Journal of Pest Science

, Volume 90, Issue 4, pp 1257–1268 | Cite as

Halyomorpha halys mortality and sublethal feeding effects following insecticide exposure

  • Theresa M. Cira
  • Eric C. Burkness
  • Robert L. Koch
  • W. D. Hutchison
Original Paper


The brown marmorated stink bug, Halyomorpha halys (Stål), is a highly polyphagous invasive pest. Increased use of broad-spectrum insecticides to manage H. halys has resulted in secondary pest outbreaks and disruptions to integrated pest management (IPM) programs. We evaluated H. halys mortality, molting, and feeding after exposure to insecticides in the laboratory. Five insecticides (four active ingredients), considered less risky to natural enemies, were compared to a pyrethroid insecticide and an untreated control. Compared to the control, only azadirachtin + pyrethrins significantly reduced egg hatch, while all insecticides caused significant direct mortality to 1st and 2nd instars 5 days after hatch (DAH). Bifenthrin quickly caused complete mortality of adults, and the only insecticide to statistically match this level of mortality was sulfoxaflor at 14 days after treatment (DAT). Azadirachtin + pyrethrins and sulfoxaflor significantly reduced the proportion of 1st instars that molted compared to the control. Adults that survived sulfoxaflor exposure produced significantly fewer feeding sites than the control. However, when taking into consideration both lethal and sublethal effects, all insecticides, except pyrethrins, resulted in significant reductions in feeding sites/individual compared to the control. This more complete estimate of efficacy (i.e., reduction in injury/insect), confirms the potential of several insecticides to reduce crop injury without the necessity of high direct mortality to H. halys.


Brown marmorated stink bug Azadirachtin + pyrethrins Spinosad Bifenthrin Salivary flange 

Key message

  • Insecticide efficacy is often determined by direct mortality, but sublethal effects, particularly on feeding, are also important.

  • Very little is known about sublethal effects of insecticides on H. halys feeding.

  • Adults that survived sulfoxaflor exposure exhibited high levels of feeding reductions.

  • Combining direct mortality and sublethal feeding effects provided a more detailed metric of insecticide efficacy (i.e., feeding sites/individual).

  • Azadirachtin + pyrethrins, spinosad, organic-certified spinosad, and sulfoxaflor did not cause complete mortality, but significantly reduced feeding sites/individual.


The brown marmorated stink bug, Halyomorpha halys (Stål), a highly polyphagous and invasive insect in North America and Europe, causes economic injury to many high-value crops such as apples, soybeans, and corn (Rice et al. 2014). Management of H. halys in the mid-Atlantic region of the USA, where its pest status is severe, has relied mainly on chemical control since 2010 (Rice et al. 2014). Broad-spectrum insecticides such as pyrethroids, neonicotinoids, carbamates, and organophosphates are currently used against H. halys because they cause high, direct mortality (e.g., Funayama 2002; Nielsen et al. 2008; Leskey et al. 2012a). However, these insecticides are also lethal to a wide range of other species, including natural enemies (e.g., Theiling and Croft 1988). The suppression of natural enemy populations through broad-spectrum insecticide applications can cause secondary pest outbreaks (Ripper 1956; Hardin et al. 1995), as has been observed after broad-spectrum insecticide use on H. halys (Leskey et al. 2012a; Lee et al. 2014). Moving forward, integrated pest management (IPM) plans for H. halys will need to balance risks to natural enemies with reducing pest populations and crop injury (Rice et al. 2014).

Insecticide efficacy is often characterized by the amount of mortality in pest populations within a given time period (Stark and Banks 2003; Ioriatti et al. 2006; He et al. 2013; Adams et al. 2016). However, insecticides can significantly affect population dynamics through non-lethal means as well. In fact, in a review of the population-level effects of pesticides on arthropods, Stark and Banks (2003) state that characterizing the sublethal effects of insecticides, in addition to direct mortality, will produce a more accurate estimate of overall insecticide efficacy. Currently, many insecticides tested on H. halys do not cause a high proportion of direct adult mortality, even at the highest labeled rates (e.g., Leskey et al. 2012a, b). High mobility of adult H. halys allows for quick dispersion from treated areas, which may further limit exposure to lethal doses (Lee et al. 2014; Morrison et al. 2017), but these insecticides may still have important sublethal impacts. Therefore, measuring sublethal effects, in addition to lethal effects of insecticides, could lead to more accurate assessments of insecticide efficacy for H. halys.

