An egg parasitoid interferes with biological control of tomato leafminer by augmentation of Nesidiocoris tenuis (Hemiptera: Miridae)

Accepted Manuscript An egg parasitoid interferes with biological control of tomato leafminer by augmentation of Nesidiocorus tenuis (Hemiptera: Miridae) Mohammad Ali Mirhosseini, Yaghoub Fathipour, Niels Holst, Mahmoud Soufbaf, J.P. Michaud PII: DOI: Reference:

S1049-9644(18)30574-7 https://doi.org/10.1016/j.biocontrol.2019.02.009 YBCON 3931

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

12 August 2018 1 February 2019 16 February 2019

Please cite this article as: Mirhosseini, M.A., Fathipour, Y., Holst, N., Soufbaf, M., Michaud, J.P., An egg parasitoid interferes with biological control of tomato leafminer by augmentation of Nesidiocorus tenuis (Hemiptera: Miridae), Biological Control (2019), doi: https://doi.org/10.1016/j.biocontrol.2019.02.009

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An egg parasitoid interferes with biological control of tomato leafminer by augmentation of Nesidiocorus tenuis (Hemiptera: Miridae) Mohammad Ali Mirhosseini1, Yaghoub Fathipour*1, Niels Holst2, Mahmoud Soufbaf 3, J.P. Michaud4 1

Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

2

Department of Agroecology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark

3

Agricultural, Medical and Industrial Research School, Karaj, Iran

4

Department of Entomology, Kansas State University, Agricultural Research Center–Hays, 1232

240th Ave., Hays, KS 67601, USA

*Corresponding author: Yaghoub Fathipour E-mail: [email protected] P.O. Box: 14115-336 Phone number: +98 21 48292301

Running head: Biological Control of Tomato Leaf Miner

Abstract

Tomato leaf miner (TLM), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is a serious pest of tomato production in many parts of the world. The TLM has demonstrated capacity to evolve resistance to insecticides, and residues of these on tomato fruit pose hazards to human health, making biological control solutions an urgent priority. We assessed the biological control potential of the predatory bug Nesidiocoris tenuis (Reuter), in combination with the egg parasitoid Trichogramma brassicae Bezdenko at various release rates (0, 10 or 30 females/m2/week). Predators were released either 10 days before, or 10 days after, pest establishment. The predator lowered pest density only when it was released before the pest, but 1

not to levels likely to retain the population below economic threshold. The parasitoid had no direct effect on pest density, but negatively affected the predator's impact on the pest, likely by reducing prey suitability and shifting feeding behavior toward more herbivory and/or cannibalism. Both pest and predator displayed negative density dependence; their population growth rates declined with increasing conspecific density. Our results indicate that N. tenuis should be augmented using a predator-in-first approach, and without simultaneous releases of egg parasitoids. Augmentation of N. tenuis will require integration with other tactics to provide adequate control of TLM, but has the potential for ancillary impacts on other tomato pests such as whiteflies and spider mites.

Keywords: augmentation, competition, Tuta absoluta, Trichogramma brassicae, zoophytophagy

1. Introduction

The tomato leaf miner (TLM), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is native to South America and one of the most destructive invasive pests of tomato around the world (Desneux et al., 2010). Tomato-producing regions of the world infested with TLM have increased from 3% to 60% over the last 10 years (Biondi et al., 2018). Collectively, China, Mexico and the United States produce 42% of the world's tomatoes, and remain at high risk of invasion by this serious pest. In addition to tomato, TLM may attack other solanaceous plants including potato, pepper, and eggplant (Desneux et al., 2010). Chemical insecticides are widely used against TLM, but most research emphasis remains focused on other pest management methods (e.g. biological control) due to problems of insecticide resistance evolution (Roditakis et al., 2018) and the risks posed by pesticide residues on tomato fruit. Zoophytophagus mirid bugs are omnivorous predators which have come to be recognized as effective biological control agents in various crops, including tomato (e.g., Molla et al., 2011). Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) occurs naturally in tomato crops in the Mediterranean region and is widely used, either alone or in combination with other agents, in augmentation biological control tactics targeting tomato pests, including TLM (Calvo et al., 2009; Calvo et al., 2012a; Urbaneja et al., 2012). Both augmentation and conservation of this predator have been considered as approaches to improve biological control of TLM (Campos et 2

