DOI: 10.5061/dryad.1g1jwstrt
title: Native generalist natural enemies and an introduced specialist parasitoid together control an invasive forest insect

description: Specialized natural enemies have long been considered a major force
 driving the population dynamics of outbreaking forest insects. While
 research has traditionally focused on the role of specialist parasitoids,
 recent studies and reviews reflect an appreciation of complex interactions
 among many regulatory factors. The sources suggest that specialist
 parasitoids and generalist predators can each inflict strong top‐down
 effects and that specialists and generalists can interact to regulate
 insect herbivore populations. Here we use the model study organism winter
 moth (Operophtera brumata) in its invasive range in the northeast United
 States to investigate interactions between the introduced, host-specific
 tachinid parasitoid Cyzenis albicans and native, generalist pupal
 predators. Prior research in Canada showed that predation of winter moth
 pupae increased after C. albicans establishment. To explain this
 phenomenon, the following hypotheses have been suggested: (1) parasitoids
 suppress the winter moth population to a density that can be maintained by
 generalist predators, (2) unparasitized pupae are preferred by predators
 and thus experience higher mortality rates, or (3) C. albicans sustain
 higher predator populations throughout the year more effectively than
 winter moth alone. We tested these hypotheses by deploying winter moth
 pupae over six years spanning 2005 to 2017 and by modeling pupal predation
 rates as a function of winter moth density and C. albicans establishment.
 We also compared predation rates of unparasitized and parasitized pupae
 and considered additional mortality by a native pupal parasitoid. We found
 support for the first hypothesis; we detected both temporal and spatial
 density dependence, but only in the latter years of the study when winter
 moth densities were lower. We found no evidence for the latter two
 hypotheses. Our findings suggest that pupal predators have a regulatory
 effect on winter moth populations only after populations have been
 reduced, presumably by the introduction of the host-specific parasitoid C.
 albicans.
description: <i>Study organisms </i> Winter moth, <i>Operophtera
 brumata</i> L., is a geometrid moth native to Europe and northern
 Asia and can be an important defoliator, particularly in its invasive
 ranges in coastal North America (Nova Scotia, British Columbia, and New
 England) (Roland and Embree 1995, Elkinton et al. 2015) and in sub‐arctic
 birch forest in northern Fennoscandia (Jepsen et al. 2008). Winter moth
 caterpillars hatch in synchrony with bud-break of their host plants, a
 broad range of deciduous trees, particularly oak and maple. The
 caterpillars feed on the foliage in early spring before dropping to the
 soil in mid-late May to pupate. Winter moth has a long pupal period (6–7
 months during the summer, representing most of its life) and it pupates in
 the top layer of soil or leaf litter. They emerge as adults in early
 winter, from early November through early January at which point they mate
 and lay their eggs in bark cervices. These eggs overwinter and the cycle
 repeats with one generation per year. <i>Cyzenis </i>is a
 tachinid parasitoid that co-evolved with winter moth. It deposits its eggs
 on leaves, and any subsequent caterpillars that feed on those
 leaves ingest the eggs. The
 fly maggot develops inside the caterpillar, eventually killing it, and
 pupates inside its host’s pupa. Like winter moth, <i>Cyzenis
 </i>has one generation per year, but the adult flies emerge the