A sublethal effect particularly pertinent to insecticide efficacy is a reduction in feeding. Salivary flanges and sheaths produced by several stink bug species have been shown to be a good predictor of crop injury and/or damage (Bowling 1979, 1980; Viator et al. 1983; Barbour et al. 1990; Bundy et al. 2000; Zeilinger et al. 2015). Halyomorpha halys feeding sites, in particular, have been correlated with feeding injury that results in impacts on crop yields and quality and economic losses (Leskey et al. 2012b; Owens et al. 2013; Cissel et al. 2015). When H. halys and other phytophagous Pentatomidae feed, a viscous saliva is secreted and solidifies around the labellar lobes on the surface of the food source. This hardened saliva is called a salivary flange and remains on the plant after the insect has stopped feeding. Inside of the food source, more viscous saliva is produced to create a hardened salivary sheath around the stylets (Chapman 2013). These dried salivary flanges and sheaths can be easily visualized for quantification by staining (Bowling 1979, 1980).

Risks to agriculturally important natural enemies have previously been studied for certain active ingredients (AIs), including pyrethrins, an Organic Materials Review Institute (OMRI)-certified organic insecticide, derived from Chrysanthemum spp. flowers; azadirachtin, also an option for organic growers in the USA, derived from the neem tree, Azadirachta indica A. Juss.; sulfoxaflor, a relatively new insecticide with broad-spectrum efficacy on sap-sucking insects (Zhu et al. 2011); and spinosad, a United States Environmental Protection Agency (EPA) registered “reduced risk” product, available with or without OMRI organic-certification, obtained by fermentation of the soil bacterium Saccharopolyspora spinosad. A reduced risk pesticide is defined by the EPA’s Office of Pesticide Programs as a pesticide which poses “less risk to human health and the environment than existing conventional alternatives” (United States Environmental Protection Agency 2016). The aforementioned AIs pose less risk (i.e., direct mortality) to generalist natural enemy species (Viñuela et al. 2000; Kraiss and Cullen 2008a, b; Garzón et al. 2015; Pezzini and Koch 2015; Colares et al. 2016; Tran et al. 2016; Fernández et al. 2017), and reviews of azadirachtin and spinosad concluded that both AIs displayed qualities that align well with the tenets of IPM (Schmutterer 1990; Williams et al. 2003). Conversely, bifenthrin, commonly used in many cropping systems (National Pesticide Information Center 2011), shows high toxicity in the laboratory to multiple natural enemy species, including Harmonia axyridis (Pallas) (Galvan et al. 2005), Orius insidiosus (Say), and Encarsia citrina Crawford (Frank 2012).

To date, research on the effects of insecticides on H. halys has largely focused on direct mortality with few studies characterizing sublethal effects. We were therefore motivated to conduct experiments to assess metrics that directly or indirectly contribute to reducing the potential crop injury caused by an insect after insecticide exposure. We compared the effects of six insecticides (five AIs) on H. halys by measuring direct mortality within an acute time period (i.e., 1–14 days), and two sublethal metrics: development (i.e., the ability of H. halys to molt) and feeding (i.e., changes in quantity of feeding flanges) in the laboratory. Our aim was to obtain a more accurate estimate of overall insecticide efficacy based on lethal and sublethal metrics. In addition, by focusing on insecticidal products that could be less detrimental to natural enemy populations, our work can contribute to more sustainable IPM plans, balancing risks to natural enemies, and reductions in crop injury through lethal and sublethal effects on pest populations.

Materials and methods


Halyomorpha halys were sourced from a laboratory colony at the University of Minnesota. This colony was founded from egg masses obtained in spring of 2012 from a laboratory colony at the University of Maryland and was supplemented with field-collected eggs from Virginia in the summer of 2013. Insects were maintained at ~25 °C, 16L:8D in walk-in growth chambers. Within the growth chambers, insects were kept in 38 × 38 × 61 cm mesh cages (BioQuip, Rancho Dominguez, CA) and provisioned with potted snap bean plants (Phaseolus vulgaris L. cv “Romano Bush”), fresh organic snap bean pods, dried raw organic sunflower (Helianthus annus L.) seeds and soybean (Glycine max (L.) Merr.) seeds ad libitum. Cages and plants were misted with water every weekday to maintain humidity. Determination of instars was based on morphological traits outlined in Hoebeke and Carter (2003), and determination of sex was based on visual inspection of the ventral, apical section of the abdomen (Rice et al. 2014).

