Survey for Winter Moth (Lepidoptera: Geometridae) in ... - PubAg - USDA

FORUM

Survey for Winter Moth (Lepidoptera: Geometridae) in Northeastern North 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 6 AND NICHOLE K. CAMPBELL

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, Operophtera brumata (L.) (Lepidoptera: Geometridae), a new invasive defoliator from Europe in eastern New England. The traps also attracted Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera: Geometridae), native to North America. We distinguished between the two species by examining male genitalia and sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene, the DNA barcoding 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, and southern 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 coastal Maine, 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, in New Brunswick and the interior areas of Maine, New Hampshire, and New York. Winter moth and Bruce spanworm differed in the COI sequence by 7.45% of their nucleotides. The prevalence of intermediate genitalia in the zone of overlap suggested that hybridization between the two species may be occurring. To conÞrm the presence of hybrids, we sequenced the nuclear gene, glucose-6phosphate dehydrogenase (G6PD). We identiÞed six nucleotides that routinely distinguished winter moth and Bruce spanworm, of which three were always diagnostic. We showed that eggs produced by hybridizing the two species in the laboratory contained copies of both species at these six sites. We found that most of the moths collected in the Þeld with intermediate genitalia had winter moth CO1 and G6PD sequences and thus were not hybrids (or at least F1 hybrids). We found three hybrids out of 158 moths with intermediate genitalia in the region where both species were caught. We conclude that hybrids occur in nature, but are not as common as previously reported. Introgression of genes between the two species may still be signiÞcant. KEY WORDS forest defoliator, hybridization, invasive species, pheromone trap survey, DNA barcoding

In eastern Massachusetts, widespread defoliation by geometrid larvae in coastal areas north and south of Boston has been reported since the early 1990s (DeThis research may not necessarily express APHISÕ views. 1 Corresponding author: Department of Plant, Soil, and Insect Sciences, University of Massachusetts, Amherst, MA 01003 (e-mail: [email protected]). 2 Graduate Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA 01003. 3 Massachusetts Department of Agricultural Resources, University of Massachusetts, Amherst, MA 01003. 4 Maine Forest Service, Maine Department of Conservation, Insect and Disease Laboratory, 50 Hospital St., Augusta, ME 04330. 5 USDAÐARS, Bldg. 007, BARC-West, 10300 Baltimore Blvd., Beltsville, MD 20705. 6 PSS CT/MA/RI USDAÐAPHISÐPPQ, 900 Northrop Rd., Suite C, Wallingford, CT 06492.

borah Swanson, personal communication) and was assumed to be caused by a native species such as the fall cankerworm, Alsophila pometaria (Harris) (Lepidoptera: Geometridae), which occasionally exhibits outbreaks that last several years (Baker 1972). The fall cankerworm is one of several species of geometrid moths that feed in early spring and produce ßightless adult females that emerge, attract winged males, and lay eggs in November. An examination of larvae and male moths in 2002 revealed that the outbreak was either caused by winter moth, Operophtera brumata (L.) (Lepidoptera: Geometridae), which is native to Europe, or its North American congener, the Bruce spanworm, Operophtera bruceata (Hulst) (Lepidoptera: Geometridae). DeÞnitive identiÞcation of this outbreak species as winter moth and not Bruce span-

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

136

ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA

worm was provided in December 2003 by David Wagner (personal communication) at the University of Connecticut and Richard Hoebeke (personal communication) at Cornell University. Invasions of winter moth have occurred at other sites in North America, namely, Nova Scotia, Canada, in the 1930 Ð1950s (Embree 1966, 1991) and in the PaciÞc Northwest in the 1970s (Roland 1986). In each case, a decade-long outbreak has been successfully and permanently controlled by the introduction of a tachinid parasitoid, Cyzenis albicans (Fallen), from Europe (Embree 1966, Roland 1986, Roland and Embree 1995). This species is highly specialized on winter moth and efforts are currently underway (by J.S.E.) to get it established in Massachusetts. Beginning in November 2005, we worked with a team of cooperators in seven northeastern states (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, and Rhode Island) to deploy a grid of pheromone-baited sticky traps to delineate the extent of the winter moth infestation across the region. In November 2006, we expanded the survey to include Quebec City, Canada; Nova Scotia, Canada; and coastal regions of Maine and New Brunswick, Canada. We also trapped in Vermont, southeastern New Hampshire, and New York (Long Island and the Hudson Valley), and we sent a small number of traps to cooperators in Minnesota; New Jersey; Ontario, Canada; Wisconsin; and Michigan. We also sent traps to cooperators in Great Britain and Austria to obtain winter moths from European populations for DNA comparison. In 2007, we deployed traps in areas of the northeastern United States not surveyed the previous 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 the 3-yr period. Materials and Methods Pheromone Synthesis. The pheromone of the winter moth, (1,3Z,6Z,9Z)-1,3,6,9-nonadecatetraene (Roelofs et al. 1982), was prepared following the approach by Bestmann et al. (1982). In 2005, a 90:10 mixture of 3Z and 3E isomers (1,3Z,6Z,9Z-19:H and 1,3E,6Z,9Z19:H) was prepared from the Þnal Wittig reaction using (3Z,6Z)-3,6-hexadecadienyltriphenylphosphonium bromide and butyl lithium as a base. We included the E-isomer because Underhill et al. (1987) indicated it might suppress the trapÕs attractiveness to the Bruce spanworm, but not winter moth. This turned out not to be the case, and in 2006 Ð2008, a 95:5 mixture of 1,3Z,6Z,9Z-19:H and 1,3E,6Z,9Z-19:H was obtained by using sodium bis (trimethylsilyl) amide as a base in the Wittig reaction. The lures were analyzed by gas chromatography (GC) on an 6890N Network GC system (Agilent Technologies, Santa Clara, CA) equipped with a ßame ionization detector and 30- by 0.25-mm (i.d.) DB-210 capillary column (Þlm thickness, 0.25 ␮m) that provided a partial separation of 1,3Z,6Z,9Z-19:H and 1,3E,6Z,9Z-19:H geometric isomers (Underhill et al.