 following spring rather than in the winter when winter moth adults
 emerge.   <i>Pupa Deployment
 </i> Pupae
 were deployed in 2005 and yearly from 2013 through 2017 to estimate
 mortality from predation over the winter moth pupal period. We deployed
 unparasitized winter moth pupae and <i>Cyzenis </i>puparia
 (winter moth pupae parasitized by <i>Cyzenis</i>), both reared
 from spring larval collections. Methods for pupal collection, rearing, and
 storage are described in Appendix 1. Pupae were deployed in eastern
 Massachusetts at eight study sites used for long-term assessment of winter
 moth population dynamics (Elkinton et al. 2015) in either three or five
 consecutive rounds per year, from mid-June until end of October. Five
 deployments (one every three weeks) were completed in 2005, 2013, and
 2014; three deployments (one every six weeks) were completed in 2015 -
 2017. In 2005, sets of 40 winter moth pupae were deployed, while in all
 the subsequent years (2013 – 2017) sets of 100 winter moth pupae and 50
 <i>Cyzenis </i>puparia were deployed. To evaluate season-long
 mortality as compared to the cumulative mortality calculated from the
 consecutive deployments, we deployed an additional 200 winter moth pupae
 at two sites (Table 1, sites B and D) from 12 Jun. - 20 Oct. 2014. To
 evaluate mortality of <i>Cyzenis </i>puparia over the winter,
 we deployed a set of 100 puparia at sites B and D from 26 Oct. 2013 - 3
 Apr. 2014.  Deployed pupae were attached to
 small burlap squares (one per square) using beeswax, as per Broadley et
 al. (2018). Pupae on burlap were placed haphazardly 2.5 cm below the soil
 surface under the drip line of a red oak (<i>Quercus
 rubra</i>) at the study sites (Table 1, Appendix S1: Figure S2). The
 deployment depth was chosen to mimic natural pupa placement (Holliday
 1977). Study sites coincided with winter moth long-term study sites and
 reflected a range of winter moth and <i>Cyzenis
 </i>establishment histories (Elkinton et al. 2014). All sites were
 in mixed hardwood forests dominated by red oak (<i>Q.
 rubra</i>) and red maple (<i>Acer rubrum</i>). Due to
 limited supply, only select sites received deployments of <i>Cyzenis
 </i>puparia (Table 1). To test for an effect of our deployment
 method as compared to previously published mortality estimates using a
 wire tag method (Buckner 1969, East 1974, Raymond et al. 2002, Horgan and
 Myers 2004), in 2005, we compared mortality of pupae attached to wire tags
 as compared to pupae attached to burlap squares. We buried 30 wired pupae
 and 30 burlap-attached pupae at two sites for two weeks.
   <i>Estimating site pupal densities and
 parasitism by </i>Cyzenis To estimate winter moth pupal
 density and percent <i>Cyzenis </i>parasitism at each site, 16
 plastic buckets (16 cm wide x 28 cm long x 10 cm high) were filled 3 cm
 deep with sifted peat moss and placed under each study tree in late May,
 before pre-pupal winter moth caterpillars began to spin down from the tree
 canopies. Each bucket was placed at a randomly selected distance between
 the tree stem and the edge of the tree canopy, along one of eight
 evenly-spaced directions radiating from the tree stem. Parasitism rates on
 winter moth by <i>Cyzenis </i>were estimated both from the
 proportion of <i>Cyzenis</i>-parasitized pupae from these
 pupal bucket collections and from collections of 100 to 500 late-instar
 larvae collected at each site.  The collections were made one week prior
 to the onset of pupation. The larvae were reared in groups of up to 500 in
 20 liter buckets with screen tops. In July, after
 <i>Cyzenis</i> had completed development, we dissected all the
 cocoons and scored them for the presence of fly puparia.