Insecticide application

Insecticides were applied using a Teejet 8002EVS flat-fan nozzle in a motorized spray chamber. The sprayer was calibrated to deliver the equivalent of 220 L water/ha at 0.24 MPa (23.5 gallons of water/acre at 35 psi). This delivery rate optimized the area of the Petri dish covered and post-spray dry time. The spray tank was rinsed three times between treatments. Table 1 details the six insecticides used in our experiments. The same application rates were used to treat eggs and adults, though pyrethrins and conventional spinosad were only tested on adults. A randomized complete block design, with date of exposure as the block, was used. Within a block, the experiment was replicated three times for eggs and six times for adults. This resulted in totals of 12 egg mass replications (295.4 ± 7.00 (SEM) eggs/treatment) and 30 adult replications/treatment. Experiments were conducted between March 25, 2013, and January 13, 2014.
Table 1

Insecticides and application rates used in bioassays of H. halys. All information pertains to labels for use of the product in the USA

Active ingredient

Trade name and manufacturer

Application rate

Insects and crops labeled for the tested rate

Azadirachtin + Pyrethrins

Azera®, MGK, Minneapolis, MN

4.09 L product/ha (56 oz. product/ac)

High populations of adult insects, such as H. halys, on a wide variety of row and orchard crops


PyGanic® EC 1.4II, MGK, Minneapolis, MN

4.69 L product/ha (64 oz. product/ac)

Highest labeled rate for pentatomids in row and orchard crops

Spinosad (Organic-certified)

Entrust® SC, Dow AgroSciences LLC, Indianapolis, IN

0.73 L product/ha (10 oz. product/ac)

Highest labeled rate in citrus and pome fruits for lepidopteran pests and thrips (Thysanoptera); product is not labeled for pentatomids


Blackhawk®, Dow AgroSciences LLC, Indianapolis, IN

231.18 g product/ha (3.3 oz. product/ac)

Highest labeled rate in corn and legume vegetables for lepidopteran pests; product is not labeled for pentatomids


Transform® WG, Dow AgroSciences LLC, Indianapolis, IN

192.65 g product/ha (2.75 oz. product/ac)

Highest labeled rate in root and tuber vegetables for hemipteran pests (e.g., Cicadellidae and Aleyrodidae)


Brigade® 2EC, FMC Corporation, Philadelphia, PA

0.47 L product/ha (6.4 oz. product/ac)

Pentatomids in a variety of crops, including field and sweet corn, cucurbits and grapes

Insecticide exposure

Halyomorpha halys eggs were exposed to insecticides topically. Egg masses were removed from snap bean leaves, along with a small amount of leaf tissue to which they were attached, approximately 4 days after oviposition. After removal, and before insecticide treatment, eggs that were not damaged and had developed red eye spots and a black egg burster were considered to be viable (personal observation). Any eggs that were not viable were noted and not included in the final analyses. Each egg mass averaged 24.63 ± 1.31 viable eggs. Individual egg masses were placed in the center of a Petri dish (100 × 15 mm, VWR, Radnor, PA, USA) and then sprayed. After 24 h, a half-sheet of moistened filter paper (4.5 cm diameter; Fisher Scientific, Pittsburgh, PA, USA) was placed in the treated Petri dish with the eggs, and Parafilm (Bemis Inc. Neenah, WI, USA) was used to seal approximately 2/3 of the circumference of the Petri dish to maintain humidity, the paper being re-wetted when dry. Upon hatching, 1st instars were left in the treated dishes for 5 days after hatching (DAH) to avoid excessive handling, which may injure 1st instars. Hatching of eggs within a given H. halys egg mass is synchronized, with eggs hatching within 24 h of each other; therefore, DAH for each egg in an egg mass was considered the same. No food or water was added at this stage; 1st instars are thought to only feed on the egg chorion or symbionts on the egg chorion (McPherson 1982; Taylor et al. 2014).

Adult H. halys were exposed to insecticides residually. No topical exposure was applied to this life stage because of their ability to fly out of the dish during the spray. Age of adults was not standardized: Individuals were randomly assigned across insecticide treatments and the control in each block. One adult was added to each treated Petri dish (100 × 15 mm) that had been previously sprayed and allowed to dry completely (approximately 1 h dry time). After 24 h of exposure to the dried residue (with no food or water), individuals were removed and placed individually in clean, lidded tubs (Translucent 473 mL, Consolidated Plastics Stow, OH), provisioned with three dry organic soybean seeds (Glycine max (L.) Merr. and a cotton ball soaked in water. Cotton balls were re-wetted as needed. Soybean seeds were removed and replaced 7 and 14 days after treatment (DAT) and saved for feeding analysis (see below). An equal ratio of females to males was tested. Each individual adult was considered an independent replicate; exposure to treatments occurred in individual arenas and individuals remained separated throughout the experiment.

All blocks also included an untreated control, which consisted of individuals placed in unsprayed Petri dishes and clean tubs as described above. The Petri dishes and clean tub arenas were kept in growth chambers (Percival Scientific, Inc., Perry, Iowa) at 25 °C, 16L:8D.