Vol. 103, no. 2

1987). The analyses were conducted in a split mode (50:1) by using H2 as a carrier gas at 1 ml/min. The oven was programmed from 100⬚C (5 min) to 240⬚C at 10⬚C/min, the injector temperature was 260⬚C, and the 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 with 1,000 ␮g blend of 90% (Z,Z,Z)-1,3,6,9-nonadecatetraene (the pheromone) with 10% (E,Z,Z)-1,3,6,9nonadecatetraene (the Bruce spanworm inhibitor). The trapping extended throughout Connecticut, Rhode Island, eastern and central Massachusetts, southeastern New Hampshire, and coastal Maine in a grid pattern with a spacing of ⬇15 km between traps. Traps were deployed in late November and recovered in late December or early January. Additional traps were placed on Long Island and in the Hudson Valley in New York, and in New Jersey. In 2006 and 2007, we switched to large capacity Universal Moth Traps (Great Lakes IPM, Inc., Vestaburg, MI) instead of sticky traps, so that the moths collected would be in better condition for subsequent analysis. Traps were baited with the pheromone only, because we captured Bruce spanworm in all traps in 2005, despite presence of the inhibitor (except for traps that were quickly saturated with winter moths at sites within the outbreak area). We used the software ARCGIS 9.3 (ESRI, Redlands, CA) to produce maps of the moth capture data. Identification of Moths and DNA Extraction. Collected specimens were sent to the Elkinton laboratory at the University of Massachusetts, Amherst, MA. Initial identiÞcation of male winter moths was based on dissection of the male genitalia by G.H.B. (Eidt et al. 1966, Troubridge and Fitzpatrick 1993) because wing patterns and other characteristics are unreliable and due to the poor condition of moths in sticky traps. In 2005, we removed genitalia from 10 moths from each trap (⬇1,500 moths examined) and measured the uncus (Fig. 1) for three characters: 1) general shape, 2) width of the uncus (in micrometers) near its tip, and 3) width of the uncus shaft at its widest point. We also extracted DNA from individual moths and sequenced the cytochrome oxidase subunit 1 (COI) mitochondrial gene (the barcoding region) to conÞrm our ability to distinguish Bruce spanworm from winter moth. We used two commonly used methods for DNA extraction. In 2005, we used a standard CTAB protocol followed by phenol:chloroform extraction. Beginning in 2006, we used the DNeasy Blood and Tissue kit (catalog no. 69504, QIAGEN GmbH, Hilden, Germany) for DNA extraction following manufacturerÕs instructions. Aliquots of genomic DNA were diluted either to 1:10 or 1:20 and stored at ⫺20⬚C for a subsequent use in polymerase chain reaction (PCR) ampliÞcations. PCR Amplification. For the PCR ampliÞcation of COI, we used diluted genomic DNA as the template and commercially synthesized primers (Integrated DNA Technologies, Inc., Coralville, IA). For the ampliÞcation of COI gene, the 20-␮l reaction mixture