   <i>Pupal mortality
 estimates</i> After each deployment, pupae were
 retrieved and stored in a growth chamber (Percival Scientific, Inc.) at
 12°C in dark until analysis. We characterized the fate of the pupae as
 intact, predated, parasitized, or diseased. Predation was assumed for
 pupae that had been removed from the burlap square and for pupae with only
 the crushed cuticle remaining, holes chewed in them, and evidence of teeth
 or claw marks in the wax, as shown in Broadley et al. (2018). Parasitism
 by native <i>Pimpla</i> wasps was assumed for pupae with
 characteristic wasp emergence holes and pupae that yielded wasps as shown
 in Broadley et al. (2019). No data on <i>Pimpla</i> were
 recorded in 2005 and 2013 because we had not yet identified the
 characteristic emergence holes these wasps leave in the pupal cuticle, and
 we did not hold the pupae to allow for wasp development. We excluded
 diseased pupae in our mortality estimates since the desiccation or mold
 likely occurred as a result of rearing conditions; these pupae accounted
 for a small proportion of mortality (&lt; 6%). To allow for the
 development of pupal parasitoids, seemingly intact pupae were stored in an
 growth chamber over the winter, as outlined in Broadley et al. (2019), and
 were re-examined in the spring. Predation rate and parasitism were
 calculated for each pupal deployment. The proportion parasitized was
 calculated as the number of pupae parasitized by <i>Pimpla</i>
 wasps divided by the number of pupae that remained after predation. This
 method of calculating percent parasitism estimates the true, underlying
 mortality rate of contemporaneous mortality factors and incorporates the
 fact that parasitism rates can be obscured by predation rates because
 predation typically occurs on the pupae whether or not they were
 parasitized (Elkinton et al. 1992). Because previous research found no
 relationship between the proportion of pupae parasitized by
 <i>Pimpla</i> and the proportion lost to predation (Broadley
 et al. 2019), we assumed the estimate of <i>Pimpla</i>
 parasitism determined after predation acted on the deployment will be the
 same as that prior to predation. Annual cumulative (life stage long)
 survivorship values were calculated as the product of successive
 survivorships of each pupal deployment (e.g., S<sub>c</sub> =
 S<sub>1a</sub> x S<sub>1b</sub> x
 S<sub>2</sub> x S<sub>3</sub>) and the cumulative
 mortality values (P<sub>c</sub>) were calculated as
 (P<sub>c</sub> = 1−S<sub>c</sub>). To analyze
 mortality rates of deployments across years and months, even when the
 number of days deployed varied (from 19 to 45), we standardized mortality
 proportions to the mean deployment duration of 31 days. We estimated the
 daily survival rate (S) =
 S<sub>i</sub><sup>1/n</sup> where n is the
 variable number of days the pupae were deployed (ranged from 19 to 45
 days, mean of 31 days). The daily survivorship (S) was raised to an
 exponent of 31 for the expected survivorship over a standardized 31 days
 (S<sub>31</sub> =
 [(S<sub>i</sub>)<sup>1/n</sup>]<sup>31</sup>). To estimate pre-experimental mortality of the pupae (S<sub>1a</sub>), we used the estimated daily survival of the first deployment (S<sub>1a</sub> = S<sub>1b</sub><sup>1/n</sup>) where n is the number of days the first deployment was out and raised this to the estimated number of days of the winter moth pupal period that elapsed before to the onset of the study. Based on prior research (Elkinton et al. 2015), we estimated the start of the pupal period to be 1 June.   <i>Predator exclusion and community experiments </i> In 2013 and 2014, we used a combination of predator exclusion studies and pitfall traps to identify predators in the community and to evaluate their relative contribution to pupal mortality. We ran six deployments of a predator exclusion experiment. Using two sites for each deployment (Table 1, sites A and D), we ran one deployment in 2013 (12 Aug. - 20 Oct.) and two in 2014 (26 Jun. - 11 Aug. and 18 Aug. - 30 Oct.). For each deployment, 100 pupae were deployed in a 100 m by 100 m grid with one pupa placed every 1 m<sup>2</sup> in the array. The pupae were attached to burlap squares and these squares were secured to the bottom of one of three cage treatments—cages with 3.2 mm (1/8”), 6.4 mm (1/4”), or 12.7 mm (1/2”) square openings or a control (just the wire mesh bottom of one of the other three cages). The cages were placed 2 to 3 cm into the ground and covered with soil and leaf litter. At the end of each deployment, the pupae were characterized as intact, predated, parasitized, or diseased. In 2013, two pitfall traps were placed at each study site and checked weekly. The pitfall traps were each made of a 0.91 L (24 oz) plastic cup, buried flush to the surface, covered with a lid elevated five centimeters above with a wire stand, and partially filled with 70% ethyl alcohol solution. All beetle larvae, adult staphylinid and carabid beetles, and any small mammal by-catch were counted.