Austria, and the rest from North America.e The number in the upper right hand corner indicates the starting nucleotide position of the CO1 gene obtained from the complete

mitochondrial genome of the geometrid Phthonandria atrilineata (NC_010522). The starting position of the CO1 gene within the genome wasidentiÞed, located and conÞrmed with COI sequence of Charissa crenulata (AJ870408) obtained from the Barcode of Life website, http://www.barcodinglife.com.

Fig. 4. Male genitalia measurements for 424 Bruce span-worm (BS, open) and winter moths (WM, closed). Uncuswidth at tip versus shape (tip width /midshaft width).


spring may play a role. Tenow and Nilssen (1990)demonstrated that winter moth eggs become less coldtolerant in the weeks before hatch. Whatever the lifestage affected, limitation by winter temperatures mayalso explain why there has never been, to our knowl-edge, a winter moth outbreak in Maine, even thoughwe have shown that winter moth occurs in coastalMaine where temperatures are warmer (Fig. 3). It isonly when winter moths became established in east-ern Massachusetts and southeastern New Hampshirethat they encountered winter temperatures compara-ble with those that exist in maritime Nova Scotia. Innorthern Scandinavia, global climate change has pro-duced warmerwintersandasaresult therangeofwintermoth outbreaks has expanded into colder areas at higheraltitude or further from the coast (Jepson et al. 2008).Warming climate may thus play an important role in thefuture distribution of winter moth in North America.

Of course, it is possible that the observed corre-spondence of the distribution of winter moth with thewarmer cold-hardiness zones is caused by the otherfactors correlated to winter temperatures. For exam-ple, the prevalence of acceptable host plants may bean important factor. Deciduous trees such as oaks andmaples are the dominant forest trees in Massachusetts,southern Maine, and New Hampshire, in contrast tothe Picea (spruce)ÐAbies (Þr) forests of northernMaine and New Brunswick. Nova Scotia, however, isalso dominated by spruceÐÞr forests (Rowe 1959),despite the warmer winter temperatures similar tothose of Massachusetts. Winter moth there survives onpockets of deciduous trees such asQuercus rubraL. or inabandoned apple (Malus spp.) orchards (Embree 1965).

Our DNA analysis largely conÞrmed the use of gen-ital characters to distinguish winter moth and Bruce

spanworm males. Both methods for species identiÞ-cation had pros and cons. Our uncus measurementsincluded winter moth specimens from Europe, whereBruce spanworm does not exist, suggesting that theoverlap in shape is not due to introgression of genitaliatraits via hybridization. We were therefore not alwaysable to determine species based on examination ofgenitalia. IdentiÞcation of the two species via DNAsequence was more reliable, but much more costly.Also, when pheromone-baited traps were left in theÞeld for several weeks, or Þlled with rain, the mothsoften decayed and as a result we were unable toextract DNA, yet the uncus was always in good con-dition. Furthermore, the COI sequences can onlyidentify the maternal line and cannot distinguish hy-brids. We are currently developing a robust, yet lessexpensive, DNA identiÞcation method for these twospecies and their hybrids, based on restriction enzymetests that will avoid the expense of DNA sequencing.

The occurrence of hybrids has important implica-tions for the future spread of winter moth and thesuccess of the ongoing biological control effort againstwinter moth. Our survey shows that Bruce spanwormis able to persist in areas with much colder wintersthan winter moth. Although we do not know thegenetic basis of this difference, it is possible that what-ever traits that confer this ability could pass fromBruce spanworm to winter moth via hybridization,allowing winter moth to expand into northern areas ofeastern North America. Also, we are currently releas-ing the parasitoidC. albicans to control winter moth inNew England. This parasitoid was very successful inpermanently reducing outbreak populations of wintermoth in Nova Scotia in the 1950s and in the PaciÞcNorthwest in the 1980s. According to Roland and

Table 2. Nucleotide sequence of a portion the nuclear gene G6PD for winter moth, three haplotypes of Bruce spanworm, hybrid eggsfrom parents mated in the laboratory, and two haplotypes of hybrids recovered in New England

Spa Hapb Nc 1

WM h1 63 GAGTTGCTCA ATCAATCCAT CAGCAAAAGT GAAAAAGGAC CTGTGGCGAABS h1 58 .......... .......... T.T....... .......... ..........BS h2 33 .......... .......... T.T....R.. .......... ..........BS h3 6 .......... .......... T.T....G.. .......... ..........Hyb eggs 8 .......... .......... Y.K....... .......... ..........Hyb h1 3 .......... .......... Y.K....... .......... ..........51CAGGATATTT TACCTAGCAG TGCCACCCAC TGTATTTGAA GAAGTGACCG TCAACATTAG.......... .....G.... .......... .......... ........T. .................... .....R.... .......... .......... ........Y. .................... .......... .......... .......... .......... .................... .....R.... .......... .......... ........Y. .................... .....R.... .......... .......... ........Y. ..........111d