al., 2017). Establishment and colonization of N. tenuis can be improved by releasing it onto tomato seedlings prior to transplantion (Calvo et al., 2012b) and providing it with alternative food, e.g., eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), a tactic that has been used to successfully control TLM and other pests in greenhouses (Urbaneja et al., 2012). Trichogrammatid wasps are among the most widely used natural enemies in augmentation biocontrol and inundative applications against agricultural pests (Li et al., 1994; Wajnberg and Hassan, 1994). The advantages of these parasitoids include a low cost of rearing, their ability to utilize alternative hosts, and elimination of the pest in the egg stage before any feeding occurs (Smith, 1996; Parra and Zucchi, 2004). Various Trichogramma spp. have been assessed for their ability to control TLM (Cabello et al., 2009; Chailleux et al., 2012). Trichogramma brassicae Bezdenko is among the most commonly reared Trichogramma species in many countries (Ebrahimi et al., 1998; Lundgren et al., 2002). Mohamadi et al. (2017) evaluated the effects of different fertilization regimes on the population growth parameters of T. brassicae parasitizing T. absoluta eggs. Safaeeniya et al. (2017) concluded that the use of T. brassicae was compatible with a source of host plant resistance to T. absoluta and Alsaedi et al. (2017) tested it in combination with Bacillus thuringiensis var. kurstaki. Only one study, Guven et al. (2017), has tested T. brassicae in combination with N. tenuis and found that use of the bug alone yielded the fewest TLM-infested fruit. Although simultaneous augmentaation of an egg parasitoid and an omnivorous predator could lead to improved biocontrol of TLM, there exists the possibility of negative interactions between them (e.g., Chailleux et al., 2017). The concurrent augmentation of two natural enemies may not be more effective than either one alone if intraguild predation occurs, such as predator consumption of parasitized eggs (Cabello et al., 2012; Cabello et al., 2015). We hypothesized that a suitably timed release of N. tenuis, combined with periodic releases of T. brassicae, could potentially synergize biological control of TLM. In the present study, we compared two different predator release timings (before or after TLM establishment on greenhouse tomato) when combined with three release rates of T. brassicae to determine the most effective approach for augmentation biological control of the pest.

2. Materials and methods 3

2.1. Insects Adults of TLM and N. tenuis were collected from tomato fields in the Varamin region of Iran (GPS coordinates 35°18′49″ N, 51°37′46″) in September, 2015, and reared separately in gauze-covered wooden cages (100 × 100 × 100 cm, 1 mm2 mesh size) on cherry tomato in a greenhouse under controlled conditions (27 ± 3 °C, 55 ± 5 % RH and a 16:8 h L:D photoperiod). Cotton balls soaked in a honey/water solution (20% by volume) were provided for adult moths and ad libitum eggs of E. kuehniella were placed on tomato leaves as food for N. tenuis. Individuals of T. brassicae were obtained from a colony maintained by the Iranian Research Institute of Plant Protection and were reared on eggs of E. kuehniella in a climate-controlled growth chamber set to 25 ± 1 °C, 60 ± 5 % RH and a 16:8 h L:D photoperiod. Fresh eggs of E. kuehniella were glued to strips of white cardboard (1.0 × 8.0 cm), ca. 500 per strip, using a diluted honey solution (10% by volume). Each cardboard strip was then placed into a glass tube (10.0 x 2.0 cm diam) containing 30-40 females of T. brassicae. Adult wasps were removed after 48 h and egg sgtrips were held in tubes until wasp emergence.

2.2. Experimental design The experiment was carried out in a plastic greenhouse under controlled conditions (25 ± 3 °C, 55 ± 5 % RH) inside metal-framed cages (100 × 100 × 150 cm) covered with fine gauze (mesh size 0.5 mm2) and were placed on the ground in which tomato plants were established. Four cherry tomato seedlings were transplanted from germination trays directly into the soil under each cage and were allowed to grow, without any amendments of pesticides or fertilizers, to a height of 35-40 cm before the experiment was begun. Egg parasitoids were released at three rates (0, 10 or 30 females per week until end of experiment), with or without releases of N. tenuis (one pair of three d-old adults, either before or after pest establishment), and cages were each provisioned with a diluted honey solution on cotton balls (as above), refreshed as needed. All cages were infested with four pairs of three d-old T. absoluta, either ten days before or after predator release (Table 1). Each treatment was replicated four times, for a total of 36 cages arranged in a randomized block design. In all predator-first treatments, ad libitum eggs of E. kuehniella were provided on tomato leaves as an alternative food source for the predator prior to pest infestation. Weekly monitoring of insect populations was initiated one month after pest release. All live pest and predator adults were counted in each cage for 25 weeks from April to 4

November 2016. While plants remained small, insects were counted by visual inspection, but once plants grew to more than one meter in height, a small vacuum collection device was used to aspirate the insects, which were then counted and returned to their respective cages.