March 2010

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

137

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

contained the following: 10 ␮l of HotStarTaq Master Mix (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⬘-TAA ACT TCA GGG TGA CCA AAA AAT CA-3⬘), 0.8 ␮l of 25 mM MgCl2, 3 ␮l of template DNA, and 2.2 ␮l of sterile distilled H2O. The cycling program was: initial activation for 15 min at 95⬚C; 30 cycles of denaturation for 30 s at 94⬚C, annealing for 30 s at 56⬚C, extension for 1 min at 72⬚C; and Þnal extension for 10 min at 72⬚C. For the ampliÞcation of the nuclear gene glucose6-phosphate dehydrogenase (G6PD), two sets of primers were used, one for a regular PCR ampliÞcation and the other for a nested ampliÞcation. The Þrst set of primers was designed by Dr. Baiqing Wang for Colias and Papilio butterßies following the methods of Jiggins (2003), based on sequences in GenBank and Soto-Adames et al. (1988). These were, for the forward primer (G6PD-F), 5⬘-TGC CAA AAG TA/ ideoxyl/ ATG AAA GTT CGG-3⬘, and for the reverse primer (G6PD-R), 5⬘-TTT CCC AAG TAA TGA TCG ATT CTG-3⬘. The nested ampliÞcation, designed for this work, used forward primer (G6PD-nested-F) 5⬘ACT TAT CAC GCT GGT GCC TAC GAT-3⬘ and reverse primer (G6PD-nested-R) 5⬘-ACT TCG CTG AGC TTA CAT CAT CCC-3⬘. The PCR reaction mixtures contained the same reagents as for COI, with the annealing time of 45 s at 50⬚C for 35 cycles for the initial G6PD ampliÞcation, and 45 s at 59⬚C for 30 cycles for the nested ampliÞcation. The ampliÞed DNA products were resolved electrophoretically on 1.5 or 2.0% agarose gels and visualized by ethidium bromide staining. Samples with the correct fragment size were sent out for sequencing to Laragen, Inc. (Los Angeles, CA), and the DNA sequences 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. In previous laboratory studies, Smith and Ring (unpublished, cited in Underhill et al. 1987) bred winter moth females with Bruce spanworm males in cages and produced viable progeny to the F2 generation. Bruce spanworm females did not mate with male winter moths, so any hybrids would have had winter moth mitochondrial genes. To determine whether winter moth was hybridizing with Bruce spanworm in our survey, we Þrst determined whether any specimens had intermediate genitalia, then sequenced the nuclear gene G6PD for these and a sample of morphologically unambiguous moths, including winter moths from Europe. We compared the sequences to identify nucleotides in the G6PD gene that uniquely and reliably distinguished the two species. Hybrids (at least F1 hybrids) would be expected to have both. We conÞrmed this in 2007 by extracting DNA from eggs produced by mating female winter moths collected in British Columbia with male Bruce spanworm collected with pheromone traps in western Massachusetts. Individual mating pairs were held at 16⬚C and a photoperiod of 12:12 [L:D] h, and females were allowed to oviposit on paper strips. Given the small size of the eggs, we pooled all eggs from each female before DNA extraction. Hybrid families exhibit a heterozygous genotypic pattern, i.e., overlapping peaks in the chromatogram, at the species-speciÞc sequence sites in these pooled samples. In 2007, we ran an eastÐwest transect along Route 2 in northern Massachusetts, with traps spaced every three km to further delineate the boundary of the winter moth infestations. Near the boundary of the winter moth infestation that we had mapped in 2005, we decreased the spacing between traps to 1.5 km to provide a more exact delineation of the boundary between the populations. We speculated that in this region we would be most likely to collect hybrids, because in areas further east where winter moth was in outbreak phase, the number of winter moths would vastly outnumber Bruce spanworm, which has re-

138


Vol. 103, no. 2

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

mained at low density everywhere in New England throughout our study. Ground Survey. In May and June 2005, we conducted a ground survey of towns in eastern Massachusetts by car, where we recorded presence and absence of observable defoliation by winter moth. We conÞrmed that defoliation was caused by winter moth by noting the presence of caterpillars. After the caterpillars had pupated in June, we also surveyed defoliation by examining the damage (holes in leaves) caused by the very early feeding of winter moth on unexpanded buds. This winter moth damage is unique from that of other common defoliators such as gypsy moth, Lymantria dispar L., or forest tent caterpillars (Malacosoma disstria Hu¨ bner), which were also present in the region. Results Winter moths were trapped at sites that stretched from eastern Long Island, the southeastern corner of Connecticut, all of Rhode Island, eastern Massachusetts, coastal New Hampshire, and southern coastal

Maine in 2005. They were initially identiÞed based on male genitalia (Fig. 2). In contrast, defoliation by winter moth was observed only in Massachusetts within a region surrounding Boston and on Cape Cod (Fig. 2). We caught winter moths in areas that were at least 50 km from any areas known to be defoliated by winter moths. Traps further west and north caught exclusively Bruce spanworm. These include other traps in New Jersey and the Hudson Valley in New York that are not shown on the map. In 2006, we conÞrmed that both winter moth and Bruce spanworm were present in Nova Scotia and in coastal Maine, but only Bruce spanworm was recovered in coastal New Brunswick, Pennsylvania, Vermont, and Quebec City (Fig. 3A). We also recovered Bruce spanworm but not winter moths in Minnesota; Wisconsin; Michigan; and Sault Ste. Marie, ON, Canada. In 2007, there were no winter moths collected anywhere in New Brunswick or the interior areas of Maine, New Hampshire, or New York, whereas traps in all these locations captured Bruce spanworm (Fig. 3A). The resulting distribution of winter moths in the Northeast has a striking correspondence to the winter