Direct mortality assessment

Eggs were monitored daily for hatch. At 1 DAH, the number of hatched eggs was divided by the number of total viable eggs (counted pre-treatment) to determine the proportion of unhatched eggs. For all subsequent life stages, mortality was defined by an inability to walk when gently prodded with a soft bristle brush. Assessment of 1st and 2nd instars was completed at 5 DAH. Halyomorpha halys has the ability to recover after an initial knockdown from insecticide treatments (Leskey et al. 2012a); therefore, even individuals that appeared dead remained in containers and were monitored for the full observation period.

Sublethal feeding assessment

Adults received no food or water while in insecticide-treated Petri dishes, so feeding quantification began 24 h after treatment. To quantify feeding of adults, an acid fuchsin solution was used to stain salivary flanges left on the collected seeds, following the methods of Bowling (1979). Briefly, seeds were immersed in staining solution for approximately 2 min and then rinsed gently with water. After air-drying on filter paper, stained salivary flanges on each seed were counted under 8× magnification. The number of feeding sites/individual insect was recorded. The number of feeding sites/day alive for each individual was calculated by dividing the number of feeding flanges in a given time period (e.g., week one, two, or the total of both weeks) by the number of days alive. Fifteen of 210 individuals died between 7–11 DAT or 11–14 DAT, where we did not have daily observations. We interpolated those individuals to have died at the midpoint between the days of observation.

Combined mortality and feeding metric

The number of feeding sites produced after a specific insecticide treatment was divided by the total number of individuals receiving that insecticide treatment (n = 30 individuals) to give the expected mean number of feeding sites/individual. By including both living and dead individuals in the denominator, this metric incorporates direct mortality and sublethal feeding changes to produce a more complete estimate of insecticide efficacy.
Fig. 1

Proportion mortality of H. halys adults after being exposed residually to insecticides for 24 h. Each symbol represents the raw mean across five blocks. Different letters within a day indicate significant differences of least squares means (α = 0.05). Mortality was determined by an inability to walk

Fig. 2

Number of feeding sites/day alive for adult H. halys at a 2–7; b 7–14; c 2–14 days after 24-h exposure to insecticide residues. Each box represents the raw median and 1st and 3rd quartiles, whiskers extend 1.5 × the inter-quartile range from the median, and points indicate outliers. Different letters within a graph indicate significant differences of the modeled medians based on a nonparametric Dunn’s test with Holm’s multiple comparisons correction (α = 0.05)


R version 3.3.2 (R Core Team 2016) and RStudio Desktop version 0.99.902 (RStudio Team 2015) were used for all statistical analyses. All plots were constructed using R [packages and commands: ggplot2, ggplot, (Wickham 2009), Rmisc, summarySE (Hope 2013)], except for Fig. 3, which was made in Sigma Plot (Systat Software Inc.). Where complete separation prohibited accurate calculation of maximum likelihood estimates, a dummy variable was added so that treatments could be compared. For example, all eggs treated with sulfoxaflor hatched, so in each sulfoxaflor block one unhatched egg was added to the raw data. These dummy variables are not presented in tables and figures. In one instance (i.e., bifenthrin 2nd instars at 5 DAH), complete separation occurred and the sample size was less than 5; in this case, the treatment was not included in post hoc comparisons. All post hoc comparisons were conducted on the least squares means or modeled medians.
Fig. 3

Proportion mortality and number of feeding sites/individual for adult H. halys a 7 days after treatment (DAT) and 2–7 DAT, respectively, and, b 14 DAT and 2–14 DAT, respectively. Each symbol represents the raw mean across five blocks. Different letters within a graph indicate significant differences of least squares means of feeding sites/individual (α = 0.05). Dashed lines are plotted at 50% mortality. Mortality was determined by an inability to walk

Treated eggs

A single egg mass in a Petri dish was considered the independent experimental unit. For each egg mass, we counted the number of unhatched eggs, the number of dead nymphs (both 1st and 2nd instars) at 5 DAH, and proportion of 2nd instars at 5 DAH. The proportion of 2nd instars was calculated by dividing the number of 2nd instars by the number of living nymphs (both 1st and 2nd instars) at 5 DAH. Generalized linear mixed effects models [lme4, glmer (Bates et al. 2015), afex, mixed (Singmann et al. 2016)] with binomial error distributions were used to test the fixed effects of insecticide on the three aforementioned variables. Block was included as a random effect in all three models. Likelihood ratio tests were used to calculate P values. Tukey’s HSD [multcomp, cld, glht (Hothorn et al. 2008)] was used to determine significant differences (α = 0.05) between treatments.

