Parasitism of the Invasive Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae), by the Native Parasitoid, Trichopoda pennipes (Diptera: Tachinidae)

The invasive brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), has been an important agricultural pest in the Mid-Atlantic United States since its introduction in 1996. Biological control by native species may play an important role in suppressing H. halys populations and reduce reliance on chemical control. We collected H. halys adults in agricultural areas of five Pennsylvania counties over two years to examine the extent and characteristics of adult stink bug parasitism by Trichopoda pennipes (Diptera: Tachinidae), a native parasitoid of hemipterans. The overall parasitism rate (in terms of T. pennipes egg deposition) was 2.38 percent. Rates differed among counties and seasons, but not between years. Instances of supernumerary oviposition were evident, and eggs were more commonly found on the ventral side of the thorax, although no differences in egg deposition were found between males and female hosts. T. pennipes has begun to target H. halys adults in Pennsylvania and has the potential to play a role in regulating this pest. Adult parasitism of H. halys by T. pennipes should continue to be monitored, and landscape management and ecological pest management practices that conserve T. pennipes populations should be supported in agricultural areas where H. halys is found.


Introduction
The invasive brown marmorated stink bug, Halyopmorpha halys Stål (Hemiptera: Pentatomidae), was first found in Allentown, PA in the United States in 1996 [1] and has since spread to many states throughout the country, as well as to Canada, Europe and South America [2,3]. H. halys, which is native to Eastern Asia, has become an economically important pest of many crops in the U.S., especially tree fruit in the Mid-Atlantic states [3,4]. For example, in Eastern U.S. apples and peaches, H. halys was shown to damage up to 25% of fruit per tree [5], and losses in apple production exceeded $37 million in 2010 alone [3]. As a result, the frequency and intensity of insecticide applications to control H. halys in orchards has also increased substantially [6]. There are relatively few insecticides that provide reliable control of H. halys adults, which fly into the orchards from adjacent managed and unmanaged habitats in the late summer and fall close to harvest for apples and peaches. This further restricts insecticide control options to those with short pre-harvest label restrictions. The majority of insecticides have limited residual activity against the adults, whereas eggs and nymphs are easier to control [7]. As an agricultural pest, H. halys is a promising candidate for biological regulation by predators and parasitoids, since it spends the majority of the fruit-growing season in unmanaged, wooded habitats rather than in agricultural crops where pesticides can limit biological control [8].
In commercial agriculture settings, as well as other ecosystems, the decoupling of invasive pests from their native biocontrol agents is often thought to be the most probable mechanism by which invasive species become extremely successful pest species [9]. Classical biological control programs seek to correct this imbalance by importing co-evolved natural enemies from the countries of origin, but this technique has been called into question for impacts on non-target arthropods and further disruption of natural ecosystems [10][11][12][13]. It has been argued that invasion biologists and ecologists should consider responses of native biological control agents (parasitoids and predators) to invasive host species [14]. Native species of predators and parasitoids may play an important role in regulating populations of not only native host species, but also other related invasive and exotic host species. Such shifts in the preference of native natural enemies may influence the population dynamics and long-term establishment of exotic species [14]. Classical biological control may have the advantage of much more rapid control of the co-evolved exotic prey than native predators or parasitoids, which may take many years to adapt to new prey, but native biological control agents have the advantage of already being adapted to local environmental conditions and ecosystems.
While classical biological control of exotic pests in agriculture has met with some notable successes in the past, there have also been some failures where the introduced species negatively impacted non-target species [12]. It is these failures that have led to more stringent regulatory constraints in releasing exotic biological control agents [15]. Many exotic biological control introductions have been made with little knowledge or consideration of the biological control factors already present in the introduced ecosystem. Due to a tendency to focus on exotic biological control agents of exotic prey, there is often less information on the importance of native natural enemies in controlling populations of exotic species. However, in the area of tree fruit integrated pest management (IPM) in the U.S., there are several key examples in which the primary biological control agents of exotic pests -such as codling moth [Cydia pomonella (L.)], Oriental fruit moth [Grapholita molesta (Busck)], spotted wing drosophila [Drosophila suzukii (Matsumura)], and European red mite (Panonychus ulmi Koch)-are native predators or parasitoids that adapted to these exotic hosts over time [16][17][18].
This study was part of a large, multi-state group effort to survey for and characterize the impact of indigenous arthropod natural enemies that were attacking H. halys [19]. In the Mid-Atlantic region, there are several parasitoids of stink bugs that could potentially contribute to H. halys control. Here, we focus on field parasitism by Trichopoda pennipes Fab. (Diptera: Tachinidae) that was previously noted to parasitize this introduced pest in caged studies [20]. This common and widely distributed fly is found throughout North America and is reported to have three geographically isolated host strains that are important in the biological control of several species of Coreidae, Pentatomidae and Largidae [21]. While most parasitoids target the egg stage of stink bugs, T. pennipes targets the adults (and late-stage nymphs), a life stage that is more difficult to control chemically. In this study, we document and provided baseline data on adult H. halys parasitism rates (in terms of egg deposition on the host) in the field by T. pennipes in several counties of Pennsylvania. In addition, both the sex of the host and the location of the tachinid eggs on the host were recorded, since the location of where the eggs are laid is important to the successful penetration of the host cuticle by tachinids [22], and there has been speculation that the ventral thoracic pheromone glands of the male stink bugs are likely the main host finding mechanism [20].