March 2010


139

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

hardiness zones (Cathey 1990; Fig. 3B). Winter moths were recovered in areas corresponding to hardiness zone 5b (minimum temperature, ⫺26 to ⫺23⬚C) but not 5a (⫺29 to ⫺26⬚C). Analysis of COI gene sequences demonstrated very clear differences between the winter moth and Bruce spanworm (Table 1). We sequenced 124 winter moths and 106 Bruce spanworms and found that they differed by 7.4% of the 679 bp in our sequences. The entire 679 bp sequence (GenBank accessions GQ424954 and GQ424955) was identical for all winter moths we analyzed, whereas there was some variation among the Bruce spanworm (Table 1). There was also some variation in the six winter moths we analyzed from Austria and the United Kingdom (Table 1). Measurements of male genitalia were found to be intermediate in the region where both species were captured, suggestive of possible hybrids. Although Bruce spanworm unci were generally longer and thinner (Fig. 1); we identiÞed specimens as intermediates if they fell within the region where measurements overlapped (Fig. 4). We found that 24% of moths in the band where the two populations came together in

Massachusetts and Rhode Island had intermediate genitalia, suggesting that they might be hybrids (Fig. 2). When we sequenced G6PD (Table 2), we found that only 3/158 (1.9%) were unmistakable hybrids. These three moths all came from traps near the center of the 2007 eastÐwest transect in Massachusetts indicated by the closely spaced line of traps (Fig. 3A, inset). Two of these hybrids had winter moth mothers as indicated by their CO1 sequences and one had a Bruce spanworm mother. As with our analysis of CO1, all winter moths that we analyzed had identical G6PD sequences (Table 2), whereas Bruce spanworm showed heterozygosity at some sites. We found six nucleotides that routinely distinguished winter moth from Bruce spanworm (Table 2), but only the Þrst two and the sixth (sequence numbers 21, 23, 141, Table 2) were invariably homozygous in Bruce spanworm. Our DNA analyses revealed that most of moths with intermediate genitalia had winter moth rather than mixed ancestry genotypes. If these intermediates did indeed have mixed ancestry, then they would have originated from backcrosses extending one or more

140


Vol. 103, no. 2

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

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 uncus width (Fig. 4), we discovered a broad range of overlap between the two species, indicating that uncus shape was an imperfect tool for distinguishing the two species. The range of overlap included moths from Europe, where there has been no possibility of hybridization between winter moth and Bruce spanworm. Discussion The COI sequences show that DNA analysis is a deÞnitive method to distinguish these two moth species. It also conÞrms that we have recovered winter moths from near Worcester, MA, ⬇50 km west of any known winter moth infestation. We also caught winter moths at various places along the coast of Maine (Figs. 2 and 3A). Thus, we cannot rule out the possibility that winter moth spread along the coast from Nova Scotia to Massachusetts. However, to our knowledge, there has never been an outbreak of winter moth in Maine or New Brunswick. Review of the Canadian Forest Insect Survey for New Brunswick for 1970 Ð1990 (Natural Resources Canada 2008) conÞrms that winter

moth has not been recovered in interior New Brunswick, but it is sometimes recovered along the coast. It is possible that male moths in coastal Maine and New Brunswick may have blown across the Bay of Fundy from Nova Scotia. Furthermore, in New England we have not recovered either of the two parasitoids [Cyzenis albicans (Falle´ n) and Agrypon flaveolatum (Gravenhorst); J.S.E., unpublished data] that control winter moth in Nova Scotia, from the collection and rearing of several thousand mature winter moth larvae from various sites. These facts may suggest a separate introduction of winter moth to New England instead of spread along the coast, although the source of the populations might still have been Nova Scotia. We found that the COI sequences were identical for all winter moths that we analyzed in North America including moths from Nova Scotia, British Columbia, and New England. The lack of polymorphism in winter moth, in contrast to Bruce spanworm, suggests a possible founder effect from an initial invasion of one or very few individuals and the spread of winter moth from Nova Scotia to New England and to British Columbia. We are currently collecting more winter moths from Europe in hopes of identifying the source population of North American winter moths.

March 2010


141

Table 1. Nucleotide sequences for the barcoding fragment of the mitochondrial gene Cytochrome oxidase subunit 1 (CO1) for all North American winter moths (first line), six winter moths from Europe, and Bruce spanworm from various locations in the northeastern United States Spa Locb Hapc Nd

1,515e

WM-NAmer 1 WM-UK 1 WM-UK 2 WM-UK WM-AUS BS NAmer 1 BS NAmer 2 BS NH BS MA BS VT 1,576 TAAGTTTATT .......... .......... .......... .......... .......... .......... .......... .......... .......... 1,646 TATTGTTACA .......... .......... .......... .......... .......... .......... .......... .......... .......... 1,716 AATTGATTAG .......... .......... .......... .......... ........G. ........G. ........G. ........G. ........G. 1,786 GATTATTACC .......... .......... .......... .......... .......... .......... .......... .......... .......... 1,856 AACTGTTTAC .......... .......... .......... .......... .........T .........T .........T .........T .........T 1,926 CTTCATTTAG .......... ..........