Treated adults

A single adult in a Petri dish was considered the independent experimental unit. Generalized linear mixed effects models [lme4, glmer (Bates et al. 2015), afex, mixed (Singmann et al. 2016)] with binomial error distributions were used to test the fixed effects of sex, insecticide treatment, and their interaction on mortality at 7 and 14 DAT. Block was included as a random effect in both models. Likelihood ratio tests were used to calculate P values and backwards elimination (stepwise removal of nonsignificant parameters at P < 0.05, starting with the most complex parameters) was used to determine the final model parameters. Tukey’s HSD [multcomp, cld, glht (Hothorn et al. 2008)] was used to determine significant differences (α = 0.05) between treatments.

When testing the effects of block, sex, insecticide, and their two- and three-way interactions on the number of feeding sheaths/day alive during the first week, second week, and both weeks combined, data were found to violate model assumptions. No appropriate transformations were found, so a nonparametric Kruskal–Wallis rank sum test was used. A Dunn’s test [dunn.test, dunn.test (Dinno 2016)] with a Holm’s multiple comparisons adjusted α (α = 0.05) was used to determine significant differences between treatments.

Generalized linear mixed effects models [lme4, glmer (Bates et al. 2015), afex, mixed (Singmann et al. 2016)] with Poisson error distributions were used to test the fixed effect of insecticide treatment on the mean number of feeding sites/individual in the first week and across the first and second weeks. Block was included as a random effect in both models. Likelihood ratio tests were used to calculate P values. Tukey’s HSD [multcomp, cld, glht (Hothorn et al. 2008)] was used to determine significant differences (α = 0.05) between treatments.


Treated eggs

Direct mortality

Insecticide treatment affected the proportion of unhatched eggs (χ 2 = 35.40, df = 4, P < 0.0001), though the only insecticide treatment different from the control was azadirachtin + pyrethrins, which significantly reduced egg hatch (Table 2). Insecticide treatments applied to eggs also affected nymphal mortality at 5 DAH (χ 2 = 734.75, df = 4, P < 0.0001). Residual exposure to all insecticides caused significantly greater early-instar mortality than the control, with organic-certified spinosad and bifenthrin treatments causing the highest degree of mortality, followed by sulfoxaflor, then azadirachtin + pyrethrins (Table 2).
Table 2

Observed proportion of unhatched H. halys eggs, mortality of nymphs, and surviving nymphs that molted to 2nd instar by 5 days after hatch (DAH) following topical insecticide exposure to eggs


Proportion ± SEM

Unhatched eggs (n)

Nymph mortality at 5 DAH (n)

Nymphs molted to 2nd instars by 5 DAH (n)


0.04 ± 0.01 (284) ab

0.15 ± 0.02 (274) a

0.83 ± 0.02 (234) a

Azadirachtin + Pyrethrins

0.09 ± 0.02 (277) c

0.67 ± 0.03 (252) b

0.13 ± 0.04 (83) b

Spinosad (Organic-certified)

0.03 ± 0.01 (297) ab

0.96 ± 0.01 (288) d

0.08 ± 0.08 (12) ab


0.00 ± 0.00 (317) a

0.83 ± 0.02 (317) c

0.02 ± 0.02 (54) b


0.06 ± 0.01 (302) bc

0.99 ± 0.01 (283) d

0.00 ± 0.00 (4) NA

Raw means and standard errors are presented, and different letters within a column indicate significant differences of least squares means (α = 0.05)

Hatch was determined by an empty egg, nymphal mortality was determined by an inability to walk, and eggs and nymphs were held at 25 °C 16L:12D

Each proportion represents the mean across four blocks

Numbers in parentheses indicate sample size (number of viable eggs, hatched individuals, and surviving individuals) for a given measure


Insecticide treatment applied to eggs also affected the proportion of surviving nymphs that molted to 2nd instar by 5 DAH (χ 2 = 212.06, df = 4, P < 0.0001); azadirachtin + pyrethrins and sulfoxaflor treatment resulted in significantly fewer 2nd instars at 5 DAH than the control (Table 2). While the proportion of organic-certified spinosad-treated individuals that molted to 2nd instar was numerically low, due to a low sample size of living individuals, statistically it did not differ from the control.

