A Field Test on the Effectiveness of Milfoil Weevil for Controlling

Home , Euhrychiopsis lecontei, Myriophyllum

Hydrobiologia (2017) 800:81–97 DOI 10.1007/s10750-017-3142-2

TRENDS IN AQUATIC ECOLOGY II

A ﬁeld test on the effectiveness of milfoil weevil for controlling Eurasian watermilfoil in Wisconsin lakes

John E. Havel . Susan E. Knight . Kristopher A. Maxson

Received: 2 November 2016 / Revised: 20 February 2017 / Accepted: 24 February 2017 / Published online: 9 March 2017 Ó Springer International Publishing Switzerland 2017

Abstract We tested the effectiveness of milfoil Background weevil densities varied widely among weevils (Euhrychiopsis lecontei) for reducing biomass beds and were often greater than the densities stocked. of Eurasian watermilfoil (Myriophyllum spicatum, Weevil stocking had no significant effect on EWM or EWM) under natural lake conditions in a 3-year field native plant biomass. Nevertheless, weevil damage to experiment. In each of four lakes, we randomly chose EWM was common and its extent appeared strongly two EWM beds for stocking and two beds as controls. related to observed densities of the weevil. ANCOVA A total of ca. 40,000 weevils were added to the eight results indicated that weevil density was a significant stocked beds. During June and August 2013–2015, we predictor of EWM biomass in both June and August, measured plant diversity, biomass of EWM and native but not on growth during summer. Overall, our study plants, and weevil abundance in the 16 study beds. found that weevil density is an important factor for predicting EWM biomass, while weevil density is likely affected by a large number of environmental Guest editors: Koen Martens, Sidinei M. Thomaz, factors. This work highlights the importance of Diego Fontaneto & Luigi Naselli-Flores / Emerging carefully considering lake conditions that may influ- Trends in Aquatic Ecology II ence the efficacy of stocking for biological control.

Electronic supplementary material The online version of Keywords Aquatic invasive species Á Augmentative this article (doi:10.1007/s10750-017-3142-2) contains supplementary material, which is available to authorized users. biological control Á Euhrychiopsis lecontei Á Invasive aquatic plants Á Lake management Á Myriophyllum J. E. Havel (&) Á K. A. Maxson spicatum Department of Biology, Missouri State University, 901 S. National Avenue, Springﬁeld, MO 65897, USA e-mail: [email protected]

S. E. Knight Introduction UW Trout Lake Station, Center for Limnology, University of Wisconsin-Madison, Boulder Junction, WI 54512, USA Numerous species of invasive aquatic plants grow to densities that cause widespread ecological and eco- Present Address: nomic effects (Carpenter & Lodge, 1986; Pimentel K. A. Maxson et al., 2005). One of the most damaging species is the Illinois River Biological Station, Illinois Natural History Survey, Prairie Research Institute, University of Illinois at exotic Eurasian watermilfoil (Myriophyllum spicatum Urbana-Champaign, Havana, IL 62644, USA L., EWM), which is widespread in the United States 123 82 Hydrobiologia (2017) 800:81–97 and Canada (48 states and 3 provinces, Pfingsten et al., whorled watermilfoil (M. verticillatum L.) (Creed, 2016) and prevalent in lake-rich regions, such as 1998; Solarz & Newman, 2001). In laboratory pref- Wisconsin (635 lakes, SWIMS, 2014). Rapid spring erence tests, the weevil adults prefer EWM over other growth allows this submersed plant to form dense milfoils (Creed & Sheldon, 1993; Solarz & Newman, surface mats, reducing diversity of native plants 2001). Thus, the milfoil weevil may focus feeding on (Madsen et al., 1991; Boylen et al., 1999) and EWM when it is abundant and use these natives as invertebrates and fish (Keast, 1984; Cheruvelil et al., alternate hosts when EWM is rare. Adult weevils feed 2001). These dense mats also interfere with human on the stems and leaves (Creed & Sheldon, 1993) and, uses, resulting in significant loss to property values once water temperature warms, females lay eggs on (Zhang & Boyle, 2010). the apical meristem. Under ideal conditions, each Agencies and lake associations use a variety of female can lay an average of two eggs per day, with methods to control EWM densities, including both total fertility up to 562 eggs over a lifespan of herbicides (Nault et al., 2014) and a variety of physical 162 days (Sheldon & O’Bryan, 1996b). Following methods: bottom barriers, water drawdown, hand hatch, the larvae feed on the meristem before pulling, and machine harvesting (McComas, 1993). burrowing through these soft tissues and into the These methods are costly, labor intensive, and require stem. The larvae cause the greatest damage to the plant repeated application (Madsen, 2000). Furthermore, through their consumption of the cortex, eventually physical and chemical control can have untoward side causing the stem to lose buoyancy and collapse (Creed effects on non-target species, including natural herbi- et al., 1992). Field surveys in Vermont indicate that vores (Madsen, 2000; Newman & Inglis, 2009). As an several generations are produced each summer (Shel- alternative to physical and chemical control, efforts don & O’Bryan, 1996b) and results from laboratory have been underway to find a suitable biological experiments suggest that 4–5 generations are possible control agent. Surveys in Asia for natural enemies of each year in Minnesota lakes (Mazzei et al., 1999). In EWM found a wide range of insects feeding on EWM, fall, the adults migrate to shore, where they overwinter but these species either caused limited damage or else in the leaf litter, then return to the milfoil bed the lacked sufficient host specificity and so could not be following spring (Newman et al., 2001). Thus, this released from quarantine (Buckingham, 1998). Sev- weevil has the potential to produce large populations eral insects already occurring in North America have during the summer, as well as maintain a permanent promise for controlling EWM (reviewed in Newman, local population. 2004): larval stages of the native milfoil midge Where they are abundant, milfoil weevils can (Cricotopus myriophylli Oliver), a naturalized pyralid control EWM biomass (Newman, 2004). Field surveys moth (Acentria ephemerella Denis & Schiffermu¨ller), in several lakes have shown a strong correlation and the native milfoil weevil (Euhrychiopsis lecontei between elevated densities of the milfoil weevil and Dietz). The midge and moth have been linked to EWM natural declines in EWM (Sheldon & Creed, 1995; declines in some lakes (Painter & McCabe, 1988; Lillie, 2000; Newman & Biesboer, 2000). Although MacRae et al., 1990; Johnson et al., 1998; Parsons natural weevil densities can be high on occasion (2–4/ et al., 2011) but have not been associated with EWM stem) (Creed & Sheldon, 1995; Newman & Biesboer, declines in well-studied lakes in Vermont and Min- 2000), surveys from 82 lakes indicate that weevil nesota (Creed, 1998; Newman, 2004). The milfoil densities are ordinarily quite low (means of 0.15–0.67 weevil is the most promising control agent and has weevils per stem, Jester et al., 2000; Tamayo et al., been the subject of extensive research (reviewed in 2004; Reeves et al., 2008), suggesting that conditions Newman, 2004). are often not favorable for sustaining high weevil The milfoil weevil is native to large areas of the populations. Augmenting existing weevil populations northern United States and Canada (Creed, 1998; may allow weevils to control nuisance levels of EWM. Cline et al., 2013), and in some regions that have been Experiments in lake enclosures (Creed & Sheldon, surveyed (e.g., Wisconsin: Jester et al., 2000), the 1995) and in cattle tanks (Newman et al., 1996) weevils are found in most lakes. Native host plants for indicated that added weevils can grow in abundance the milfoil weevil include northern watermilfoil and cause a reduction in both stem and root biomass, (Myriophyllum sibiricum Komarov) and probably also as well as cause stem collapse. But will they work 123 Hydrobiologia (2017) 800:81–97 83 under natural conditions? Although some companies The four study lakes are all moderate-sized kettle have provided stocking of milfoil weevils as a lakes (Fig. 1; Table 1), with drainage hydrology, a professional service (Reeves et al., 2008), little is public boat launch, and extensive littoral zones. known about the degree to which this intervention is Although we did not regularly monitor temperature successful. Several reports have been published on in these study lakes, a nearby lake of similar size open-water releases of milfoil weevils directly into (Allequash Lake, Vilas County, Wisconsin) that is EWM beds (Sheldon, 1997; Jester et al., 2000; Reeves regularly monitored had mean surface temperatures et al., 2008; Parsons et al., 2011). Unfortunately, the during June–August ranging from 21.5 to 22.6 °C design of each experiment does not allow discrimi- during the three study years. One of the study lakes nating weevils from other confounding variables as (Long) was stained to a degree that reduced trans- possible causes of EWM declines (see ‘‘Discussion’’ parency. Most of the lakes were mesotrophic. The section below). Clearly, the conditions that favor exception was the shallowest lake (Boot), which was control of EWM through weevil augmentation remain often turbid with suspended sediments and phyto- poorly understood. plankton (J. Havel and S. Knight, pers. obs.). Eurasian The goal of the current study was to answer the watermilfoil had frequency of occurrence in the question: ‘‘Under ordinary conditions in lakes, can littoral zone ranging from 6 to 28% of littoral sites augmented weevils control EWM?’’ We conducted a sampled (Havel et al., in review). controlled field experiment in four lakes in northern At least 4 well-defined beds of EWM were in found Wisconsin. Specifically, we hypothesized that increas- each lake (Fig. 1), and we selected beds that were ing weevil densities would result in a decline in EWM large enough for study, but small enough (\0.25 ha) biomass and subsequently an increase in native plant that we could practically stock with weevils (Table 1). biomass and diversity. Our experiment followed a We attempted to select EWM beds that were likely to randomized complete block design over the 3-year have the least boat traffic and hence remain undis- study, with four lakes, two treatments (augmented or turbed. Most beds were nearshore, with the exception control) within each lake, and two replicate EWM of Manson bed C, which was near an island but far beds per treatment within each lake. We measured or (300 m) from mainland. derived the following response variables: biomass of After the project began, we learned that genetic EWM, biomass of native plants, EWM % biomass, analysis of EWM plants from Little Bearskin Lake EWM stem density, native plant species richness, indicated that this population consisted of a hybrid of weevil density, and % of EWM plants damaged by Eurasian and northern watermilfoil (M. Nault, Wis- weevils. consin Department of Natural Resources, pers. com., October 14, 2016). Since the frequency of hybrids in northern Wisconsin lakes is not fully known, we will Materials and methods continue to refer to all of our study populations as EWM. Study site Field surveys The experiment was conducted in four lakes from the Northern Highland Lakes District of Wisconsin To determine areas of coverage, we surveyed each of (Fig. 1). The study lakes had to meet several criteria: the EWM beds in September 2012, 2014, and 2015. (1) they had well-defined beds of EWM; (2) the lakes Using two spotters, we visualized the margins of each had not been treated with chemical herbicides within bed, marked the margin with buoys, and then took the last 5 years before the study began; and (3), GPS coordinates (precision ± 3 m). Areas were members of lake associations agreed to not use determined from GPS points using polygons in chemical treatments and to minimize disturbance to ArcGIS 10.0. the study beds until the field study was completed For sampling weevils and plants (details below), we (September 2015). The final choice of study lakes visited each EWM bed during early-mid June and followed preliminary surveys of seven lakes in 2012 again in late August, during each of 3 years and the experiment began in June 2013. (2013–2015). Before sampling each bed, we used 123 84 Hydrobiologia (2017) 800:81–97