AAGATATTGG .......... .......... .......... .......... .......... .......... .......... .......... ..........

AACTTTATAC .......... .......... .......... .......... .........T .........T .........T .........T .........T

TTTATTTTTG .......... .......... .......... .......... .....C.... .....C.... .....C.... .....C.... .....C....

GAATTTGAGC .......... .......... .......... .......... .......... .......... .......... .......... ..........

CGGTATAATT .......... .........C .........C .......... T.....G... T.....G... T.....G... T.....G... T.....G...

GGAACTTCAC .......... .......... .......... .......... ........TT ........TT ........TT ........TT ........TT

AATTCGAGCT .......... .......... .......... .......... .......... .......... .......... .......... ..........

GAATTAGGTA .......... .......... .......... .......... ........A. ........A. ........A. ........A. ........A.

ACCCTGGTTC .......... .......C.. .......C.. .......... .T..A..... .T..A..... .T..A..... .T..A..... .T..A.....

TTTAATTGGG .......... .......... .......... .......... .......... .........A .......... .......... ..........

GATGACCAAA .......... .......... .......... .......... .......... .......... .......... .......... ..........

TTTACAACAC .......... .......... .......... .......... .......... .......... .......... .......... ..........

GCACATGCTT .......... .......... .......... .......... ........C. ........C. ........C. ........C. ........C.

TTATTATAAT .......... .......... .......... .......... .......... .......... .......... .......... ..........

TTTTTTTATA .......... .......... .......... .......... .........G .........G .........G .........G .........G

GTTATACCAA .......... .......... .......... .......... .......... .......... .......... .......... ..........

TTATAATTGG .......... .......... .......... .......... .......... .......... .......... .......... ..........

AGGATTTGGT .......... .......... .......... .......... .......... .......... .......... .......... ..........

TACCTTTAAT .......... .......... .......... .......... .....C.T.. .....C.T.. .....C.T.. .....C.T.. .....C.T..

ACTTGGAGCT .......... .......... .......... .......... ......G... ......G... ......G... ......G... ......G...

CCTGATATAG .......... .......... .......... .......... .......... .......... .......... .......... ..........

CTTTCCCCCG .......... .......... .......... .......... .......... .......... .......... .......... ..........

AATAAATAAT .......... .......... .......... .......... T......... T......... T......... T......... T.........

ATAAGATTTT .......... .......... .......... .......... .....T.... .....T.... .....T.... .....T.... .....T....

TCCCTCTATT .......... .......... .......... .......... ...G...... ...G...... ...G...... ...G...... ...A......

ACTCTTTTAA .......... .......... .......... .......... ..A..A.... ..A..A.... ..A..A.... ..A..A.... ..A..A....

TTTCTAGAAG .......... .......... .......... .......... .......... .......... .......... .......... ..........

AATTGTAGAA .......... .......... .......... .......... .......... .......... .......... .......... ..........

AATGGGGCAG .......... .....A.... .....A.... .......... .......... .......... .......... .......... ..........

GAACTGGATG .......... .......... .......... .......... .......... .......... .......... .......... ..........

CCCCCTTTAT .......... .......... .......... .......... ..G..CC.G. ..G..CC.G. ..G..CC.G. ..G..CC.A. ..G..CC.A.

CTTCTAATAT .......... .......... .......... .......... .......... .......... .......... .......... ..........

TGCCCATGGA .......... .......... .......... .......... C........G C........G C........G C........G C........G

GGAAGATCCG .......... .......... .......... .......... ........T. ........T. ........T. ........T. ........T.

TAGATCTAGC .......... .......... ........A. .......... .......... .......... .....T.... .......... ..........

TATCTTTTCT .......... .......... .......... .......... ...T.....C ...T.....C ...T.....C ...T.....C ...T.....C

CTGGTATTTC .......... ..........

CTCAATTTTA .......... ..........

GGTGCAATTA .......... ..........

ACTTTATTAC .......... ..........

CACTATTATC .......... ..........

AATATACGAT .......... ..........

31 2 2 1 1 16 9 1 1 1

Continued on following page

142 Table 1. .......... .......... .......... .......... .......... .......... .......... 1996 TAAATAATAT .......... .......... .......... .......... .......... .......... .......... .......... .......... 2,066 ATTGTCATTA .......... .......... .......... .......... .C.A...... .C.A...... .C.A...... .C.A...... .C.A...... 2,136 TTCGATCCTG .......... .......... .......... .......... ..T..C.... ..T..C.... ..T..C.... ..T..C.... ..T..C....


Vol. 103, no. 2

Continued .......... .......... .......... .......... .......... .......... ..........