Treated adults

Direct mortality

Insecticide treatment (χ 2 = 95.74, df = 6, P < 0.0001) affected mortality at 7 DAT. Insecticide treatment (χ 2 = 92.99, df = 6, P < 0.0001) and sex (χ 2 = 5.21, df = 1, P = 0.02) affected mortality at 14 DAT, but pooled sexes are presented in Fig. 1. Sex was not a significant predictor at 7 DAT, and the interactions of insecticide treatment and sex were not significant predictors in either model (P > 0.05); sex therefore was removed from the final models. Adult mortality increased over time in all treatments except for pyrethrins and azadirachtin + pyrethrins (Fig. 1). These two insecticides resulted in initial knockdown, where individuals appeared dead or moribund, but subsequent assessments of mortality showed they recovered. For pyrethrins-treated individuals, this temporary knockdown was minimal; one individual at 1 h post-treatment and one individual at one DAT were moribund, but by two DAT, all individuals were able to walk. Azadirachtin + pyrethrins-treated individuals showed a greater knockdown. At 1 h post-treatment, the proportion of adults that appeared dead or moribund was 0.66, but at 1 DAT the proportion appearing dead or moribund was only 0.33, and by 2 DAT the proportion of adults that were dead or moribund was reduced to 0.10. At 7 DAT, conventional spinosad, sulfoxaflor, and bifenthrin (Mean ± SEM: 0.43 ± 0.09, 0.53 ± 0.09, and 1.00 ± 0.00, respectively) caused significantly higher mortality than the untreated control (0.03 ± 0.03) (Fig. 1). By 14 DAT, the two spinosad formulations, sulfoxaflor, and bifenthrin (0.50 ± 0.09, 0.43 ± 0.09, 0.70 ± 0.09, and 1.00 ± 0.00, respectively) caused significantly higher mortality than the untreated control (0.10 ± 0.06) (Fig. 1).

Sublethal feeding

Insecticide treatment significantly affected the number of feeding sheaths/day alive in the first week (χ 2 = 26.38, df = 5, P < 0.0001), second week (χ 2 = 31.49, df = 5, P < 0.0001), and total across both weeks (χ 2 = 31.4, df = 5, P < 0.0001). The median number of feeding sheaths/d alive of untreated adults in the first week was 0.50 ± 0.00, 1.50 (median ± first and third quartile), in the second week 2.00 ± 0.00, 2.50, and across both weeks 1.27 ± 0.00, 2.00 (Fig. 2). The only insecticide treatment which significantly reduced the median number of feeding sheaths/day alive was sulfoxaflor. In every time period, the median was 0.00 ± 0.00, 0.00 (Fig. 2).

Combined direct mortality and feeding

After pooling surviving and dead individuals, insecticide treatment significantly affected the mean number of feeding sites/individual in the first week (χ 2 = 768.43, df = 6, P < 0.0001) and across the first and second week combined (χ 2 = 864.45, df = 6, P < 0.0001). The mean number of feeding sites/individual for untreated adults averaged 6.23 ± 1.41 within the first week after exposure and 15.97 ± 2.10 within the first and second week combined (Fig. 3). Insecticide treatments that reduced the mean number of feeding sites/individual were azadirachtin + pyrethrins, organic and conventional spinosad, sulfoxaflor, and bifenthrin in the first week (2.47 ± 0.86, 1.50 ± 0.77, 2.33 ± 0.91, 0.03 ± 0.03, 0.00 ± 0.00, respectively) and across both weeks (9.27 ± 2.35, 10.23 ± 2.62, 12.70 ± 3.24, 0.10 ± 0.07, 0.00 ± 0.00, respectively) (Fig. 3).


Foundational to IPM is the concept of the economic injury level (EIL), or “the lowest population density [of a pest] that will cause economic damage,” where economic damage is the amount of injury that justifies the cost of control measures (Stern et al. 1959). Pedigo et al. (1986) outlined an EIL formula commonly used for insect pests; one of the key components in the formula is injury units/insect. They define injury as an effect, often negative, that an insect has on host physiology. By focusing on injury, as opposed to pest mortality, the EIL does not necessitate complete eradication of an insect population, or that pests must be dead in order to achieve the desired injury units/insect. Our study examined the potential effects that different insecticides could have on three metrics relevant to injury units/insect: direct mortality, molting, and quantity of feeding sites. Changes in feeding physiology and behavior are particularly important factors to consider for H. halys. Their feeding causes both direct crop injury (Leskey et al. 2012b; Owens et al. 2013; Cissel et al. 2015) and indirect injury, via transmission of plant pathogens, such as yeast (Brust and Rane 2013).

Similar to previous studies (Funayama 2002; Nielsen et al. 2008; Funayama 2012; Leskey et al. 2012a; Lee et al. 2013; Leskey et al. 2014; Morrison et al. 2017), we found that bifenthrin quickly caused high mortality to adult H. halys. The other insecticides we evaluated were unable to statistically match the level of direct mortality bifenthrin caused to adults, except for sulfoxaflor at 14 DAT (Fig. 1). Lee et al. (2014) observed higher mortality among adult H. halys when residually exposed to pyrethrins and spinosad than our study. However, they used a pyrethrins rate labeled for ornamentals, nearly double the rate we used based on recommendations for row crops. Higher rates of product would be expected to cause greater mortality, but may be cost prohibitive in some cases.