Fig. 1 Lake locations and lake maps showing Eurasian watermilfoil study beds. Further description of lakes and beds in Table 1. TLS—University of Wisconsin Trout Lake Station

GPS points from the September 2012 survey to ﬁnd EWM plants in the laboratory. In order to avoid the perimeter, which we marked with colored buoys. introducing different strains of EWM into the lakes, During all sampling, we used ‘‘best boating prac- these EWM for transplanting were collected from the tices’’ to minimize the chance of disturbance to the same lake as would receive the weevils (on EWM). EWM bed and spreading fragmented EWM any Eurasian watermilfoil stems with intact meristems further in the lake or among lakes: we avoided use were cut to 13 cm length and bunched into ‘‘bou- of the boat motor and instead rowed while in the bed; quets’’ of stems in lab. Each bouquet of ten stems was we collected all stray EWM from the water; back at the placed in a small plastic cup with sand substrate and 15 launch, we removed all visible plant fragments from cups were placed in each laboratory aquarium at the boat and trailer and discarded on land; and we ambient temperature (13–17°C), with aeration and steam-washed the boat once back at the ﬁeld station. full-spectrum aquarium lights. Weevil adults were All EWM processed on station were later discarded in collected both from Little Bearskin Lake (near site C, the trash after double bagging. Fig. 1) and another water body in the region (Grand Rapids Flowage, Marinette County, Wisconsin), Weevil rearing and stocking where weevils occurred in high density. About 100 adults were introduced to each of the 20 aquaria, Milfoil weevils were supplied by a private company where they mated and laid eggs. Adults were later (http://enviroscienceinc.com/) for stocking each of the removed and refrigerated until other plants were eight EWM beds designated for augmentation (Fig. 1; available in other aquaria. Weevil abundance (eggs Table 1). The weevils were raised by inoculating and larvae) for stocking each bed was estimated from a 123 Hydrobiologia (2017) 800:81–97 85

Table 1 Description of the Study lakes Little bearskin Boot Manson Long study lakes and areas of the Eurasian watermilfoil County Oneida Vilas Oneida Iron (EWM) beds in September WBIC 1,523,500 1,619,100 1,517,200 2,303,500 2012 Longitude -89.6982 -89.3268 -89.6327 -90.0251 Latitude 45.7110 45.9674 45.5631 46.2470 Surface area (ha) 74 116 96 150 Secchi depth (m) 1.8 1.5 4.8 0.9 Maximum depth (m) 8.1 4.5 16.2 9 All the lakes have drainage Color Light brown Light brown Clear Medium brown hydrology and public boat Conductivity (lS) 109 55 66 98 launch. Location of lakes -1 Alkalinity (mEq l )35292642 and EWM beds shown in Fig. 1. Source of physical pH 7.7 8.4 7.3 7.5 data: Surface Water Trophic state Mesotrophic Eutrophic Mesotrophic Mesotrophic Resources Inventory, Area (ha) of Eurasian watermilfoil beds in September 2012 Wisconsin Department of Natural Resources; bed Beds Little bearskin Boot Manson Long areas courtesy of Onterra, Inc. WBIC is a unique lake A 0.073 0.113 0.085 0.231 identifier used by B 0.166* 0.202* 0.041* 0.097 Wisconsin DNR C 0.113* 0.146* 0.057 0.121* * Beds augmented with weevils D 0.049 0.227 0.036* 0.089* sample of plants from each aquarium. Bouquets were had not yet flowered. On each of the sampling dates transported to the target lake and, using zip ties, the (early June and late August) in 2014 and 2015, we bouquets were attached to EWM in the lake beds collected at least 50 stems from each of the 16 beds; in chosen for augmentation. During June 19–July 3, 2013, only 20 stems were collected from each bed. 2013, ca. 40,000 total weevils (eggs and larvae) were Stems were collected by rowing across the long axis of distributed among the eight augmented beds, with the bed and collecting single stems from each of 50 numbers scaled to bed area. In order to estimate the locations (20 in 2013). All stems were stored together weevil densities stocked (as number per EWM stem), in 1–2 large Ziploc baggies, kept in a cooler, then we used the following calculation from other variables transferred to a refrigerator at 5°C. Plants were estimated below: processed within 1 day of collection. In order to Number of weevils added examine population dynamics of milfoil weevils in our study lakes, we collected additional samples of EWM densityðÞÂ stems mÀ2 EWM bed area ðm2Þ weevils every 10–14 d during early June–late August In order to follow the recommended procedures 2014 in one lake (Boot Lake). from Enviroscience that weevils be stocked in three Plants were first examined for signs of weevil successive years (C. Marquette, pers. com.), we had damage. Stems were scored as damaged by weevils if planned to stock weevils again in the second and third the stem was hollowed, with inner cortex gone, years. However, Enviroscience discontinued service making the stem appear transparent. Damaged stems to Wisconsin and another vendor was unavailable; also sometimes had missing tips or had blast holes, thus we could only stock weevils in one year. places where the pupa chewed through the stem to escape. These blast holes often appeared as a dark Weevil sampling and estimation of abundance circles, with a transparent section appearing as a ‘‘bubble’’ in the stem. Milfoil weevils were sampled by collecting EWM Plants were then checked for abundance of weevils stems from a boat, using a short-handled rake. We in each of four stage classes: egg, larva, pupa, adult. collected stems that were at least 50 cm in length and For weevil abundance, we counted weevils only from