2.3. Data analysis We calculated the area under the curve (AUC in 'insect weeks') for pest and predator population trajectories in each cage and used this as the response variable for both species in a generalized linear regression (R glm function with log link); means were separated using Tukey’s test (α = 0.05) (R CoreTeam, 2017). The AUC effectively summarizes the amount of insect pressure over a specific time period as a single value (Ruppel, 1983). Population growth rates of pest and predator were calculated as:

where r, Nt and t are population growth rate (weekly), population size (no. of adults) and time (in weeks), respectively. The population growth rate was regressed on population size (Nt in eq. 1) and on treatment (T), including their interaction, for both pest and predator:

(eq. 2)

where subscript i indicates either pest (p) or predator (pr). For rp, T had nine levels (all treatments), whereas there were only six levels for rpr (only treatments with predators). For a more generic analysis of the predator-prey dynamics, regressions on rp and rpr were carried out on population densities across all treatments (pooling the 9 levels for rp and 6 levels for rpr):

where subscript i indicates either pest (p) or predator (pr), and P denotes the presence or absence of parasitoids. 5

For eqs. 2 and 3, a linear mixed model (R lme estimated by maximum likelihood) with temporal autocorrelation was run to check for correlation in the residuals between weeks. However, the autocorrelation coefficient was consistently small (

) , so autocorrelation

was excluded and only repeated measures for each cage was retained as a random effect. For all regression tests, model residuals were checked graphically for homogeneity against predicted values and for normality in a Q-Q plot. Full regression models were simplified, first by leaving out non-significant terms, then by pooling treatment levels that were not significantly different. The R anova function was used to test the validity of each step in model simplification. The xintercept in eq. 2 indicates the carrying capacity of pest and predator, which could vary among treatments. The zero isoclines for rp and rpr were determined from eq. 3 and used for a graphical stability analysis of the predator-parasitoid-prey interaction (Rosenzweig and MacArthur, 1963).

3. Results

The area under the curve (AUC) for both pest and predator were clearly affected by treatment (Table 2). The pest AUC was reduced by the predator, but only in the predator-first treatments, whereas the parasitoid had no effect on the pest, neither alone, nor when released together with the predator. However, the parasitoid reduced predator AUC, in both predator-first and pest-first treatments. The pest population increased sharply in treatments without N. tenuis, but not in treatments that included the predator (Fig. 1), its growth rate (rp) being density-dependent (Fig. 2). The regression analysis generated by eq. 2 yielded a non-significant interaction term which was discarded. The reduced model yielded three treatment groups: those that were predator-free, those in which the pest infested first, and those in which predators were released first (Table 3). The carrying capacity in predator-free treatments was calculated to be 17.5 adult T. absoluta per m2. Regression slopes, which indicate the degree of density-dependence, were only marginally different (P = 0.053) between the predator-free and pest-first groups, but were seven times greater in the predator-first group compared to the predator-free group (P < 0.001). Thus the predator had a pronounced negative effect on the pest population, but only when it established prior to pest infestation. 6

The predator population also exhibited density-dependence (Fig. 3), but augmentation of the parasitoid reduced the regression slope by over 30% (Table 4), indicating that the parasitoid had a negative effect on the predator population. The interaction term (eq. 2) was again nonsignificant and hence discarded. The carrying capacity in parasitoid-free treatments was calculated at 9.9 adult T. absoluta per m2. The responses of pest and predator population growth rates to population densities determined with eq. 3 were in line with the treatment responses depicted by the AUC and all regression terms were significant, including the interactions between pest and predator (Table 5). The pest population growth rate (rp) was unaffected by parasitoid presence, whereas the predator population growth rate (rpr) was significantly reduced by the presence of the parasitoid (Table 5). The predator isocline was almost horizontal across the range of pest densities that were well supported by the data (i.e., < 30 T. absoluta per m2, Fig. 4), suggesting that factors other than T. absoluta availability were limiting N. tenuis population growth. The carrying capacity of the pest without predators was 13.4 adults per m2 (Fig. 5) and the isoclines generated by eq. 3 resembled the classic isoclines of Rozenweig and MacArthur (1963). Whereas the presence of N. tenuis reduced the equilibrium pest population substantially, the degree of reduction was diminished by the presence of the parasitoid, which reduced the equilibrium density of the predator. Both equilibrium points can be considered stable, as they lie to the right of the hump-shaped pest isocline (Rosenzweig and MacArthur, 1963).