Field Collection
A total of 43 samples ranging from 21 to 868 H. halys adults were collected from orchards, vegetables, field crops and overwintering sites in buildings during the 2012 and 2013 growing seasons as part of an effort to establish laboratory colonies for pesticide bioassay work. Samples were taken from 11 locations spread across five Pennsylvanian counties: Adams, Allegheny (2013 only), Berks (2012 only), Franklin and Lancaster. Field-collected adults were maintained in screened cages where they were allowed to mate and lay eggs to start new colonies for bioassays.

Oviposition Patterns
Spent adults were placed in plastic petri dishes and then examined for the presence of tachinid eggs and any resulting tachinid flies that emerged from these cadavers were collected and identified. For each parasitized individual, the sex of the host and the location of the tachinid eggs on the host were recorded. Records related to location of egg deposition indicated whether eggs were found on the dorsal or ventral surface, and whether eggs were on the thorax or the abdomen (in only rare instances was an egg found on the head of the host). Because multiple eggs on a single host may occur-despite only a single parasitoid ultimately surviving [23]-the number of eggs on each host was also recorded.
T. pennipes eggs on H. halys hosts were identified according to the original description and drawings [24], photographs and descriptions [20], and from adults reared from parasitized hosts. Parasitized H. halys and reared T. pennipes adults are preserved and stored at the Penn State Frost Entomological Museum, University Park, PA or the Penn State Fruit Research and Extension Center, Biglerville, PA, U.S.

Data Analysis
Chi-square contingency tests were conducted to examine independence of parasitism frequency between years, among counties, and among seasons. Collection dates within years spanned three seasons-spring (Mar 20-Jun 20), summer (Jun 21-Sept 21), and fall (Sept 22-Dec 20)-and were grouped accordingly for seasonal analysis. The Holm's sequential Bonferroni method was used for post hoc comparisons in parasitism rates among counties and seasons. One-sample t-tests were conducted to examine if parasitism rates (i.e., percent of tachinid eggs deposited on H. halys) were significantly different from the expected value of 50 percent for: (1) males and females; (2) dorsal and ventral surface; and (3) thorax and abdomen. Since t-tests were based on percentage values, samples with small numbers of total eggs found (i.e., ≤5) were excluded from the analysis. Lastly, a descriptive analysis of frequency of supernumerary oviposition was conducted.

Results
During the two-year study, a total of 7857 H.