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EWM stems with intact apical meristem; other stems bias with respect to plant density. Samples were were omitted, even if the stems included intact side always collected from an agreed-upon direction from meristems. This process allowed counting all stage each buoy location. In order to hold boat position, a classes associated with the same plant, but underes- long pole was pushed into the bottom sediments and timated abundance of both larvae and pupae relative to held from one end of the boat, while the person stems lacking intact meristems (results below, details handling the oars either did the same (in shallow in Havel & Knight, 2016). Each EWM stem was first water) or slowly rowed against the wind. examined for adults, both in a tray of water over a light At each of the sample locations where plant box and then as the stem was processed under the biomass samples were collected, EWM stem density dissecting microscope. Densities of weevil adults was estimated by counting tips from the boat. Multiple tended to be quite low (see ‘‘Results’’ section below). tips arising from the same stem to a depth of B50 cm Although it is possible that our method of collection were counted as a single stem, those deeper as multiple from the field caused some adults to be lost from our stems. A 0.25 m2 quadrat square was placed near the samples, we observed most adults in the lab to remain water surface to determine which plants were within closely attached to the EWM. Furthermore, a com- the area. If the water was stained or turbid (Long and panion study that employed underwater collection of Boot lakes), a ‘‘Viewing scope’’ was used to see to the EWM with SCUBA also found low adult densities 50 cm depth. (Maxson, 2016). Other life stages were counted only Plant biomass samples at each of the ten random under the dissecting microscope. Eggs were generally locations were collected from the boat using a observed attached to the apical meristem. Larvae and 2-headed steel bow rake (34 cm wide) attached to a pupae were counted in the stem, which required long pole. Turning in a complete circle captured an slicing open the stem. Larvae were generally observed area = 0.0908 m2. Each sample involved turning the in regions that were visibly weakened, where the rake two complete turns (Hauxwell et al., 2010). cortex had been eaten by the larva. Pupae were found Although small plants in low density are likely in pupal chambers, which appeared as enlarged underestimated with this technique, most of our transparent bubbles within the stem. Voucher speci- samples contained large plants that were easily mens of each milfoil weevil stage class were deposited captured by the rake. Samples were washed in a in the insect collection at the Department of Entomol- colander, then stored in separate Ziploc baggies (1- or ogy, University of Minnesota (http://insectcollection. 2-gallon size). Once in the lab, each plant sample was umn.edu/). sorted under well water in plastic wash basins. Roots All statistics reported below for weevil densities are were cut off and discarded. Native plants were based on using the average from all the stems identified to species (Crow & Helquist, 2000a, b)to examined from each bed as a single replicate provide species occurrence by bed. All natives from measurement. each sample were pooled together in a tared pan for weighing. EWM plants were placed in a separate tared Plant sampling and estimation of density pan. Following sorting, all remaining plant material and biomass was double bagged and disposed in the trash. All the biomass samples were dried at 60 oC for at least 2 d, On the same dates as the weevil collections (early June cooled in a dry room, and then weighed to ±0.01 g and late August, 2013–2015), we counted EWM stem with an AND GF-10 K top loading balance. Thus each density in each bed and collected samples of plants for bed provided ten replicate measures each of EWM and determining biomass of EWM, biomass of native native plant biomass. submerged plants, and species composition and richness of native submerged plants. Statistical analysis We established ten random locations within the bed by haphazardly tossing marker buoys of a different Based on the initial design of the manipulative field color than those marking the boundary of the bed. The experiment, for each response variable, we ran a buoys were tossed to include all regions of the bed and randomized complete block ANOVA (in Minitab ver. the distance of each toss presumably prevented any 17), with treatment (augmented or control) crossed 123 Hydrobiologia (2017) 800:81–97 87 against year and lake (as block effect), with two estimation of weevil densities. All other sites and dates replicate beds per treatment. Variables failing had enough EWM for complete samples. ANOVA assumptions were transformed as needed to Weevil densities were generally higher in June than satisfy the homogeneous variance assumption. For a August (Fig. 2), despite weevil stocking that occurred couple tests, transformation did not satisfy assump- in half the EWM beds after the June sampling. This tions. However, because ANOVA tends to be robust to June-to-August reduction in density was marginally violation of these assumptions (Whitlock & Schluter, significant in 2013 (Wilcoxon matched-pairs test, 2009), we proceeded with each analysis. W = 23, P = 0.069), but highly significant in both Because the weevil augmentation treatment was 2014 and 2015 (P = 0.001 and 0.003), with a median generally weak and with no significant effects (results reduction (across all 16 beds) of 1.03 and 0.52 weevils below), we used the inherent variability in weevil per stem, respectively. densities among EWM beds to look at patterns from an Stage structure of the weevils was dominated by observational study. We ran ANCOVA on the plant eggs and larvae, particularly in June. A similar trend biomass data, with lake and year as factors and weevil occurred in all lakes and all years. For example, in density in June as covariate, and graphically examined June 2015 and pooling across all lake beds, the weevils associations of other response variables with field consisted of 46% eggs, 51% larvae (L), 1% pupae (P), observations of weevil density. Because weevil den- and 2% adults (A). Because we were concerned that sities in August were generally quite low and with this skewed stage structure might be an artifact of our little variation among beds (results below), a similar sampling method (weevils counted only from stems ANCOVA using the August densities was less with ‘‘tips’’ [intact meristems]), we collected addi- informative. tional samples in June 2016, comparing weevil densities from stems with and without tips (403 total stems) collected from two EWM beds (Little Bearskin Results bed B and Long bed A). As expected, egg densities were considerably higher on stems with tips than those In our study lakes, a wide variety of insects and other without tips (on average, 2.26 times higher). Larval macroinvertebrates were found associated with EWM densities from stems with tips were 26% lower than plants, with chironomids being most abundant (Max- those without tips, indicating that we underestimated son 2016). Although the milfoil midge (C. myrio- larvae in the broader study from 2013 to 2015. Pupae phylli) is difficult to distinguish from other were only found in stems without tips, but were not chironomids, we found no evidence of chironomids very abundant in any of the samples (3–14% of each feeding on the EWM meristem or of the silk cases population). These 2016 data suggest that pupae were characteristic of this species (MacRae & Ring, 1993). underestimated in the broader study, but we cannot say We observed larvae of aquatic moths, but these tended to what degree. Nevertheless, in June 2016, pupae to be rare and there was no evidence of the feeding or were only a small fraction of the population. The low constructed cases typical of Acentria ephemerella adult densities in the broader study were consistent (Johnson & Blossey, 2002). Thus, the most important with another study that employed SCUBA and under- herbivore of EWM in our study lakes was likely the water capture (Maxson, 2016), suggesting the low milfoil weevil. adult densities in the broader study were real. How many generations of weevils occur in these Weevil abundance study lakes during summer? We intensively sampled one lake to examine this question more closely. Eurasian watermilfoil was sufficiently abundant to Collections from Boot Lake on eight sampling dates in allow estimating total weevil density (all life stages, 2014 (ESM Appendix 11) indicated highest densities including eggs) from most beds on each of the six of eggs in June (0.96/stem), a small second cohort in sample dates (Fig. 2). The exception was Long Lake July, and a sharp decline of both eggs and larvae after bed C in June 2014, when there were no live EWM late July. Pupae and adults were never abundant evident. In June 2015, Boot Lake beds A and B had (maximum 0.05/stem). These data suggest at most two limited number of EWM (n = 4 and ten plants) for generations were present in Boot Lake, with the first 123 88 Hydrobiologia (2017) 800:81–97