4. Discussion

Only the predator-in-first treatments suppressed the pest population in these experiments. However, parasitoid releases reduced the number of predators, thereby diminishing its biocontrol impact. The stability analyses (Figs. 4 and 5) revealed that the T. absoluta population was limited by negative density dependence to a carrying capacity of 13.4 adults per m2 under these experimental conditions, probably due to reduced host plant quality at higher pest densities and/or conspecific crowding. Predator impact on the pest was positively dependent on predator density and the expected pest density was reduced by just over 40% at equilibrium, but only by about 36% when parasitoids were added to the system. The fact that the predator population reached an equilibrium density of 7-8 adults per m2 with a negligible effect of TLM density (in 7

the range of TLM densities well supported by the data) suggests that the impact of N. tenuis was likely limited by factors other than TLM availability. To the extent that bugs also do some plant feeding, this may have diverted some predatory behavior. Calvo et al. (2012a, b) reported that early releases of N. tenuis could control TLM in greenhouse tomatos, and that the addition of egg parasitoids did not improve control, nor did egg parasitoids control TLM in the absence of N. tenuis. The provision of alternate food (eggs of E. kuehniella) provides a source of protein sufficient to stimulate N. tenuis reproduction prior to pest infestation, and is thus a critical component of the approach. In contrast, other studies have shown that an egg parasitoid with or without a predator can effectively control TLM (e.g., Desneux et al., 2010; Chailleux et al., 2013b; Oliveira et al., 2017). These different results may reflect the sensitivity of egg parasitoids to environmental factors, such as temperature, humidity (Moezipour et al., 2008), or plant architecture (Gingras and Boivin, 2002), or perhaps different rearing histories (Cascone et al., 2015). However, our findings are consistent with those of Guven et al. (2017) who obtained better control of TLM with N. tenuis alone than when it was combined with T. brassicae and provide some explanation for their results. The observed negative impact of the egg parasitoid on the predator was most likely due to competition. Nesidiocorus tenuis attacks both eggs and young larvae and may be able to discern and avoid parasitized eggs. For example, the mirid Macrolophus pygmaeus Rambur preferentially preys on unparasitized or recently parasitized TLM eggs (Chailleux et al., 2013a), and another mirid, Dicyphus hesperus (Knight) will not consume nymphs of Bactericera cockerelli (Sulc) containing later instars of Tamarixia triozae (Burks) (De Lourdes Ramirez Ahuja et al., 2017). Changes in the morphology or chemistry of parasitized prey may result in their reduced acceptability, or interfere with extra-oral digestion (Hoelmer et al., 1994). If parasitized eggs of TLM are distasteful or repellent to N. tenuis, encounters with them may shift predator feeding behavior more towards herbivory, or to cannibalism; the latter would reduce predator population density and thus the equilibrium level of pest control obtained. For example, cannibalism of juvenile D. hesperus by adult females increases under conditions of prey limitation and plant features that impede foraging behavior such as dense leaf hairs (Laycock et al., 2006). In addition, mirid colonies reared to specialize on prey over herbivory tend to have higher cannibalistic tendencies (Dumont et al., 2017). 8

Augmentation of N. tenuis resulted in an expected pest density of 8-9 TLM adults per cage, which translates into two TLM adults per plant. Given that the economic threshold for TLM is between two to three larvae per plant (Shiberu and Getu, 2017, 2018), and considering that each female can lay up to 300 eggs, it is unlikely that the release rates of N. tenuis tested in the present study will be able to provide acceptable control of TLM without integration with other control tactics. Thus, early establishment of N. tenuis in the tomato crop will improve biocontrol of TLM, but will probably be insufficient to maintain the pest below economic threshold without integration with other control measures. Because N. tenuis also attacks whiteflies and spider mites (Urbaneja et al., 2003; Calvo et al., 2012a), it can utilize these other pests as alternative prey and simultaneously contribute to their control. Attempts at biological control of TLM with egg parasitoids have yielded highly variable results, but the present findings indicate that they should not be included in TLM biocontrol programs utilizing N. tenuis.

