Evidence for adaptation of Rhagoletis pomonella (Diptera: Tephritidae) on large-thorn hawthorn, Crataegus macracantha, in Okanogan County, Washington State, USA.

The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), was introduced from eastern North America into western North America via infested apples (Malus domestica Borkhausen) about 44 yr ago, where it subsequently adapted to 2 hawthorn species, Crataegus douglasii Lindley and C. monogyna Jacquin. Here, we test whether R. pomonella has also adapted to large-thorn hawthorn, Crataegus macracantha Loddiges ex Loudon, in Okanogan County, Washington State, USA. In 2020, 2021, and 2022, fruit of C. macracantha were shown to ripen in late September and were infested at rates from 0.7% to 3.0%. In laboratory rearing studies, large-thorn hawthorn flies from C. macracantha eclosed on average 9-19 days later than apple flies from earlier ripening apple (August-early September), consistent with large-thorn hawthorn flies having adapted to the later fruiting phenology of its host. In a laboratory no-choice test, significantly fewer (64.8%) large-thorn hawthorn than apple flies visited apples. In choice tests, greater percentages of large-thorn hawthorn than apple flies resided on and oviposited into C. macracantha versus apple fruit. Large-thorn hawthorn flies were also smaller in size than apple flies. Our results provide further support for the recursive adaptation hypothesis that R. pomonella has rapidly and independently specialized phenologically and behaviorally to different novel hawthorn hosts since its introduction into the Pacific Northwest of the USA, potentially leading to host race formation.

Sensitivities to Chill Durations and No-Chill Temperatures Regulating Eclosion Responses Differ Between Rhagoletis zephyria (Diptera: Tephritidae) and its Braconid Parasitoids (Hymenoptera: Braconidae).

Seasonal temperatures select for eclosion timing of temperate insects and their parasitoids. In western North America, the fruit fly Rhagoletis zephyria Snow (Diptera: Tephritidae) is parasitized by the hymenopterous wasps Utetes lectoides (Gahan), an egg parasite, and Opius downesi Gahan, a larval parasite (both Braconidae). Eclosion of wasps should be timed with the presence of susceptible fly stages, but reports indicate U. lectoides ecloses in the absence of flies under no-chill conditions. Based on this, we tested the hypotheses that chill durations and no-chill temperatures both differentially regulate eclosion times of R. zephyria and its parasitic wasps. When fly puparia were chilled at ~3°C for 130-180 d, U. lectoides and O. downesi always eclosed on average later than flies. However, after 180-d chill, flies eclosed on average earlier than after 130- and 150-d chill, whereas eclosion times of U. lectoides and O. downesi were less or not affected by chill duration. When fly puparia were exposed to 20-22°C (no chill), U. lectoides eclosed before flies, with 88.9% of U. lectoides versus only 0.61% of flies eclosing. Taken together, findings show that eclosion times of flies are more sensitive to changes in chill duration than those of wasps. Flies are less sensitive than wasps to no-chill in that most flies do not respond by eclosing after no-chill while most wasps do. Our results suggest that shorter winters and longer summers due to climate change could cause mismatches in eclosion times of flies and wasps, with potentially significant evolutionary consequences.

Testing the potential contribution of Wolbachia to speciation when cytoplasmic incompatibility becomes associated with host-related reproductive isolation.