Fig. 2 Milfoil weevil densities in the 16 EWM study beds over EWM bed areas. On the graph, augmented densities are shown 3 years. Control beds in solid gray, augmented beds cross as additions to the densities observed from weevils collected hatched. Background weevil densities (in gray) include all life earlier in the month. In June 2014, Long Lake bed C had no live stages (egg to adult). Estimated densities augmented in eight EWM, and hence no weevils were sampled; and in June 2015, beds (shown in black) are based on numbers of weevils stocked weevil estimates in Boot Lake beds A and B were based on in late June 2013 and estimates of EWM stem densities and n = 4 and 10 EWM stems sampled, respectively generation (recruitment from overwintering females) evident during June, when weevil densities were being the strongest. highest (Fig. 2). The variability among beds within Background weevil densities were highly variable single lakes is evident in Long Lake in June 2013, in our study lakes (Fig. 2). Over the 3-year period, when background weevil densities ranged from 0.05 total weevil densities (including eggs) showed no (bed D) to 3.15 weevils per stem (bed B) (Fig. 2). On signiﬁcant difference among study lakes (Table 2); an areal basis, these sites had densities ranging from the signiﬁcant lake-by-year interaction effect suggests 2.1 to 54.2 weevils m-2 (ESM Appendix 8). In that any differences among lakes in weevil popula- Manson Lake, two of the beds (B and D) had natural tions depended on study year. For instance, June populations (background) of weevils that were mod- densities were highest in Long Lake in 2013 (prior to erately abundant and were also (by chance) augmented stocking), but highest in Little Bearskin in 2015 with additional weevils, while the other two beds had (Fig. 2). Much of the variation in weevil densities was densities of 0 (Fig. 2). Overall, after accounting for among beds within single lakes, a feature that is other factors (Table 2), 33% of the variation in weevil

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Table 2 ANOVA results on the effects of lake (L), treatment (T), and year (Y) on three different response variables Source DF Weevil density (#/stem) in June EWM biomass in August (g m-2) Native species richness in summer SS MS F- P- SS MS F- P- SS MS F- P- value value value value value value

Lake 3 2.414 0.805 1.770 0.180 6.177 2.059 10.110 <0.001 519.729 173.243 17.880 <0.001 Treatment 1 0.270 0.270 0.590 0.448 0.143 0.142 0.700 0.411 4.687 4.687 0.480 0.493 Year 2 1.046 0.523 1.150 0.333 1.575 0.788 3.870 0.035 12.792 6.396 0.660 0.526 L*T 3 4.326 1.442 3.180 0.043 1.165 0.388 1.910 0.155 67.063 22.354 2.310 0.102 L*Y 6 7.396 1.233 2.720 0.038 1.431 0.239 1.170 0.354 20.708 3.451 0.360 0.899 T*Y 2 0.928 0.464 1.020 0.375 0.067 0.033 0.160 0.850 30.125 15.063 1.550 0.232 L*T*Y 6 0.593 0.099 0.220 0.967 0.061 0.010 0.050 0.999 8.375 1.396 0.140 0.989 Error 24* 10.432 0.454 4.885 0.204 232.500 9.687 Total 47 27.694 15.503 895.979 P-values in bold are below the 0.05 cutoff for statistical significance Total weevil density includes eggs. EWM biomass (g m-2) was log(x ? 1) transformed * 23 df for weevil density (#/stem) in June. Weevil density data were unavailable for Long bed C in 2014 due to lack of EWM plants densities was due to differences among beds. Inspec- During a preliminary survey of our study lakes in mid tion of Fig. 2 shows no clear indication that particular June 2012 and again in 2015, EWM had already beds were consistently good for finding high weevil matted on the surface in most of the lake beds and densities. many of the plants had begun flowering. In contrast, Weevil abundance estimates from 2013 (Fig. 2) June surveys in both 2013 and 2014 showed EWM indicate that our treatment addition of weevils was plants still well below the surface in most lakes (with weak. In only one EWM bed (Long bed D), were the exception of flowering plants in Little Bearskin added weevils much higher than background; in all Lake beds B and C). other beds they added a minor component (e.g., Little To examine if phenology was related to weather, Bearskin beds B and C) (Fig. 2). The lack of a we computed the number of degree days between ice significant treatment effect (Table 2) indicates that the out and June 1st for each year of our study using daily weevil augmentation did not result in significantly observations of ice condition in five area lakes and higher weevil densities above background levels. In measurements of water temperature at 1 m depth in a other words, our manipulation created a weak treat- nearby reference lake monitored by the LTER pro- ment of augmented weevils to this study system. The gram (Allequash Lake, https://lter.limnology.wisc. significant lake-by-treatment interaction reflects the edu/). In 2012, area lakes warmed early (average ice fact that some lakes (e.g., Manson) by chance had out day among lakes Julian day 80 = March 20) and, augmentation of pre-existing populations and little or by June 1, Allequash Lake had accumulated 836 no weevils in their control beds, while other lakes had degree days (above 0 °C) since ice out. In contrast, in larger initial populations in their control beds than in both 2013 and 2014, the spring thaw was late (days those augmented with weevils (Fig. 2). 130 and 127, respectively) and, by June 1, had accumulated only 253 and 308 degree days since ice out, Biomass of Eurasian watermilfoil and native plants respectively. Ice out in 2015 was again early (day 105) and, by June 1, 597 degree days had accumulated. Phenology and biomass differences among years Despite these large year-to-year differences in lake heating and plant growth, EWM biomass in June was

In our study lakes, EWM typically grew to the surface quite similar across years (F2,24 = 0.07, P = 0.934). during June, increased in biomass during the summer, Biomass of EWM did grow considerably during the and reached its maximum in August. Nevertheless, we summer. Biomass in August was typically about observed large differences in phenology among years. double the biomass in June (medians 31.2 and 123 90 Hydrobiologia (2017) 800:81–97

16.7 g m-2, for August and June, respectively; ESM significant interaction between bed and year. We have Appendix 2) (Wilcoxon signed-rank test, W 908.5, already demonstrated no significant lake*year inter- P \ 0.001). Because peak biomass offers the greatest action (Table 2, F = 1.57, P = 0.200), indicating that challenge to lake management, we focus most of our differences among lakes were similar each year. To attention on August biomass. In contrast to the June get at the bed*year interaction, we reran ANOVA data, EWM biomass in August showed significant using EWM biomass from the ten rake samples as differences among years (Table 2), being about 45% separate replicates within each bed. Sources of higher in August 2013 than in August 2014 and 2015 variation included bed and year. Results for EWM (ESM Appendix 2). In contrast to EWM, August biomass showed highly significant bed X year inter- biomass of native plants showed no significant vari- actions (F24, 432 = 2.75, P \ 0.001 for June; and ation among years (F2,24 = 0.85, P = 0.441; ESM F = 4.04, P \ 0.001 for August). So, the answer to Appendix 3). Similarly, %EWM (of total submerged the question appears to be no; beds with highest EWM plant biomass) showed no significant difference biomass in one year do not tend have highest biomass among years in August (F2, 24 = 0.98, P = 0.391; in other years. To summarize, at the level of individual ESM Appendix 4), indicating that dominance of EWM lakes, high EWM lakes tended to remain high was similar for all years in our study. throughout our 3-year study. In contrast, EWM biomass within individual beds tended to wax and Variation among beds wane independently of other beds. None of the biomass response variables showed any EWM biomass in August showed large differences significant effect from weevil treatment (e.g., for -2 among beds (range 0–333 g m ) and ANOVA indi- EWM biomass, treatment F1,24 = 0.59, P = 0.448). cated highly significant differences among lakes Given the small treatment imposed relative to back- (Table 2). The lake means (across beds and years) ground variation in weevil densities (Fig. 2), this ranged from 3.9 g m-2 in Boot Lake to 170.8 g m-2 result is not surprising. We thus cannot demonstrate in Manson Lake. August values of % EWM also varied any causality between weevil stocking and decline in substantially among beds. For instance, in August EWM biomass (or %EWM). Below we use the high 2015 % EWM ranged from 0% (Boot beds A and B) to variation in weevil abundance among beds along with 99.4% (Long bed D). ANOVA indicated highly an ANCOVA model to examine the independent significant variation among lakes in % EWM effects of lake, year, and weevil density (covariate) on