.......... .......... T......... T......... T......... T......... T.........

.......... .......... ..A....... ..A....... ..A....... ..A....... ..A.......

.......... .......... .T........ .T........ .T........ .T........ .T........

.......... .......... T........T T........T T........T T........T T........T

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

ATTTTTTGAC .......... .......... .......... .........T .........T .........T .........T .........T .........T

CAATTACCAT .......... .......... .......... .......... .......... .......... .......... .......... ..........

TATTTGTTTG .......... .......... .......... .......... .......... .......... .......... .......... ..........

AGCTGTAGGA .......... .......... .......... .......... ...A...... ...A...... ...A...... ...A...... ...A......

ATCACAGCAT .......... .......... .......... .......... ..T....... ..T....... ..T....... ..T....... ..T.......

TTTTACTTTT .......... .......... .......... .......... .....T.A.. .....T.A.. .....T.A.. .....T.A.. .....T.A..

CCAGTATTAG .......... .......... .......... .......... .......... .......... .......... .......... ..........

CGGGAGCTAT .......... .......... .......... .......... .T..G..... .T..G..... .T..G..... .T..G..... .T..G.....

TACTATATTA .......... .......... .......... .......... .......... .......... .......... .......... ..........

TTAACAGATC .......... .......... .......... .......... .......... .......... .......... .......... ..........

TACATCATTT .......... .......... .......... .......... .......... .......... .......... .......... ..........

CTGGGGGGGG .......... ....A..... ....A.... ......... ....A..... ....A..... ....A..... ....A..... ....A.....

AGATCCTATT .......... ..........

CTTTATCAAC .......... ..........

ACTTATTTTG .......... ..........

GAAATTTAAA .......... .......... .......... .......... .......... .......... .......... .......... .......... 2194 ATTTTTTGG ...... ......

G......... G......... G......... G......... G.........

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

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

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

a

Sp, species; WM, winter moth; BS, Bruce spanworm. Location code: NAmer, North America; UK, United Kingdom; AUS, Austria; NH, New Hampshire; ME, Maine; MA, Massachusetts; PA, Pennsylvania; VT, Vermont. c Hap, haplotype. d N, 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 was identiÞed, located and conÞrmed with COI sequence of Charissa crenulata (AJ870408) obtained from the Barcode of Life website, http:// www.barcodinglife.com. b

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

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

(Cathey 1990) provides a possible explanation as to why winter moth became common in Nova Scotia in the 1930s (Embree 1965) but did not spread beyond that region until recently. The spread may have been blocked by unfavorable winter conditions in New Brunswick. Macphee (1967) and Tenow and Nilssen (1990) have shown that winter moth eggs are killed by temperatures below ⫺36⬚C, but winter temperatures rarely get that low in many parts of Maine and New Brunswick where winter moths are absent (Fig. 3B). A similar pattern exists in the birch forests of northern Scandinavia (Jepson et al. 2008), where the distribution of winter moth is seemingly limited by cold winter temperatures in areas that never get as low as ⫺36⬚C. Winter moth has a life cycle that depends on adult ßight and oviposition in late fall and early winter. It is possible that temperatures during this period in New Brunswick and interior Maine may be inimical to this behavior. It is also possible that temperatures in the

March 2010


143

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

1

WM h1 63 BS h1 58 BS h2 33 BS h3 6 Hyb eggs 8 Hyb h1 3 51 CAGGATATTT .......... .......... .......... .......... .......... 111d AAATGCTTGT .......... .......... .......... .......... ..........

GAGTTGCTCA .......... .......... .......... .......... ..........

ATCAATCCAT .......... .......... .......... .......... ..........

CAGCAAAAGT T.T....... T.T....R.. T.T....G.. Y.K....... Y.K.......

GAAAAAGGAC .......... .......... .......... .......... ..........

CTGTGGCGAA .......... .......... .......... .......... ..........

TACCTAGCAG .....G.... .....R.... .......... .....R.... .....R....

TGCCACCCAC .......... .......... .......... .......... ..........

TGTATTTGAA .......... .......... .......... .......... ..........

GAAGTGACCG ........T. ........Y. .......... ........Y. ........Y.

TCAACATTAG .......... .......... .......... .......... ..........

GTATCCATTA .C........ .Y........ .......... .Y........ .Y........

AAGGATACAC .......... .......... .......... .......... ..........

CCGAGTCATT T......... T......... T......... Y......... Y.........

ATTGAAAAAC .......... .......... .......... .......... ..........

CATTTGGA ........ ........ ........ ........ ........