Endosymbiont-induced cytoplasmic incompatibility (CI) may play an important role in arthropod speciation. However, whether CI consistently becomes associated or coupled with other host-related forms of reproductive isolation (RI) to impede the transfer of endosymbionts between hybridizing populations and further the divergence process remains an open question. Here, we show that varying degrees of pre- and postmating RI exist among allopatric populations of two interbreeding cherry-infesting tephritid fruit flies (Rhagoletis cingulata and R. indifferens) across North America. These flies display allochronic and sexual isolation among populations, as well as unidirectional reductions in egg hatch in hybrid crosses involving southwestern USA males. All populations are infected by a Wolbachia strain, wCin2, whereas a second strain, wCin3, only co-infects flies from the southwest USA and Mexico. Strain wCin3 is associated with a unique mitochondrial DNA haplotype and unidirectional postmating RI, implicating the strain as the cause of CI. When coupled with nonendosymbiont RI barriers, we estimate the strength of CI associated with wCin3 would not prevent the strain from introgressing from infected southwestern to uninfected populations elsewhere in the USA if populations were to come into secondary contact and hybridize. In contrast, cytoplasmic-nuclear coupling may impede the transfer of wCin3 if Mexican and USA populations were to come into contact. We discuss our results in the context of the general paucity of examples demonstrating stable Wolbachia hybrid zones and whether the spread of Wolbachia among taxa can be constrained in natural hybrid zones long enough for the endosymbiont to participate in speciation.

Distinct Adult Eclosion Traits of Sibling Species Rhagoletis pomonella and Rhagoletis zephyria (Diptera: Tephritidae) Under Laboratory Conditions.

Closely related phytophagous insects that specialize on different host plants may have divergent responses to environmental factors. Rhagoletis pomonella (Walsh) and Rhagoletis zephyria Snow (Diptera: Tephritidae) are sibling, sympatric fly species found in western North America that attack and mate on plants of Rosaceae (~60 taxa) and Caprifoliaceae (three taxa), respectively, likely contributing to partial reproductive isolation. Rhagoletis zephyria evolved from R. pomonella and is native to western North America, whereas R. pomonella was introduced there. Given that key features of the flies' ecology, breeding compatibility, and evolution differ, we predicted that adult eclosion patterns of the two flies from Washington State, USA are also distinct. When puparia were chilled, eclosion of apple- and black hawthorn-origin R. pomonella was significantly more dispersed, with less pronounced peaks, than of snowberry-origin R. zephyria within sympatric and nonsympatric site comparisons. Percentages of chilled puparia that produced adults were ≥67% for both species. However, when puparia were not chilled, from 13.5 to 21.9% of apple-origin R. pomonella versus only 1.2% to 1.9% of R. zephyria eclosed. The distinct differences in eclosion traits of R. pomonella and R. zephyria could be due to greater genetic variation in R. pomonella, associated with its use of a wider range of host plants than R. zephyria.

Rapid and repeatable host plant shifts drive reproductive isolation following a recent human-mediated introduction of the apple maggot fly, Rhagoletis pomonella.

Ecological speciation via host-shifting is often invoked as a mechanism for insect diversification, but the relative importance of this process is poorly understood. The shift of Rhagoletis pomonella in the 1850s from the native downy hawthorn, Crataegus mollis, to introduced apple, Malus pumila, is a classic example of sympatric host race formation, a hypothesized early stage of ecological speciation. The accidental human-mediated introduction of R. pomonella into the Pacific Northwest (PNW) in the late 1970s allows us to investigate how novel ecological opportunities may trigger divergent adaptation and host race formation on a rapid timescale. Since the introduction, the fly has spread in the PNW, where in addition to apple, it now infests native black hawthorn, Crataegus douglasii, and introduced ornamental hawthorn, Crataegus monogyna. We use this "natural experiment" to test for genetic differentiation among apple, black, and ornamental hawthorn flies co-occurring at three sympatric sites. We report evidence that populations of all three host-associations are genetically differentiated at the local level, indicating that partial reproductive isolation has evolved in this novel habitat. Our results suggest that conditions suitable for initiating host-associated divergence may be common in nature, allowing for the rapid evolution of new host races when ecological opportunity arises.

Geographic and Ecological Dimensions of Host Plant-Associated Genetic Differentiation and Speciation in the Rhagoletis cingulata (Diptera: Tephritidae) Sibling Species Group.