(F3,24 = 12.34, P \ 0.0005), with a lake-wide aver- different biomass response variables, as well as age of 9.7% in Little Bearskin Lake and up to 84.3% in examine some correlations with weevil density. Long Lake). Biomass of native plants in August also showed significant variation among lakes (F3, 24 = 16.30, EWM biomass dependence on weevil abundance P \ 0.0005). Lake means across beds and years ranged from 3.0 g m-2 in Long Lake to 328.3 g m-2 Locations where weevils were abundant tended to in Little Bearskin Lake. Although we did not measure have high amounts of weevil damage to EWM stems their separate weights, the native submerged plants and also reduced EWM biomass (Fig. 3). During June, that appeared to have the highest biomass in our study when weevils were most abundant, prevalence of stem beds included coontail (Ceratophyllum demersum L.) damage (%of EWM plants) ranged from 0 to 85% and fern leaf pondweed (Potamogeton robbinsii among the study beds (median 28%) and appeared to Oakes) (S. Knight and J. Havel, pers. obs.). We have a strong positive relationship with weevil density detected these native species in all the study beds in (Fig. 3A). Stem damage was lower in August (10%) most years (ESM Appendix 1). and significantly different from June (Kruskal–Wallis, Do beds with highest EWM biomass in 1 year tend H = 6.73, P = 0.009). EWM biomass in June have high biomass in other years? This question asks if appeared to have a strong negative relationship with differences among beds in EWM biomass are similar weevil density in June (Fig. 3B). Because multiple in each year, and the question can be formally factors may be influencing biomass, we employed evaluated with ANOVA by looking for a lack of ANCOVA to tease out their independent effects. 123 Hydrobiologia (2017) 800:81–97 91

After factoring out possible lake and year effects, EWM biomass was strongly dependent on variation in June weevil density among beds (Table 3). This result was present for EWM biomass measured in both June and August, as well as for EWM biomass measured relative to total plant biomass (%EWM). Although EWM biomass varied substantially from bed to bed, no consistent trends occurred with respect to lake or year (Table 3). The absence of significant interaction effects indicates that the effect of weevils on EWM biomass was not greater in particular lakes or years. The lower weevil densities in August of each year (Fig. 3) had less of an influence on EWM; analysis of covariance indicated no significant effect of August weevil density on either EWM biomass (P = 0.948) or %EWM (P = 0.923). These latter analyses also revealed significant differences among lakes in both EWM biomass (P = 0.003) and %EWM (P = 0.019), a result concordant with the earlier factorial ANOVA results (Table 2). During August of 2013 and 2014, Manson Lake tended to have much higher biomass of EWM (mean 164.6 g m-2) than did the other 3 lakes (mean 29.9 g m-2, Appendix 2). In contrast, EWM biomass in August 2015 showed no significant difference among lakes. Interestingly, growth of EWM over the summer Fig. 3 Associations of EWM with density of milfoil weevil in (%change from June-to-August) was unaffected by June, 2013–2015. A Percentage of EWM stems showing weevil June weevil density (Table 3). This result suggests damage versus weevil density. B EWM biomass versus weevil that, despite differences in EWM biomass among density. Weevil density includes all life stages, including eggs. Each point represents a different EWM bed and year beds, growth in EWM biomass was independent of weevil density. Although weevils seriously damaged

Table 3 Summary from ANCOVAs, with weevil density in June as covariate Source DF F-statistics and P-value by response variable EWM-June %EWM-June EWM-Aug %EWM-Aug Aug–June% change

Weevils/stem 1 8.84 (0.007) 7.65 (0.011) 6.33 (0.019) 7.76 (0.010) 1.62 (0.218) Lake 3 2.83 (0.061) 1.03 (0.397) 3.27 (0.040) 2.72 (0.068) 0.96 (0.430) Year 2 3.26 (0.057) 1.06 (0.362) 0.61 (0.554) 0.78 (0.471) 1.21 (0.319) W*L 3 0.03 (0.993) 0.35 (0.787) 1.49 (0.244) 0.97 (0.426) 1.11 (0.370) W*Y 2 (0.686) 0.38 0.40 (0.673) 0.81 (0.456) 0.50 (0.613) 1.02 (0.378) L*Y 6 0.95 (0.480) 0.10 (0.996) 0.65 (0.687) 0.15 (0.986) 1.16 (0.365) W*L*Y 6 0.49 (0.811) 0.33 0.916) 0.21 (0.971) 0.26 (0.951) 0.59 (0.733) P-values in bold are below the 0.05 cutoff for statistical significance EWM biomass values were transformed by log(x ? 1), %EWM transformed by as in (SQRT(P)); no transformation was necessary for %change Error df (23) reduced because of one missing value for weevils per stem (June 2014, Long bed C) 123 92 Hydrobiologia (2017) 800:81–97 the plants, weevils could not control subsequent and roots (Newman et al., 1996). Based on his review growth of EWM biomass in our study lakes. of available literature, Newman (2004) concluded that densities C1 weevils per stem can control EWM and Diversity of submerged native plants in EWM beds densities \0.1 probably cannot control the plant. (Here, ‘‘weevils’’ refer to the total of larvae, pupae, Identification of native plants from our rake samples and adults (LPA), i.e., not including eggs; R. Newman, (ESM Appendix 1) allowed assessing diversity of pers. com., 27 May 2016). Nevertheless, extensive lake submerged native plants in each of the EWM beds. surveys from several regions suggest that natural Native species richness varied highly among the beds, weevil densities in most lakes with EWM are generally with a range from two in Long Lake beds A and C up quite low, with a median density of ca. 0.2 LPA weevils to 18 in Little Bearskin Lake bed C. Analysis of per stem (n = 127 lakes) and only 9% of lakes Variance indicated highly significant differences exceeding the one weevil per stem threshold (Jester among lakes (F3, 24 = 16.80, P \ 0.0005); on aver- et al., 2000; Tamayo et al., 2004; Reeves et al., 2008; age, Little Bearskin Lake had the highest species Havel & Knight, 2016 chapter 3). This pattern suggests richness (mean 12.9 species per bed) and Long Lake that conditions in many lakes are not favorable for the lowest (4.2 species per bed). sustaining high weevil populations. While some com- Did the weevil stocking treatment result in higher panies have provided weevil stocking as a professional native species richness? Weevil stocking had no service (Reeves et al., 2008), little is known about the statistically significant effect on species richness degree to which this intervention is successful. (Table 2). Such a result agrees with our other obser- In the current study, we attempted to test under vation that stocking had no significant effect on EWM realistic lake conditions whether or not increased biomass. densities of milfoil weevils can result in control of Are beds with high EWM biomass also places with Eurasian watermilfoil. Stocking was done in only a a low species richness in native plants? Although a single year. The experiment failed to significantly negative association might be expected, we found no increase weevil densities and subsequently did not significant correlation in our dataset between species show a treatment effect on EWM biomass or any of the richness and EWM biomass (Spearman correlation, other response variables. Nevertheless, our detailed rS = 0.035, 46 df, P = 0.811). surveys of 16 EWM beds over three summers provide information on the variation of weevil densities at different spatial scales (beds, lakes) and further Discussion evidence that high densities of the weevils are associated with high amounts of weevil damage and Field surveys in several lakes have shown a strong low biomass of EWM. Results from our field exper- correlation between elevated densities of the milfoil iment, together with analysis of prior published field weevil and natural declines in EWM (Sheldon & experiments, allow a fresh perspective on biocontrol Creed, 1995; Lillie, 2000; Newman & Biesboer, 2000). of EWM and other invasive aquatic plants. Here we Do weevils cause these declines? Although such discuss the following: (1) design of field experiments correlations cannot by themselves indicate causation testing biocontrol of nuisance weeds in lakes, using (Whitlock & Schluter, 2009), the combination of field examples from studies of milfoil weevil; and (2) data and mesocosm experiments provide compelling recommendations on lake conditions that should favor evidence that milfoil weevils can have a significant the use of milfoil weevils in biocontrol of EWM. effect in suppressing EWM populations (Newman, 2004). Experiments in lake enclosures indicated that Design of field experiments testing biocontrol weevil additions caused a reduction in both stem and root biomass, as well as stem collapse (Creed et al., Biocontrol of EWM and other nuisance plants is best 1992; Creed & Sheldon, 1995). In a month-long cattle evaluated using field experiments. Mesocosms are a tank experiment, weevil abundances grew in each of common experimental unit in limnology, allowing the tanks and caused substantial damage to EWM randomization, replication, and control of experimen- plants and reduction of carbohydrates stored in stems tal conditions (Whitlock & Schluter, 2009). However, 123 Hydrobiologia (2017) 800:81–97 93 mesocosms do not capture the full conditions existing and thoroughly documented the number of weevils in lakes (Carpenter et al., 1995; Schindler, 1998). For stocked, as well as changes in plants, invertebrates, the EWM-weevil system, mesocosms constrain dis- and fish diets over a 7-year period (2002–2008). persal of weevils among plants in different beds and Although their study serves as a valuable case study of often fail to capture interactions with other herbivores the factors that can influence the success of weevil and fish predators. A more realistic experiment is to stocking programs (such as fish and other herbivorous augment milfoil weevils directly onto existing EWM insects), the study on impacts of weevils was uncon- beds, an approach used in the current study, as well as trolled (Parsons et al., 2011) and thus the causes for the several other reports (Sheldon, 1997; Jester et al., changes in EWM abundance and biomass cannot be 2000; Reeves et al., 2008; Parsons et al., 2011). determined. Unfortunately, the design of each of these previously Reeves et al. (2008) reported results from stocking published studies does not allow discriminating the 30 lakes by EnviroScience, Inc. The company stocked effect of weevils from other confounding variables. weevils into multiple EWM beds in each lake and In an early investigation of the effects of weevil collected data on EWM densities from these beds, as releases, Sheldon (1997) introduced large numbers of well as from one or two reference (control) beds in weevils (of unspecified stage) into EWM beds in three each lake. Description of their selection of augmented lakes. Two of the lakes lacked controls. In the one lake and control beds (Reeves et al., 2008, p. 145) suggests with controls (Lake Bomoseen), three sites each had that the process was not random, because it followed a paired strips of EWM that were protected from procedure designed to enhance the chance of weevil mechanical harvesting. One member of each pair impacts in the augmented beds that was not fully was stocked during summer and the other left as a matched in the control beds. Clearly, the conditions control. Although the actual distance was not given, that favor control of EWM through weevil augmen- based on the scale of the study, the augmented and tation remain poorly understood. control strips appear to have been quite close. Indeed, In the current study, we attempted a carefully in year two (1994) the controls were colonized from designed field experiment to assess the effects of spring migrants and so all beds were effectively weevil stocking on EWM biomass. The study system stocked with weevils. looked promising, in that we had four different lakes Jester et al. (2000) augmented EWM beds in 12 with multiple EWM beds and the cooperation from study lakes with three different densities of milfoil their lake associations. Stocking and control treat- weevils and presented results from the first year ments were randomly assigned to beds and each following augmentation. Unfortunately, the different treatment was replicated within each lake. Neverthe- density levels were in separate lakes and so other less, the experimental elevation of weevil densities in confounding variables among the study lakes could the augmented beds (Fig. 2) did not significantly not be eliminated. Furthermore, because weevil den- increase their densities over background and thus was sities had not significantly increased following aug- insufficient to affect any of our response variables. mentation (Jester et al., 2000 table 5) and random Financial considerations caused EnviroScience to year-to-year changes in EWM biomass is generally terminate their involvement in this project after the large (Johnson et al., 2000; Lillie, 2000, current study), first year and other weevil sources were not available. any post-treatment changes in response variables The single year of stocking that was completed is less could not necessarily be attributed to weevil stocking. than their recommended stocking of 3 years in Although the authors describe greater impacts from succession (C. Marquette, pers. com.). The high the highest density treatment, our reanalysis of their among-bed variation in background weevil densities data, comparing between-year change in EWM within each of the study lakes (Fig. 2) created biomass among the three groups (substituting 0 for considerable background ‘‘noise’’ in this study system. non-significant changes between years), indicates no However, our analysis of the variation in plant significant difference among groups (ANOVA, biomass indicated a significant dependence on weevil F = 2.04, P = 0.185). density. Our results suggest that differences in EWM Parsons et al. (2011) carried out a massive stocking biomass among sites were dependent on weevil effort over two summers in a single Washington lake density, although other unknown environmental 123 94 Hydrobiologia (2017) 800:81–97