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. c N, number of moths in category out of 63 winter moths, 95 Bruce spanworm and 3 hybrids sequenced. All moths came from North America. d Number 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.

spring may play a role. Tenow and Nilssen (1990) demonstrated that winter moth eggs become less cold tolerant in the weeks before hatch. Whatever the life stage affected, limitation by winter temperatures may also explain why there has never been, to our knowledge, a winter moth outbreak in Maine, even though we have shown that winter moth occurs in coastal Maine where temperatures are warmer (Fig. 3). It is only when winter moths became established in eastern Massachusetts and southeastern New Hampshire that they encountered winter temperatures comparable with those that exist in maritime Nova Scotia. In northern Scandinavia, global climate change has produced warmer winters and as a result the range of winter moth outbreaks has expanded into colder areas at higher altitude or further from the coast (Jepson et al. 2008). Warming climate may thus play an important role in the future distribution of winter moth in North America. Of course, it is possible that the observed correspondence of the distribution of winter moth with the warmer cold-hardiness zones is caused by the other factors correlated to winter temperatures. For example, the prevalence of acceptable host plants may be an important factor. Deciduous trees such as oaks and maples are the dominant forest trees in Massachusetts, southern Maine, and New Hampshire, in contrast to the Picea (spruce)ÐAbies (Þr) forests of northern Maine and New Brunswick. Nova Scotia, however, is also dominated by spruceÐÞr forests (Rowe 1959), despite the warmer winter temperatures similar to those of Massachusetts. Winter moth there survives on pockets of deciduous trees such as Quercus rubra L. or in abandoned apple (Malus spp.) orchards (Embree 1965). Our DNA analysis largely conÞrmed the use of genital characters to distinguish winter moth and Bruce

spanworm males. Both methods for species identiÞcation had pros and cons. Our uncus measurements included winter moth specimens from Europe, where Bruce spanworm does not exist, suggesting that the overlap in shape is not due to introgression of genitalia traits via hybridization. We were therefore not always able to determine species based on examination of genitalia. IdentiÞcation of the two species via DNA sequence was more reliable, but much more costly. Also, when pheromone-baited traps were left in the Þeld for several weeks, or Þlled with rain, the moths often decayed and as a result we were unable to extract DNA, yet the uncus was always in good condition. Furthermore, the COI sequences can only identify the maternal line and cannot distinguish hybrids. We are currently developing a robust, yet less expensive, DNA identiÞcation method for these two species and their hybrids, based on restriction enzyme tests that will avoid the expense of DNA sequencing. The occurrence of hybrids has important implications for the future spread of winter moth and the success of the ongoing biological control effort against winter moth. Our survey shows that Bruce spanworm is able to persist in areas with much colder winters than winter moth. Although we do not know the genetic basis of this difference, it is possible that whatever traits that confer this ability could pass from Bruce spanworm to winter moth via hybridization, allowing winter moth to expand into northern areas of eastern North America. Also, we are currently releasing the parasitoid C. albicans to control winter moth in New England. This parasitoid was very successful in permanently reducing outbreak populations of winter moth in Nova Scotia in the 1950s and in the PaciÞc Northwest in the 1980s. According to Roland and

144


Embree (1995), C. albicans does not attack Bruce spanworm under Þeld conditions, although it will attack Bruce spanworm in the laboratory. If this functional immunity to C. albicans has a genetic basis, then this trait also could pass from Bruce spanworm to winter moth via hybridization. In Nova Scotia and British Columbia it seems that winter moth remains under good biological control, but research by Roland (1990, 1994) in British Columbia and Pearsall and Walde (1994) in Nova Scotia suggests that it is predators and not C. albicans that maintains winter moth at innocuous densities. Indeed, the rates of parasitism in both of these regions by C. albicans are much lower now than the rate reported by Embree (1965) after the initial introduction. These previous researchers assumed that this decline in parasitism was caused by the lower densities of winter moth, but nothing is known about the degree of hybridization with Bruce spanworm in Canada or its possible inßuence on dynamics of winter moth.

Acknowledgments We thank the following people for deploying and retrieving the pheromone traps and for sending us the moths from the 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; New Jersey: S. Vaiciunas, R. Fine, J. Simmons; Wisconsin: A. DissTorrance, B. Schwingle, L. Williams; Michigan: D. McCullough; 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. Vanbergen, A. Watt. We thank I. Otvos and N. Condor for sending 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 Disease survey regarding winter moth in New Brunswick. We thank Gloria Witkus for help in the Þeld and with dissections, Natalie Leva for mailing traps to our cooperators, Jeremey Anderson for assisting with GenBank submissions, Jeff Ahern for assistance in initiating the DNA analysis, and Maili Page and Diana Barscz for assistance in producing maps. This research was made possible, in part, by a cooperative agreement (05-8225-0464-CA) from the U.S. Department of AgricultureÕs Animal and Plant Health Inspection Service (APHIS). We are also grateful to the USDA Forest Service (cooperative agreement 04-CA-11244225-414), and a grant from the Massachusetts State Legislature for supporting this work.