Ascertaining the causes of adaptive radiation is central to understanding how new species arise and come to vary with their resources. The ecological theory posits adaptive radiation via divergent natural selection associated with novel resource use; an alternative suggests character displacement following speciation in allopatry and then secondary contact of reproductively isolated but ecologically similar species. Discriminating between hypotheses, therefore, requires the establishment of a key role for ecological diversification in initiating speciation versus a secondary role in facilitating co-existence. Here, we characterize patterns of genetic variation and postzygotic reproductive isolation for tephritid fruit flies in the Rhagoletis cingulata sibling species group to assess the significance of ecology, geography, and non-adaptive processes for their divergence. Our results support the ecological theory: no evidence for intrinsic postzygotic reproductive isolation was found between two populations of allopatric species, while nuclear-encoded microsatellites implied strong ecologically based reproductive isolation among sympatric species infesting different host plants. Analysis of mitochondrial DNA suggested, however, that cytoplasmic-related reproductive isolation may also exist between two geographically isolated populations within R cingulata. Thus, ecology associated with sympatric host shifts and cytoplasmic effects possibly associated with an endosymbiont may be the key initial drivers of the radiation of the R. cingulata group.

Comparative Responses of Rhagoletis zephyria and Rhagoletis pomonella (Diptera: Tephritidae) to Commercial and Experimental Sticky Traps and Odors in Washington State.

Rhagoletis zephyria Snow and Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) are morphologically similar flies that attack white-colored snowberry fruit (Symphoricarpos spp.) and yellow/red or dark-colored apple/hawthorn fruit (Malus/Crataegus spp.), respectively. The two flies are caught together on traps in R. pomonella surveys in the western United States, increasing labor needed to process catches. Comparing responses of the two species with different traps could help identify best practices for reducing R. zephyria captures in these surveys and could contribute to understanding population divergence in Rhagoletis flies. In Washington State, United States, we found that R. zephyria responded most to yellow rectangles and more to white than red spheres (RSs) baited with ammonium carbonate (AC), whereas R. pomonella responded most to RSs with AC. Yellow plastic rectangles with AC were more effective in capturing R. zephyria than cardboard rectangles, as has been found for R. pomonella. R. zephyria did not respond to apple fruit volatiles associated with RSs that were attractive to R. pomonella. In contrast, R. zephyria responded more to yellow rectangles with snowberry than apple volatiles. Both species responded to AC. Our results suggest that RSs are better than yellow rectangles for surveying R. pomonella when snowberries are abundant. However, if discrimination from R. zephyria is paramount, RSs with apple volatiles should be used. Differences in the species' responses to traps appear related to odor/color cues of the flies' host fruit, while commonalties appear related to visual/olfactory stimuli associated with protein feeding, for which AC is a general attractant.

Genetic Evidence for the Introduction of Rhagoletis pomonella (Diptera: Tephritidae) into the Northwestern United States.

The apple maggot fly, Rhagoletis pomonella Walsh (Diptera: Tephritidae), is a serious quarantine pest in the apple-growing regions of central Washington and Oregon. The fly is believed to have been introduced into the Pacific Northwest via the transport of larval-infested apples near Portland, Oregon, within the last 40 yr. However, R. pomonella also attacks native black hawthorn, Crataegus douglasii Lindley (Rosales: Rosaceae), and introduced ornamental hawthorn, Crataegus monogyna Jacquin, in the region. It is, therefore, possible that R. pomonella was not introduced but has always been present on black hawthorn. If true, then the fly may have independently shifted from hawthorn onto apple in the Pacific Northwest within the last 40 yr after apples were introduced. Here, we test the introduction hypothesis through a microsatellite genetic survey of 10 R. pomonella sites in Washington and 5 in the eastern United States, as well as a comparison to patterns of genetic variation between populations of Rhagoletis cingulata Loew and Rhagoletis indifferens Curran, two sister species of cherry-infesting flies known to be native to the eastern and western United States, respectively. We report results based on genetic distance networks, patterns of allelic variation, and estimated times of population divergence that are consistent with the introduction hypothesis for R. pomonella. The results have important implications for R. pomonella management, suggesting that black hawthorn-infesting flies near commercial apple-growing regions of central Washington may harbor sufficient variation to utilize apple as an alternate host, urging careful monitoring, and possible removal of hawthorn trees near orchards.