Discussion
Native predators and parasitoids have the potential to play an important role in the regulation of invasive species populations [14]. This study provides evidence that the native tachinid fly and known parasitoid of hemipterans, T. pennipes, is targeting up to 3.3% of adult brown marmorated stink bugs (H. halys) in Pennsylvanian agricultural landscapes. Other studies have recently provided evidence of other indigenous species exploiting H. halys [19] including egg parasitoids [25,26], egg predators [27], and nymph/adult predators [28][29][30]. Our findings, in combination with these other studies, reveal that native predators and parasitoids have begun to use the invasive H. halys as a food source or host, and could help regulate populations of this economically important pest.
Although current adult parasitism rates are quite low, it is possible that the contribution of T. pennipes to the biological control of H. halys could increase over time. T. pennipes is multivoltine and a single adult female can lay hundreds of eggs. In addition, T. pennipes is already known to parasitize a range of other hemipteran pests [31]. Parasitism by T. pennipes can influence both lifetime fecundity and population growth of pests such as the southern green stink bug [Nezara viridula (L.)], although the effectiveness of T. pennipes can be limited due to the fact that the adult host is not immediately killed [32,33]. T. pennipes can also show host preference, and actually consists of a complex of biotypes or cryptic species across North America that specialize on regionally abundant pests, such as squash bugs [Anasa tristis (DeGeer)] [34] and bordered plant bugs [Largus californicus (Van Duzee)] in California [35]. It is thus worth monitoring if the exploitation of H. halys by T. pennipes increases in the Mid-Atlantic region of the U.S., and determining if this may represent the emergence of a new regional biotype that specializes on H. halys.
T. pennipes populations should be conserved as part of the larger community of indigenous natural enemies in Mid-Atlantic agricultural landscapes to encourage biological control and potentially reduce reliance on insecticide use against H. halys. Native species of stink bugs in this area, which can cause catfacing (peaches), dimpling, scarring, discoloration and internal corking on tree fruits, are considered relatively minor pests, as their populations are held in check by a complex of parasitoids and generalist predators. Since H. halys exhibits similar characteristics and often moves into cropping areas from a surrounding unmanaged habitat, the best option for long-term population suppression may come from biological control in areas outside of the crop. However, in the absence of robust natural regulation, broad-spectrum insecticides, such as pyrethroids, are often needed for H. halys control. These products can impact the natural enemy community as a whole and can be quite disruptive to current IPM programs in tree fruit [6,[36][37][38]. Similarly, pesticide drift to field margins or forest edges could negatively affect pollinators and natural enemies using these extraorchard habitats.