References Cited Baker, W. L. 1972. Eastern forest insects, pp. 335Ð336. U.S. Dep. Agric. Forest Service, Misc. Publ. No. 1175. Bestmann, H. J., T. Brosche, K. H. Koschatzky, K. Michaelis, H. Platz, K. Roth, J.S.O. Vostrowsky, and W. Knauf. 1982. Pheromone-XLII 1,3,6,9-nonadecatetraen, das sexual

Vol. 103, no. 2

pheromone des frostspanners Operophtera brumata (Geometridae). Tetrahedron Lett. 23: 4007Ð 4010. Cathey, H. M. 1990. U.S. Dep. Agric. plant hardiness zone map. U.S. Dep. Agric. Misc. Publ. No. 1475. Eidt, D. C., D. G. Embree, and C. C. Smith. 1966. Distinguishing adults of the winter moth Operophtera brumata and Bruce spanworm Operophtera bruceata. Can. Entomol. 98: 258 Ð261. Embree, D. G. 1965. The population dynamics of the winter moth in Nova Scotia, 1954 Ð 62. Mem. Entomol. Soc. Can. 46: 1Ð57. Embree, D. G. 1966. The role of introduced parasites in control of winter moth in Nova Scotia. Can. Entomol. 98: 1159 Ð1168. Embree, D. G. 1991. The winter moth Operophtera brumata in eastern Canada, 1962Ð1988. For. Ecol. Manage. 39: 47Ð54. Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek. 1994. DNA primers for ampliÞcation of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294 Ð299. Jepson, J. U., S. B. Hagen, R. A. Ims, and N. G. Yoccoz. 2008. Climate change and outbreaks of the Geometrids. Operophtera brumata and Epirrita autumnata in subarctic birch forest: evidence of a recent outbreak range expansion. J. Anim. Ecol. 77: 257Ð264. Jiggins, F. M. 2003. Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and parasite population dynamics. Genetics 164: 5Ð12. Macphee, A. W. 1967. Winter moth, Operophtera brumata Lepidoptera: Geometridae), a new pest attacking apple orchards in Nova Scotia and its coldhardiness. Can. Entomol. 99: 829 Ð 834. Natural Resources Canada. 2008. Canadian Forest Invasive Alien Species Database. Canadian Forest Service, Atlantic Forestry Centre. Fredericton, NB, Canada. (https:// afc-fr.cfsnet.nÞs.org/Þasdb/srch_rch.jsp). Pearsall, I. A., and S. J. Walde. 1994. Parasitism and predation as agents of mortality of winter moth populations in neglected apple orchards in Nova-Scotia. Ecol. Entomol. 19: 190 Ð198. Pivnick, K. A., D. L. Barton, J. G. Millar, and E. W. Underhill. 1988. Improved exclusion of the Bruce spanworm Operophtera bruceata when monitoring populations of winter moth Operophtera brumata. Can. Entomol. 120: 389 Ð396. Roelofs, W. L., A. S. Hill, C. E. Linn, J. Meinwald, S. C. Jain, H. J. Herbert, and R. F. Smith. 1982. Sex pheromone of the winter moth, a geometrid with unusual low temperature precopulatory responses. Science (Wash., D.C.) 217: 657Ð 659. Roland, J. 1986. Parasitism of winter moth in British Columbia during buildup of its parasitoid Cyzenis albicansÑ attack rate on oak v. apple. J. Anim. Ecol. 55: 215Ð234. Roland, J. 1990. Interaction of parasitism and predation in the decline of winter moth in Canada, pp. 289 Ð301. In A. D. Watt, S. R. Leather, M. D. Hunter, and N.A.C. Kidd [eds.], Population dynamics of forest insects. Intercept, Andover, United Kingdom. Roland, J. 1994. After the decline: what maintains low winter moth density after successful biological control? J. Anim. Ecol. 63: 392Ð398. Roland, J., and D. G. Embree. 1995. Biological control of the winter moth. Annu. Rev. Entomol. 40: 475Ð 492. Rowe, J. S. 1959. Forest regions of Canada. Department of Northern Affairs and National Resources, Forestry Branch, 7100, Ottawa, ON, Canada. Soto-Adames, F. N., H. M. Robertson, and S. H. Berlocher. 1988. Phylogenetic utility of partial DNA sequences of

March 2010


G6PD at different taxonomic levels in Hexapoda with emphasis on Diptera. Ann. Entomol. Soc. Am. 87: 723Ð736. Tenow, O., and A. C. Nilssen. 1990. Egg cold hardiness and topoclimatic limitations of outbreaks of Epirrita autumnata in Northern Fennoscandia. J. Appl. Ecol. 27: 723Ð734. Troubridge, J. T., and S. M. Fitzpatrick. 1993. A revision of the North American Operophtera. Can. Entomol. 125: 379Ð397.

145

Underhill, E. W., J. G. Millar, R. A. Ring, J. W. Wong, D. Barton, and M. Giblin. 1987. Use of a sex attractant and an inhibitor for monitoring winter moth and Bruce spanworm populations. J. Chem. Ecol. 13: 1319 Ð1330. Received 13 August 2009; accepted 4 November 2009.