Identification of Host Fruit Volatiles from Snowberry (Symphoricarpos albus), Attractive to Rhagoletis zephyria Flies from the Western United States.

A mixture of behaviorally active volatiles was identified from the fruit of snowberry, Symphoricarpos albus laevigatus, for Rhagoletis zephyria flies reared from snowberry fruit. A nine-component blend containing 3-methylbutan-1-ol (3%), dimethyl trisulfide (1%), 1-octen-3-ol (40%), myrcene (8%), nonanal (9%), linalool (13%), (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, 6%), decanal (15%), and ß-caryophyllene (5%) was identified that gave consistent electroantennogram activity and was behaviorally active in flight tunnel tests. In other flight tunnel assays, snowberry flies from two sites in Washington state, USA, displayed significantly greater levels of upwind oriented flight to sources with the snowberry volatile blend compared with previously identified volatile blends from domestic apple (Malus domestica) and downy hawthorn (Crataegus mollis) fruit from the eastern USA, and domestic apple, black hawthorn (C. douglasii) and ornamental hawthorn (C. monogyna) from Washington state. Selected subtraction assays showed that whereas removal of DMNT or 1-octen-3-ol significantly reduced the level of upwind flight, removal of myrcene and ß-caryophyllene, or dimethyl trisulfide alone did not significantly affect the proportion of upwind flights. Our findings add to previous studies showing that populations of Rhagoletis flies infesting different host fruit are attracted to unique mixtures of volatile compounds specific to their respective host plants. Taken together, the results support the hypothesis that differences among flies in their behavioral responses to host fruit odors represent key adaptations involved in sympatric host plant shifts, contributing to host specific mating and generating prezygotic reproductive isolation among members of the R. pomonella sibling species complex.

Hybridization and the spread of the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae), in the northwestern United States.

Hybridization may be an important process interjecting variation into insect populations enabling host plant shifts and the origin of new economic pests. Here, we examine whether hybridization between the native snowberry-infesting fruit fly Rhagoletis zephyria (Snow) and the introduced quarantine pest R. pomonella (Walsh) is occurring and may aid the spread of the latter into more arid commercial apple-growing regions of central Washington state, USA. Results for 19 microsatellites implied hybridization occurring at a rate of 1.44% per generation between the species. However, there was no evidence for increased hybridization in central Washington. Allele frequencies for seven microsatellites in R. pomonella were more 'R. zephyria-like' in central Washington, suggesting that genes conferring resistance to desiccation may be adaptively introgressing from R. zephyria. However, in only one case was the putatively introgressing allele from R. zephyria not found in R. pomonella in the eastern USA. Thus, many of the alleles changing in frequency may have been prestanding in the introduced R. pomonella population. The dynamics of hybridization are therefore complex and nuanced for R. pomonella, with various causes and factors, including introgression for a portion, but not all of the genome, potentially contributing to the pest insect's spread.

Chilling and Host Plant/Site-Associated Eclosion Times of Western Cherry Fruit Fly (Diptera: Tephritidae) and a Host-Specific Parasitoid.