Table 4 Environmental conditions favoring success from stocking weevils for biocontrol of EWM Variable Ideal state Reason and reference citation*

Shoreline habitat Natural leaf litter, with dry Enhances adult survival over wintera soil Distance of EWM bed to shore Low May enhance dispersal to and from overwintering site Density of small sunfish Low Reduce losses of adults and larvae to predationb Density of other herbivores Low Some other herbivorous insects consume apical meristemc Water temperature 10–30°C Thermal limits for egg hatching and larval development, geography and climated Phenology of weevils and plants Weevils migrate to EWM With flowering, the apical meristem is no longer available for before the plants flower easy entry of larvae into the steme EWM density (or biomass) High Should allow easy dispersal of adults and larvae among plants Location of other EWM beds Near Should allow easy dispersal of adults to other beds Northern watermilfoil Present Native host plant for milfoil weevil provides alternative food for weevils during periods of EWM declinef Chemical control of EWM None Chemicals kill host plants and have unknown effects on fish and weevils Physical disturbance to EWM beds None or low Avoiding physical disturbance allows weevil access to EWM by harvesting or boat traffic apical meristemg * Reference citations: a—Newman et al. (2001), Thorstenson et al. (2013); b–Parsons et al. (2011), Sutter & Newman (1997); Maxson (2016); c– Parsons et al. (2011); d—Mazzei et al. (1999), Creed (1998); e—Wheeler & Center (1997); f—Solarz & Newman (2001); g—Sheldon and O’Bryan (1996a)

features that are also associated with weevil density Recommendations on use of milfoil weevil could be the underlying cause. for biocontrol of EWM In contrast to other studies that either describe or predict multiple generations and growth of weevil Following review of existing literature, Newman populations during the summer (Sheldon & O’Bryan, (2004) suggested that densities C1.0 LPA weevils 1996b; Mazzei et al., 1999), our results from eight per stem can probably control EWM. The critical sampling dates during 2014 in Boot Lake indicated a feature is how to maintain the weevil densities above steady decline in density of all weevil stages over the this threshold. We suspect that a ‘‘perfect storm’’ of summer (Appendix 11) and a decline in weevil conditions must be in place to allow weevils to persist densities from June-to-August during most years in at high densities and to have access to EWM plants for all of the lakes (Fig. 2). Except for occasional high consumption (Table 4). One feature is access to weevil densities, the general pattern across beds and overwintering habitat. In order to allow survival of years was for weevil density to be low. Our observa- adults over winter, EWM beds must be near to tions are consistent with the statement of Newman shoreline habitat that is both dry and includes natural et al. (2001) that lakes having small weevil popula- leaf litter (Newman et al., 2001). Human-altered tions fail to show increases in density over the lakeshores and wetlands make poor weevil habitat. A summer. Perhaps sunfish predation on weevil adults second feature is predation of weevils. During the (Sutter & Newman, 1997) and larvae (Maxson, 2016) warm months, sunfish predators cannot be too numer- limits population growth in summer and subsequent ous; otherwise they consume both adults and larvae recruitment to later years. Further diet studies during (Sutter & Newman, 1997; Maxson, 2016). Ward & periods of highest weevil abundance, together with Newman (2006) suggested that densities of bluegill fish exclosure experiments (Ward & Newman, 2006), sunfish greater than 25–30 per 24-h trap net would are necessary to evaluate this predator-limitation negatively impact weevil populations. Prior fish sur- hypothesis. veys in two of our study lakes (Long Lake in 2006,