When tested on H. halys nymphs, all insecticides caused significantly higher mortality than the control, at times statistically equivalent to bifenthrin (Table 2). This suggests that early instars are more susceptible to insecticides than adults, which has previously been seen for H. halys (Bergmann and Raupp 2014). Conversely, when tested on eggs, only the azadirachtin + pyrethrins insecticide treatment resulted in significantly higher mortality than the control (Table 2). While there was statistical significance, the reduction in hatch rate was not dramatic, from 96% hatching in the control to 91% in the azadirachtin + pyrethrins treatment. The effect of spinosad, neem oil, and permethrins on hatch rate was previously tested for H. halys but, compared to our study, lower levels of hatching were observed (Bergmann and Raupp 2014). Differences in methods between studies, such as the age of eggs that were exposed, could explain differences in hatch results. For example, Mathews and Barry (2014) found younger H. halys eggs (i.e., 1 day after oviposition) were impacted more greatly by compost tea (i.e., “biologically-active organic matter”) than older eggs (i.e., 2–3 days after oviposition) when measuring hatch rate. We exposed egg masses approximately four days after oviposition.

Previous investigation of sublethal insecticidal effects on H. halys is limited. Changes in adult mobility in the laboratory after sublethal exposure to insecticides have been investigated (Lee et al. 2013, 2014; Morrison et al. 2017). Other studies assessed H. halys injury on insecticide-treated crops, showing that insecticides could variably affect subsequent injury, but they did not investigate whether this was due to direct mortality or sublethal effects (Funayama 2002; Leskey et al. 2012b; Funayama 2012; Aigner et al. 2015). Leskey et al. (2014) noted a reduction in feeding by living H. halys on certain insecticide-treated apples and peaches, suggesting complete mortality may not be necessary to reduce crop injury. However, without providing insecticide-free food to the insects the authors could not determine the causal mechanism for reduced feeding (Leskey et al. 2014).

By quantifying feeding of surviving individuals on insecticide-free food, we could conclude insecticide repellency was not the mechanism causing feeding reduction. We observed significant reductions in feeding by adults that survived sulfoxaflor exposure (Fig. 2). Sulfoxaflor prevented nearly all living individuals from feeding; only two males fed, for a total of only three salivary flanges between them across 2 weeks (Fig. 2). While observing individuals after insecticide treatment, we noticed sulfoxaflor-treated individuals appeared to be trying to feed. Their maxillary and mandibular feeding stylets, however, were splayed out and did not penetrate the food source (see Online Resource 1). No adults exposed to bifenthrin produced any salivary flanges, but this was due to the high mortality by 1 DAT so sublethal effects could not be measured (Fig. 2). The other insecticides (pyrethrins, azadirachtin + pyrethrins, and both formulations of spinosad) tested on adults did not significantly change feeding quantity of surviving individuals compared to the control (Fig. 2).

We found azadirachtin + pyrethrins and sulfoxaflor reduced molting of 1st instars to 2nd instars (Table 2). However, we cannot conclude whether these insecticides completely prevented molting because we did not monitor individuals until death or molt but stopped after 5 days. These results indicate, at the very least, that these insecticides delay molting to the next instar. In previous studies, exposure to azadirachtin (Schmutterer 1990; Wang et al. 2014) and sulfoxaflor (Brown and Short 2010) delayed development or inhibited molting of some insect species.

In our study, the effect of an insecticide was not solely characterized by direct mortality or the degree to which feeding was reduced, but a combination of these two factors. To summarize both direct mortality and quantity of feeding, we measured the number of feeding sites/individual, accounting for both dead and living individuals (Fig. 3). All insecticides except pyrethrins showed a significant reduction of feeding sites across the first and second weeks after insecticide exposure based on this measure. This means that, due to either death or sublethal feeding effects, exposure to azadirachtin + pyrethrins, organic-certified spinosad, conventional spinosad, sulfoxaflor, and bifenthrin reduced injury/insect compared to no insecticide treatment. When plotted against direct mortality, we get a sense of the level to which direct mortality alone does not capture changes in the amount of injury/insect; this was particularly true for sulfoxaflor (Fig. 3). Sublethal effects (e.g., developmental time, population growth rate, fertility, fecundity) of sulfoxaflor have been studied in other insect species (Garzón et al. 2015; Tang et al. 2015; Chen et al. 2016; Colares et al. 2016; Xu et al. 2016; Fernández et al. 2017; Pan et al. 2017), but our study is the first to report sublethal effects on feeding.

Cannibalism of eggs is known to occur in H. halys colonies (Iverson et al. 2016) and could potentially confound feeding on seeds if females laid and fed upon eggs while in their post-treatment containers. However, only seven out of 105 total females laid eggs during the course of the trial and eggs were removed within 24 h after being laid. Therefore, any potential effect of egg feeding on the amount of seed feeding was likely minimal, but we did not quantify feeding sites on eggs.