The western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), is an endemic herbivore of bitter cherry, Prunus emarginata (Douglas ex Hooker) Eaton, but ∼100 years ago established on earlier-fruiting domesticated sweet cherry, Prunus avium (L.) L. Here, we determined if eclosion times of adult R. indifferens from sweet and bitter cherry differ according to the phenology of their respective host plants and if eclosion times of the host-specific parasitoid Diachasma muliebre (Muesebeck) (Hymenoptera: Braconidae) attacking bitter and sweet cherry flies differ according to the eclosion phenology of their fly hosts. Fly pupae from sweet and bitter cherry fruit were collected from sympatric and allopatric sites in Washington state, and chilled at 5°C. Because timing of eclosion in R. indifferens depends on chill duration, eclosion time in wasps could also vary with chill duration. To account for this, fly pupae were chilled for 1, 2, 2.5, 3, 4, 6, or 8 mo. Both flies and wasps eclosed earlier with longer chill durations. Eclosion times of sweet and bitter cherry flies from a sympatric site in central Washington did not differ. However, at allopatric sites in northwestern and central Washington, bitter cherry flies eclosed later than sweet and bitter cherry flies at the sympatric site. Correspondingly, D. muliebre parasitizing a more isolated bitter cherry fly population eclosed later than D. muliebre parasitizing earlier-emerging sweet and bitter cherry fly populations. These results provide evidence for D. muliebre rapidly responding to changes in host plant shifts by R. indifferens.

Ammonium carbonate is more attractive than apple and hawthorn fruit volatile lures to Rhagoletis pomonella (Diptera: Tephritidae) in Washington State.

The apple maggot fly, Rhagoletis pomonella (Walsh), is an introduced, quarantine pest of apple (Malus domestica Borkhausen) in the Pacific Northwest of the United States. In the eastern United States where the fly is native, fruit volatiles have been reported to be more attractive than ammonia compounds to R. pomonella. However, the opposite may be true in the western United States. Here, we determined whether newly identified western apple and western hawthorn fruit volatiles are more attractive than ammonium carbonate (AC) to R. pomonella in apple, black hawthorn, and ornamental hawthorn trees in western Washington State. In all three host trees, sticky red sphere or yellow panel traps baited with AC generally caught more flies than traps baited with lures containing the four newly developed fruit blends (modified eastern apple, western apple, western ornamental hawthorn, and western black hawthorn) or two older blends (eastern apple and eastern downy hawthorn). Fruit volatiles also displayed more variation among trapping studies conducted at different sites, in different host trees, and across years than AC. The results imply that traps baited with AC represent the best approach to monitoring R. pomonella in Washington State.

Behavioral evidence for fruit odor discrimination and sympatric host races of Rhagoletis pomonella flies in the Western United States.

The recent shift of Rhagoletis pomonella (Diptera: Tephritidae) from its native host downy hawthorn, Crataegus mollis, to introduced domesticated apple, Malus domestica, in the eastern United States is a model for sympatric host race formation. However, the fly is also present in the western United States, where it may have been introduced via infested apples within the last 60 years. In addition to apple, R. pomonella also infests two hawthorns in the West, one the native black hawthorn, C. douglasii, and the other the introduced English ornamental hawthorn, C. monogyna. Here, we test for behavioral evidence of host races in the western United States. through flight tunnel assays of western R. pomonella flies to host fruit volatile blends. We report that western apple, black hawthorn, and ornamental hawthorn flies showed significantly increased levels of upwind-directed flight to their respective natal compared to nonnatal fruit volatile blends, consistent with host race status. We discuss the implications of the behavioral results for the origin(s) of western R. pomonella, including the possibility that western apple flies were not introduced, but may represent a recent shift from local hawthorn fly populations.

Identification of host fruit volatiles from domestic apple (Malus domestica), native black hawthorn (Crataegus douglasii) and introduced ornamental hawthorn (C. monogyna) attractive to Rhagoletis pomonella flies from the western United States.