123 Hydrobiologia (2017) 800:81–97 95

Manson Lake in 2011) revealed densities of bluegill substantially lower weevil abundance than those not sunfish well above this threshold (99 and 72.6 bluegill, recently treated (Havel et al., in review). As others respectively; Maxson, 2016). Therefore, sunfish may have pointed out, herbicides can have indirect nega- have suppressed weevil densities in at least some of tive effects on biological control agents and so these our study lakes. Managing for large populations of two control measures should be carefully coordinated piscivorous fish may enhance conditions for better (Newman et al., 1998). weevil survival through a trophic cascade (Carpenter In complex lake environments with multiple uses & Kitchell, 1993). by humans, biocontrol may be best used in combina- A related feature important to weevil population tion with other control methods (Madsen, 2000; growth is density of plants in the milfoil beds. Beds Newman, 2004). Sometimes biocontrol is not appro- that are overly choked with EWM or other plants priate, such as in lakes that are heavily used and have prevent piscivore access, releasing sunfish populations little natural shoreline (Newman et al., 2001). Simi- to survive and forage, and thus causing higher larly, lakes with a low density of EWM may be better mortality to weevils and other invertebrates. Density served with other control measures, such as hand of EWM may also influence weevil dispersal, impact- pulling. Lakes with moderate use and some natural ing their ability to find new food resources. If EWM shoreline may benefit with a mixture of biocontrol and density is too low, dispersal of adults and larvae other measures. In locations where humans have a low among plants is likely inhibited. However, the average tolerance of high density aquatic plants (e.g., near boat density of plants alone can be a bit misleading, since docks and swimming beaches) or if a rapid response is even within the EWM beds in our lakes the individual desired, physical removal, such as with diver-assisted plants were quite patchy in their distribution. Using suction harvesting, may be the best choice. In habitats the ten replicate rake samples from each bed and year, more protected from human interference [e.g., bays we quantified this within-bed patchiness in biomass by with lower boat traffic and good shoreline (Newman calculating the variance to mean ratio (V/M) within et al., 2001)] and that have high density of EWM, each bed (and year). This ratio is typically about 1.0 stocked weevils may have the chance to work. To for random distributions and greater than 1.0 for eliminate the risk of sunfish eating the weevils before patchy distributions (Ricklefs, 1990, p. 286). The V/M they can get started, initial stocking might be in fish ratio for our study beds ranged from ca. 0 to 23.1, with exclosures (Ward & Newman, 2006) that are later a median of 3.4, and 61% of the bed-year estimates of removed after the weevils reach high enough density. (V/M) exceeded 2.0. In other words, the typical bed Including signs warning boaters to avoid motoring had a highly patchy structure of EWM plants. through the area can both limit fragmenting EWM Another important environmental feature affecting (and further dispersal) and maintain the apical meris- weevil success is phenology. The timing of the weevils tem needed for the larvae to grow. A lake organization and plants must be matched so that weevil eggs and with a local rearing facility may be an effective larvae attach to the plants prior to flowering (C. approach for producing the weevils needed locally. Marquette, EnviroScience, pers. com., October 26, This process helps to avoid the problem of spreading 2016). A similar effect is seen with other biological strains of EWM among lakes. Furthermore, the use of control agents (Wheeler & Center, 1997). Also, having volunteers can lower cost of rearing and stocking the native milfoils in the EWM bed allows alternate plant weevils and provides a further benefit of increasing hosts for times when EWM may be in low abundance public awareness of complex lake ecosystems. (Solarz & Newman, 2001) or mismatched in its phenology. Finally, the beds cannot be disturbed by Acknowledgements We thank Kevin Gauthier for suggesting mechanical harvesting or by herbicides, as these the project and facilitating discussions with participating lake associations and with EnviroScience. We also thank J. Heywood control measures remove the meristem upon which and L. Morrison for helpful discussion of experimental design, the weevils depend. Others have shown that harvesting and C. Marquette for information on weevil biology and the locally (Sheldon & O’Bryan, 1996a) or in the vicinity rearing method used by EnviroScience. Onterra LLC provided (Newman & Inglis, 2009) reduces abundance of the invaluable assistance with bed mapping and area determination in 2012 and advice on area estimation in other years. We milfoil weevil. Recent survey data suggest that lakes especially thank student workers, J. Bevington, E. Fruhling, C. chemically treated in the past 5 years have Kruger, V. Jones, J. Miazga, C. Winter, and N. Winter, who 123 96 Hydrobiologia (2017) 800:81–97 assisted with field collections and laboratory analysis. Angiosperms: Monocotyledons University of Wisconsin Comments from L. Morrison and two anonymous reviewers Press, Madison. helped us refine the paper. Logistical support was provided by Hauxwell, J., S. Knight, K. Wagner, A. Mikulyuk, M. Nault, M. the UW-Madison Center for Limnology and Trout Lake Station Porzky & S. Chase, 2010. Recommended baseline moni- and financial support by a grant from the Wisconsin Department toring of aquatic plants in Wisconsin: sampling design, of Natural Resources (Grant ACE-122-12). field and laboratory procedures, data entry and analysis, and applications. Wisconsin Department of Natural Resources, Report Number PUB-SS-1068 2010, Madison, Wisconsin. References Havel, J. E. & S. E. Knight, 2016. A field test on the effectiveness of milfoil weevil for controlling Eurasian water- Boylen, C. W., L. W. Eichler & J. D. Madsen, 1999. Loss of milfoil in northern lakes. Final report to Wisconsin native aquatic plant species in a community dominated by Department of Natural Resources, grant ACE-122-12, Eurasian watermilfoil. In Caffrey, J., P. R. F. Barrett, M. Madison, Wisconsin. T. Ferreira, I. S. Moreira, K. J. Murphy & P. M. Wade Havel, J. E., S. E. Knight & J. Miazga, Abundance of milfoil (eds), Biology, Ecology, and Management of Aquatic weevil in northern lakes: potential secondary impacts from Plants. Developments in Hydrobiology. Springer, Nether- herbicide control of Eurasian watermilfoil. Lake and lands: 207–211. Reservoir Management, in review. Buckingham, G. R., 1998. Surveys for insects that feed on Jester, L. L., M. A. Bozek, D. R. Helsel & S. P. Sheldon, 2000. Eurasian watermilfoil, Myriophyllum spicatum, and Euhrychiopsis lecontei distribution, abundance, and Hydrilla, Hydrilla verticillata, in the People’s Republic of experimental augmentations for Eurasian watermilfoil China, Japan and Korea. US Army Corps of Engineers control in Wisconsin lakes. Journal of Aquatic Plant Waterways Experiment Station, Technical report A-98-5, Management 38: 88–97. Vicksburg, Mississippi, 1–131. Johnson, R. L. & B. Blossey, 2002. Eurasian watermilfoil. In Carpenter, S. R. & D. M. Lodge, 1986. Effects of submersed Van Driesche, R., B. Blossey, M. Hoddle, S. Lyon & R. macrophytes on ecosystem processes. Aquatic Botany 26: Reardon (eds), Biological Control of Invasive Plants in the 341–370. Eastern United States. US Forest Service (Publication Carpenter, S. R. & J. F. Kitchell, 1993. The Trophic Cascade in FHTET-2002-04), Morgantown: 79–90. Lakes. Cambridge University Press, Cambridge. Johnson, R. L., E. M. Gross & N. G. Hairston, 1998. Decline of Carpenter, S. R., S. W. Chisholm, C. J. Krebs, D. W. Schindler the invasive submersed macrophyte Myriophyllum spica- & R. F. Wright, 1995. Ecosystem experiments. Science tum (Haloragaceae) associated with herbivory by larvae of 269: 324–327. Acentria ephemerella (Lepidoptera). Aquatic Ecology 31: Cheruvelil, K. S., P. A. Soranno & J. D. Madsen, 2001. Epi- 273–282. phytic macroinvertebrates along a gradient of Eurasian Johnson, R. L., P. J. Van Dusen, J. A. Toner & N. G. Hairston, watermilfoil cover. Journal of Aquatic Plant Management 2000. Eurasian watermilfoil biomass associated with insect 39: 67–72. herbivores in New York. Journal of Aquatic Plant Man- Cline, A. R., B. Villegas, M. J. Pitcairn & C. W. O’Brien, 2013. agement 38: 82–88. The status of Euhrychiopsis lecontei (Dietz) (coleoptera: Keast, A., 1984. The introduced aquatic macrophyte, Myrio- Curculionidae) in California, with notes on other weevils phyllum spicatum, as habitat for fish and their invertebrate associated with milfoil. Coleopterists Bulletin 67: 75–80. prey. Canadian Journal of Zoology 62: 1289–1303. Creed Jr., R. P., 1998. A biogeographic perspective on Eurasian Lillie, R. A., 2000. Temporal and spatial changes in milfoil watermilfoil declines: additional evidence for the role of distribution and biomass associated with weevils in Fish herbivorous weevils in promoting declines? Journal of Lake, WI. Journal of Aquatic Plant Managment 38: Aquatic Plant Management 36: 16–22. 98–104. Creed Jr., R. P. & S. P. Sheldon, 1993. The effect of feeding by a MacRae, I. V. & R. A. Ring, 1993. Life history of Cricotopus North American weevil Euhrychiopsis lecontei on Eur- myriophylli Oliver (Diptera: Chironomidae) in the asian watermilfoil (Myriophyllum spicatum). Aquatic Okanagan Valley, British Columbia. The Canadian Ento- Botany 45: 245–256. mologist 125: 979–985. Creed Jr., R. P. & S. P. Sheldon, 1995. Weevils and watermil- MacRae, I. V., N. N. Winchester & R. A. Ring, 1990. Feeding foil: did a North American herbivore cause the decline of activity and host preference of the milfoil midge, Cri- an exotic plant? Ecological Applications 5: 1113–1121. copterus myriophylli Oliver (Diptera: Chironomidae). Creed Jr., R. P., S. P. Sheldon & D. M. Cheek, 1992. The effect Journal of Aquatic Plant Management 28: 89–92. of herbivore feeding on the buoyancy of Eurasian water- Madsen, J. D., 2000. Advantages and disadvantages of aquatic milfoil. Journal of Aquatic Plant Management 30: 75–76. plant management techniques, US Army Corps of Engi- Crow, G. E. & C. B. Helquist, 2000a. Aquatic and Wetland neers, Report Number ERDC/EL MP-00-1. Vicksburg, Plants of Northeastern North America, Vol. One., Pteri- Mississippi. dophytes, Gymnosperms and Angiosperms: Dicotyledons Madsen, J. D., J. W. Sutherland, J. A. Bloomfield, L. W. Eichler University of Wisconsin Press, Madison. & C. W. Boylen, 1991. The decline of native vegetation Crow, G. E. & C. B. Helquist, 2000b. Aquatic and Wetland under dense Eurasian watermilfoil canopies. Journal of Plants of Northeastern North America, Vol. Two., Aquatic Plant Management 29: 94–99.