In the future, it will be important to further investigate how the insecticides evaluated here perform in the field, measuring both direct mortality and sublethal effects. The duration of exposure in our laboratory experiments was longer than what would be expected in a field-setting, particularly for highly mobile adults. Additionally, some of these insecticides rapidly degrade in field settings (Schmutterer 1988; Katsuda 1999; Williams et al. 2003; Spurlock and Lee 2008), and when mortality of adult H. halys was compared between the laboratory and field settings, it was reduced by >35% (Leskey et al. 2014). Furthermore, caution should be exercised when relying on sublethal effects to achieve efficacy. Despite sublethal effects that may prevent an economic threshold from being reached, insects may still be able to reproduce after insecticide exposure, which in turn could increase the risk of resistance evolution. Hormesis, a phenomenon whereby low levels of exposure to toxic substances can improve pest fitness, could further complicate assessments of efficacy. Studies have found that sublethal doses of sulfoxaflor on green peach aphid, Myzus persicae (Sulzer) (Tang et al. 2015) and the small brown planthopper Laodelphax striatellus (Fallén) (Xu et al. 2016) produced hormetic effects on reproductive measures. Moreover, we looked at insecticides that are purported to cause less harm to natural enemies. In some cases, only direct mortality was used as the indicator of harm, but insecticides can result in important sublethal effects on natural enemies as well (Delpuech et al. 1998; Desneux et al. 2007; Biondi et al. 2012). Moving forward it will be important to continue studying lethal and sublethal effects of insecticides on the natural enemies in systems with H. halys.

Direct mortality alone (Fig. 1) or sublethal feeding alone (Fig. 2) did not capture the full effect of an insecticide on H. halys (Fig. 3). It was only by combining these two measures that we obtained a more comprehensive characterization of the crop-protective potential of these insecticides (Fig. 3). Perceived efficacy of an insecticide based on mortality alone may therefore be problematic. Many field-based insecticide trials already use crop injury as the primary means to compare insecticides; however, laboratory studies generally do not measure changes in feeding and are unable to measure crop injury. We suggest that in addition to direct mortality, sublethal effects of insecticides on H. halys, specifically those which affect feeding, should be considered in future insecticide efficacy trials, and when developing IPM recommendations and economic thresholds.

Currently, the most widely used tools for management of H. halys are broad-spectrum insecticides (Rice et al. 2014). The increased use of these chemicals to manage H. halys can adversely affect natural enemy populations, as evidenced by secondary pest outbreaks and disruptions to IPM programs (Leskey et al. 2012a; Rice et al. 2014). We found that several insecticides that pose less risk to natural enemies can significantly reduce H. halys feeding injury, despite lower rates of direct mortality, compared to bifenthrin, a highly lethal insecticide (Fig. 3). Having a more accurate estimate of injury/individual after insecticide exposure may allow for less disruptive insecticides (i.e., those causing less mortality to natural enemies) to be considered in management programs, which would be useful in reducing secondary pest outbreaks and for developing more sustainable IPM programs.

Author contributions

All authors conceived and designed the research. TC and EB conducted the experiments, and TC analyzed the data and wrote the manuscript. All authors read, edited, and approved the manuscript.



We would like to thank those who assisted us in making this study possible including the labs of Dr. Galen Dively (University of Maryland), Dr. Thomas Kuhar (Virginia Polytech University), and Jaana Iverson, and Wally Rich IV (University of Minnesota). Additionally, we express gratitude to Drs. Amy Morey and Rob Venette for reviewing a previous draft, and Dr. Dan Cariveau for statistical assistance. We also thank our funding sources including a United States Department of Agriculture—Minnesota Department of Agriculture Specialty Crop Block Grant, a University of Minnesota—Department of Entomology, McLaughlin Gormley King Corp. (MGK) graduate fellowship, and a University of Minnesota MnDRIVE Global Food Ventures graduate fellowship. The research was also supported by the University of Minnesota Agricultural Experiment Station.

Compliance with ethical standards

Conflict of interest

All authors declare they have no conflicts of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10340_2017_871_MOESM1_ESM.docx (10 kb)
Supplementary material 1 (DOCX 10 kb)
10340_2017_871_MOESM2_ESM.tif (1.9 mb)
Supplementary material 2 (TIFF 1964 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Theresa M. Cira
    • 1
  • Eric C. Burkness
    • 1
  • Robert L. Koch
    • 1
  • W. D. Hutchison
    • 1
  1. 1.Department of EntomologyUniversity of MinnesotaSaint PaulUSA

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