AAATGCTTGT GTATCCATTA AAGGATACAC CCGAGTCATT ATTGAAAAAC CATTTGGA.......... .C........ .......... T......... .......... .................. .Y........ .......... T......... .......... .................. .......... .......... T......... .......... .................. .Y........ .......... Y......... .......... .................. .Y........ .......... Y......... .......... ........

R, heterozygotes with A and G; Y, heterozygotes with C and T; K, heterozygotes with G and T. (GenBank accessions GQ429177, GQ429178).a Sp, species; WM, winter moth; BS, Bruce spanworm; Hyb, hybrid.b hap, haplotype.cN, number of moths in category out of 63 winter moths, 95 Bruce spanworm and 3 hybrids sequenced. All moths came from North America.dNumber at the head of each column of the left references our nucleotide sequence to the Þrst in our sequence. We found no example of

a complete G6PD sequence for any Lepidoptera in the National Center for Biotechnology Information database.


Embree (1995), C. albicans does not attack Brucespanworm under Þeld conditions, although it will at-tack Bruce spanworm in the laboratory. If this func-tional immunity toC. albicans has a genetic basis, thenthis trait also could pass from Bruce spanworm towinter moth via hybridization. In Nova Scotia andBritish Columbia it seems that winter moth remainsunder good biological control, but research by Roland(1990, 1994) in British Columbia and Pearsall andWalde (1994) in Nova Scotia suggests that it is pred-ators and notC. albicans that maintains winter moth atinnocuous densities. Indeed, the rates of parasitism inboth of these regions by C. albicans are much lowernow than the rate reported by Embree (1965) afterthe initial introduction. These previous researchersassumed that this decline in parasitism was caused bythe lower densities of winter moth, but nothing isknown about the degree of hybridization with Brucespanworm in Canada or its possible inßuence on dy-namics of winter moth.

Acknowledgments

We thank the following people for deploying and retriev-ing the pheromone traps and for sending us the moths fromthe stated locations: Massachusetts: C. Burnham, K. Gooch,G. Witkus; Maine: K. Coluzzi, J. Crowe, M. Skinner, G. Smith,W. Urquhart; Vermont: B. Burns, J. Esden, T. Greaves, T.Hanson, R. Kelly, L. Lund, T. Simmons; New York: K. Carnes,P. Jentsch, D. Gilrein, H. McGinnis; Connecticut: V. Smith,P. Trenchard, D. Ellis; Rhode Island: S. Baxter, H. Faubert, C.Sparks, D. Martin; New Hampshire: C. Tatum, J. Weaver;Pennsylvania: J. Stimmel, S. Gardosik, Sven Spichiger; NewJersey: S. Vaiciunas, R. Fine, J. Simmons; Wisconsin: A. Diss-Torrance, B. Schwingle, L. Williams; Michigan: D. Mc-Cullough; Minnesota: D. Zumeta, A. Jones; British Columbia:L. Humble, G. Zilahi-Balogh; New Brunswick: R. Webster;Ontario: Ba. Lyons, Be Lyons; Quebec: J. Regniere, P. Duval;Austria: G. Hoch; and United Kingdom: C. Tilbury, A. Van-bergen, A. Watt. We thank I. Otvos and N. Condor forsending us winter moth pupae from British Columbia; A.Liebhold D. Embree and D. Souto for critical reviews; and V.Neilis for querying the Canadian Forest Insect and Diseasesurvey regarding winter moth in New Brunswick. We thankGloria Witkus for help in the Þeld and with dissections,Natalie Leva for mailing traps to our cooperators, JeremeyAnderson for assisting with GenBank submissions, Jeff Ahernfor assistance in initiating the DNA analysis, and Maili Pageand Diana Barscz for assistance in producing maps. Thisresearch was made possible, in part, by a cooperative agree-ment (05-8225-0464-CA) from the U.S. Department of Ag-ricultureÕs Animal and Plant Health Inspection Service(APHIS). We are also grateful to the USDA Forest Service(cooperative agreement 04-CA-11244225-414), and a grantfrom the Massachusetts State Legislature for supporting thiswork.

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Received 13 August 2009; accepted 4 November 2009.


survey for winter moth (lepidoptera: geometridae) in northeastern north america with...

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