The apple maggot fly, Rhagoletis pomonella, infests apple (Malus domestica) and hawthorn species (most notably the downy hawthorn, Crataegus mollis) in the eastern USA. Evidence suggests that the fly was introduced into the western USA sometime in the last 60 years. In addition to apple, R. pomonella also infests two species of hawthorns in the western USA as major hosts: the native black hawthorn (C. douglasii) and the introduced ornamental English hawthorn, C. monogyna. Apple and downy hawthorn-origin flies in the eastern USA use volatile blends emitted from the surface of their respective ripening fruit to find and discriminate among host trees. To test whether the same is true for western flies, we used coupled gas chromatography and electroantennographic detection (GC-EAD) and developed a 7-component apple fruit blend for western apple-origin flies, an 8-component black hawthorn fruit blend for flies infesting C. douglasii, and a 9-component ornamental hawthorn blend for flies from C. monogyna. Crataegus douglasii and C. monogyna-origin flies showed similar levels of upwind directed flight to their respective natal synthetic fruit blends in flight tunnel assays compared to whole fruit adsorbent extracts, indicating that the blends contain all the behaviorally relevant fruit volatiles to induce maximal response levels. The black and ornamental hawthorn blends shared four compounds in common including 3-methylbutan-1-ol, which appears to be a key volatile for R. pomonella populations in the eastern, southern, and western USA that show a preference for fruit from different Crataegus species. However, the blends also differed from one another and from domesticated apple in several respects that make it possible that western R. pomonella flies behaviorally discriminate among fruit volatiles and form ecologically differentiated host races, as is the case for eastern apple and hawthorn flies.

Abundance of apple maggot, Rhagoletis pomonella, across different areas in central Washington, with special reference to black-fruited hawthorns.

The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), infests non-commercial apple (Malus domestica (Borkh.) Borkh.) and native black-fruited hawthorns (mostly Crataegus douglasii Lindl.) in central Washington, but little has been published on the abundance of the fly in this region. In this paper, the abundance of R. pomonella across different sites near apple-growing areas in central Washington is documented in order to assess the threat of the fly to commercial apple orchards. The fly was first detected on traps in Klickitat, Yakima, and Kittitas Counties in 1981, 1995, and 1997, respectively. From 1981-2010 in Kittitas and Yakima Counties, only 0 to 4.7% of traps on apple, crabapple, and hawthorn trees were positive for flies, whereas in Klickitat County, located farther from commercial apple orchards, 0 to 41.9% of traps were positive. In 2008, in Yakima County and Goldendale in Klickitat County, 7.8% of black-fruited hawthorn trees were infested, with 0 to 0.00054 larvae per fruit. In 2010, in Kittitas and Yakima Counties and Goldendale in Klickitat County, 25.0% of C. douglasii trees were infested, with 0.00042 to 0.00248 larvae per fruit. In 2010, in a remote forested area of Klickitat County far from commercial apple orchards, 94.7% of C. douglasii trees were infested, with 0.20813 larvae per fruit. Overall results suggest R. pomonella is unlikely to develop high populations rapidly near major commercial apple-growing areas in central Washington, including in black-fruited hawthorns, increasing chances it can be kept out of commercial orchards.

Differences in body size and egg loads of Rhagoletis indifferens (Diptera: Tephritidae) from introduced and native cherries.

The western cherry fruit fly, Rhagoletis indifferens Curran, infests introduced, domesticated sweet [Prunus avium (L.) L.], and tart cherries (Prunus cerasus L.) as well as native bitter cherry, Prunus emarginata (Douglas) Eaton. Bitter cherries are smaller than sweet and tart cherries and this could affect various life history traits of flies. The objectives of the current study were to determine 1) if body size and egg loads of flies infesting sweet, tart, and bitter cherries differ from one another; and 2) if any observed body size differences are genetically based or caused by the host fruit environment. Pupae and adults of both sexes reared from larval-infested sweet and tart cherries collected in Washington and Montana were larger than those reared from bitter cherries. In addition, flies of both sexes caught on traps in sweet and tart cherry trees were larger than those caught in bitter cherry trees and females trapped from sweet and tart cherry trees had 54.0-98.8% more eggs. The progeny of flies from naturally-infested sweet and bitter cherries reared for one generation in the laboratory on sweet cherry did not differ in size. The same also was true for progeny of sweet and bitter cherry flies reared in the field on bitter cherry. The results suggest that the larger body sizes of flies from sweet and tart cherries than bitter cherries in the field are caused by host fruit and not genetic factors.