Discussion
Native predators and parasitoids have the potential to play an important role in the regulation of invasive species populations [14]. This study provides evidence that the native tachinid fly and known parasitoid of hemipterans, T. pennipes, is targeting up to 3.3% of adult brown marmorated stink bugs (H. halys) in Pennsylvanian agricultural landscapes. Other studies have recently provided evidence of other indigenous species exploiting H. halys [19] including egg parasitoids [25,26], egg predators [27], and nymph/adult predators [28][29][30]. Our findings, in combination with these other studies, reveal that native predators and parasitoids have begun to use the invasive H. halys as a food source or host, and could help regulate populations of this economically important pest.
Although current adult parasitism rates are quite low, it is possible that the contribution of T. pennipes to the biological control of H. halys could increase over time. T. pennipes is multivoltine and a single adult female can lay hundreds of eggs. In addition, T. pennipes is already known to parasitize a range of other hemipteran pests [31]. Parasitism by T. pennipes can influence both lifetime fecundity and population growth of pests such as the southern green stink bug [Nezara viridula (L.)], although the effectiveness of T. pennipes can be limited due to the fact that the adult host is not immediately killed [32,33]. T. pennipes can also show host preference, and actually consists of a complex of biotypes or cryptic species across North America that specialize on regionally abundant pests, such as squash bugs [Anasa tristis (DeGeer)] [34] and bordered plant bugs [Largus californicus (Van Duzee)] in California [35]. It is thus worth monitoring if the exploitation of H. halys by T. pennipes increases in the Mid-Atlantic region of the U.S., and determining if this may represent the emergence of a new regional biotype that specializes on H. halys.
T. pennipes populations should be conserved as part of the larger community of indigenous natural enemies in Mid-Atlantic agricultural landscapes to encourage biological control and potentially reduce reliance on insecticide use against H. halys. Native species of stink bugs in this area, which can cause catfacing (peaches), dimpling, scarring, discoloration and internal corking on tree fruits, are considered relatively minor pests, as their populations are held in check by a complex of parasitoids and generalist predators. Since H. halys exhibits similar characteristics and often moves into cropping areas from a surrounding unmanaged habitat, the best option for long-term population suppression may come from biological control in areas outside of the crop. However, in the absence of robust natural regulation, broad-spectrum insecticides, such as pyrethroids, are often needed for H. halys control. These products can impact the natural enemy community as a whole and can be quite disruptive to current IPM programs in tree fruit [6,[36][37][38]. Similarly, pesticide drift to field margins or forest edges could negatively affect pollinators and natural enemies using these extra-orchard habitats.
Our study found different parasitism rates among locations and this may have to do with T. pennipes abundance in the surrounding landscape. Certain plants, especially members of Asteraceae and Apiaceae, are known to support populations of beneficial flies such as syrphids and tachinids in agricultural areas [39]. For example, in Pennsylvania, T. pennipes emerges in late spring and has been observed using nectar sources from host plants such as wild carrot (Dacaus carota L.) and goldenrod (Solidago spp. L.) [39]. In general, agricultural landscapes with higher levels of plant diversity and/or habitat structure can provide shelter, favorable microclimatic conditions, and alternative food sources for natural enemies [40][41][42][43]. Therefore, providing nectar-rich resources in agricultural landscapes, whether through deliberate floral plantings or setting aside unmanaged areas in field margins, could potentially result in greater numbers of parasitoids and increased biological control (i.e., "parasitoid nectar provision hypothesis") [43], and can be an important part of integrated pest and pollinator management (IPPM) programs in tree fruit orchards [44], where H. halys is an important pest and causes severe fruit infestation.
In this study, we found significantly higher parasitism in H. halys adults during the spring season. Higher incidence of parasitism detected in the spring may have been an artifact of time, as eggs may have been deposited on spring-collected H. halys either during the previous year (prior to overwintering) or during the spring collection period. We also found significant patterns related to the location of egg deposition on H. halys adults. Studies suggest that T. pennipes locates stinkbug hosts by following the thoracic aggregation pheromones secreted by males [22,45] and field collections of southern green stinkbugs (Nezara viridula) found a greater number of parasitized males than females [46]. While our results did reveal a distinct preference for egg deposition on the ventral side of the thorax of H. halys adults, we did not find a preference for males. It is possible that T. pennipes used the male pheromones to locate H. halys aggregations and then laid eggs indiscriminately among males and females. Although not included in this analysis of adult parasitism, last instar nymphs of field-collected H. halys were also found with T. pennipes eggs, which supports previous findings that members of the Phasiine endoparasitoid group of Tachinidae are attracted to nymphal defensive allomones despite attacking mostly the adult stages [20,47].

Conclusions
Here, we provide evidence that T. pennipes has begun to target H. halys adults in agricultural landscapes of Pennsylvania, although at low rates. Parasitism rates varied across seasons and location. Although other studies suggest that T. pennipes more frequently targets male hosts, we found no difference in egg deposition on male and female hosts; however, T. pennipes oviposited more frequently on the ventral side of the thorax relative to other body regions of the host. T. pennipes is an effective and regionally specialized parasitoid of other hemipteran pests throughout the U.S. and thus has potential to contribute to the biological control of H. halys. In combination with targeted floral provisions and judicious use of insecticides, biological control by T. pennipes and other native parasitoids and predators, may play an increasingly prominent role in suppressing H. halys populations as part of IPM programs in the Mid-Atlantic region of the U.S.