123 Hydrobiologia (2017) 800:81–97 97

Maxson, K. A., 2016. Trophic interactions and the efficacy of Reeves, J. L., P. D. Lorch, M. W. Kerschner & M. A. Hilovsky, milfoil weevils for biocontrol of Eurasian watermilfoil in 2008. Biological control of Eurasian watermilfoil by Wisconsin lakes. Master’s Thesis, Missouri State Univer- Euhrychiopsis lecontei: assessing efficacy and timing of sity, Springfield, Missouri. sampling. Journal of Aquatic Plant Management 46: Mazzei, K. C., R. M. Newman, A. Loos & D. W. Ragsdale, 144–149. 1999. Developmental rates of the native milfoil weevil, Ricklefs, R. E., 1990. Ecology, 3rd ed. W.H. Freeman, New Euhrychiopsis lecontei and damage to Eurasian watermil- York. foil at constant temperatures. Biological Control 16: Schindler, D. W., 1998. Whole-ecosystem experiments: repli- 139–143. cation versus realism: the need for ecosystem-scale McComas, S., 1993. Lake Smarts: The First Lake Maintenance experiments. Ecosystems 1: 323–334. Handbook. Terrene Institute, Washington, D.C. Sheldon, S. P., 1997. Investigations on the potential use of an Nault, M. E., M. D. Netherland, A. Mikulyuk, J. G. Skogerboe, aquatic weevil to control Eurasian watermilfoil. Journal of T. Asplund, J. Hauxwell & P. Toshner, 2014. Efficacy, Lake and Reservoir Management 13: 79–88. selectivity, and herbicide concentrations following a Sheldon, S. P. & R. P. Creed Jr., 1995. Use of a native insect as a whole-lake 2,4-D application targeting Eurasian water- biological control for an introduced weed. Ecological milfoil in two adjacent northern Wisconsin lakes. Lake and Applications 5: 1122–1132. Reservoir Management 30: 1–10. Sheldon, S. P. & L. M. O’Bryan, 1996a. The effects of har- Newman, R. M., 2004. Biological control of Eurasian water- vesting Eurasian watermilfoil on the aquatic weevil milfoil by aquatic insects: basic insights from an applied Euhrychiopsis lecontei. Journal of Aquatic Plant Man- problem. Archiv fu¨r Hydrobiologie 159: 145–184. agement 34: 76–77. Newman, R. M. & D. D. Biesboer, 2000. A decline of Eurasian Sheldon, S. P. & L. M. O’Bryan, 1996b. Life history of the watermilfoil in Minnesota associated with the milfoil weevil Euhrychiopsis lecontei, a potential biological con- weevil Euhrychiopsis lecontei. Journal of Aquatic Plant trol agent of Eurasian watermilfoil. Entomological News Management 38: 105–111. 107: 16–22. Newman, R. M. & W. G. Inglis, 2009. Distribution and abun- Solarz, S. L. & R. M. Newman, 2001. Variation in hostplant dance of the milfoil weevil, Euhrychiopsis lecontei,in preference and performance by the milfoil weevil, Euhry- Lake Minnetonka and relation to milfoil harvesting. Jour- chiopsis lecontei Dietz, exposed to native and exotic nal of Aquatic Plant Management 47: 21–25. watermilfoils. Oecologia 126: 66–75. Newman, R. M., K. L. Holmberg, D. D. Biesboer & B. G. Pen- Sutter, T. J. & R. M. Newman, 1997. Is predation by sunfish ner, 1996. Effects of a potential biocontrol agent, Euhry- (Lepomis spp.) an important source of mortality for the chiopsis lecontei, on Eurasian watermilfoil in experimental Eurasian watermilfoil biocontrol agent Euhrychiopsis tanks. Aquatic Botany 53: 131–150. lecontei? Journal of Freshwater Ecology 12: 225–234. Newman, R. M., D. C. Thompson & D. B. Richman, 1998. Surface Water Integrated Monitoring System 2014. Wisconsin Conservation Strategies for the Biological Control of Department of Natural Resources. http://dnr.wi.gov/topic/ Weeds. In Barbosa, P. (ed), Conservation Biological surfacewater/swims/. Accessed September 3, 2014. Control. Academic Press, New York: 371–396. Tamayo, M., C. E. Grue & K. Hamel, 2004. Densities of milfoil Newman, R. M., D. W. Ragsdale, A. Milles & C. Oien, 2001. weevil (Euhrychiopsis lecontei) on native and exotic Overwinter habitat and the relationship of overwinter to in- watermilfoils. Journal of Freshwater Ecology 19: 203–211. lake densities of the milfoil weevil, Euhrychiopsis lecontei, Thorstenson, A. L., R. L. Crunkilton, M. A. Bozek & N. a Eurasian watermilfoil biological control agent. Journal of B. Turyk, 2013. Overwintering habitat requirements of the Aquatic Plant Management 39: 63–67. milfoil weevil, Euhrychiopsis lecontei, in two central Painter, D. S. & K. J. McCabe, 1988. Investigation into the Wisconsin lakes. Journal of Aquatic Plant Management 51: disappearance of Eurasian watermilfoil from the Kawartha 88–93. Lakes. Journal of Aquatic Plant Management 26: 3–12. Ward, D. M. & R. M. Newman, 2006. Fish predation on Eur- Parsons, J. K., G. E. Marx & M. Divens, 2011. A study of asian watermilfoil (Myriophyllum spicatum) herbivores Eurasian watermilfoil, macroinvertebrates and fish in a and indirect effects on macrophytes. Canadian Journal of Washington lake. Journal of Aquatic Plant Management Fish and Aquatic Sciences 63: 1049–1057. 49: 71–82. Wheeler, G. S. & T. D. Center, 1997. Growth and development Pfingsten, I.A., L. Berent, C.C. Jacono, and M.M. Richerson, of the biological control agent Bagous hydrillaeas influ- 2016. Myriophyllum spicatum. USGS Nonindigenous enced by Hydrilla (Hydrilla verticillata) stem quality. Aquatic Species Database, Gainesville, Florida. https://nas. Biological Control 8: 52–57. er.usgs.gov/queries/FactSheet.aspx?speciesID=237. Whitlock, M. C. & D. Schluter, 2009. The Analysis of Biolog- Accessed October 31,2016. ical Data. Roberts and Company, Greenwood Village. Pimentel, D., R. Zuniga & D. Morrison, 2005. Update on the Zhang, C. & K. J. Boyle, 2010. The effect of an aquatic invasive environmental and economic costs associated with alien- species (Eurasian watermilfoil) on lakefront property val- invasive species in the United States. Ecological Eco- ues. Ecological Economics 70: 394–404. nomics 52: 273–288.

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