Acknowledgments The authors are grateful for financial and technical support provided by the Department of Entomology, Tarbiat Modares University (grant number: 9330462007).

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Figure captions

Fig. 1. Weekly population fluctuations of Tuta absoluta (black lines) and Nesidiocoris tenuis (gray lines) in different treatments. Equilibrium densities were determined from isoclines and are shown with natural enemies (dotted lines; 7.9 per m2 for N. tenuis and 8.6 per m2 for N. tenuis plus Trichogramma brassicae, which are indistinguishable) and without (dashed lines; 13.4 per m2). See Table 1 for treatment descriptions.

Fig. 2. Linear regressions of Tuta absoluta population growth rates on conspecific density in each treatment. See Table 1 for treatment descriptions.

Fig. 3. Linear regressions of Nesidiocoris tenuis population growth rates on conspecific density in each treatment. See Table 1 for treatment descriptions.

Fig. 4. Isoclines for Tuta absoluta and Nesidiocorus tenuis derived from eq. 3 regressions and depicted with observed densities (points) for the six treatments that included N. tenuis. Zero 13

isoclines shown for pest (rp), predator alone (rpr) and predator plus parasitoids (rprpz). See Table 1 for treatment descriptions.

Fig. 5. Schematic representation of zero isoclines for Tuta absoluta and its predator, Nesidiocorus tenuis. Pest and predator populations increased in the dotted and gray areas, respectively. Depression of the predator isocline by the parasitoid is indicated by the arrow.

14

Fig. 1. Weekly population fluctuations of Tuta absoluta (black lines) and Nesidiocoris tenuis (gray lines) in different treatments. Equilibrium densities were determined from isoclines and are shown with natural enemies (dotted lines; 7.9 per m2 for N. tenuis and 8.6 per m2 for N. tenuis plus Trichogramma brassicae, which are indistinguishable) and without (dashed lines; 13.4 per m2). See Table 1 for treatment descriptions.

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Fig. 2. Linear regressions of Tuta absoluta population growth rates on conspecific density in each treatment. See Table 1 for treatment descriptions.

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Fig. 3. Linear regressions of Nesidiocoris tenuis population growth rates on conspecific density in each treatment. See Table 1 for treatment descriptions.

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Fig. 4. Isoclines for Tuta absoluta and Nesidiocorus tenuis derived from eq. 3 regressions and depicted with observed densities (points) for the six treatments that included N. tenuis. Zero isoclines shown for pest (rp), predator alone (rpr) and predator plus parasitoids (rprpz). See Table 1 for treatment descriptions.

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Fig. 5. Schematic representation of zero isoclines for Tuta absoluta and its predator, Nesidiocorus tenuis. Pest and predator populations increased in the dotted and gray areas, respectively. Depression of the predator isocline by the parasitoid is indicated by the arrow.

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Table 1. Details of experimental treatments employing various releases of Trichogramma brassicae and/or Nesidiocoris tenuis. Control

pest alone

P1+Tb10

pest first*, plus 10 female T. brassicae weekly

P1+Tb30

pest first, plus 30 female T. brassicae weekly

P1+Nt

pest first, plus one pair of N. tenuis adults

P1+Nt+Tb10

pest first, plus one pair of N. tenuis adults, 10 female T. brassicae weekly

P1+Nt+Tb30

pest first, plus one pair of N. tenuis adults, 30 female T. brassicae weekly

Nt1+P2

one pair of N. tenuis adults first, pest second*

Nt1+P2+Tb10

one pair of N. tenuis adults first, pest second, plus 10 female T. brassicae weekly

Nt1+P2+Tb30

one pair of N. tenuis adults first, pest second, plus 30 female T. brassicae weekly

Pests released in all replicates were four pairs of three d-old Tuta absoluta adults, released either 10 days prior to (first), or 10 days after (second), the initial release of parasitoids or predators.

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Table 2. Mean (± SE) area under the curve (AUC, insect weeks) for Tuta absoluta and Nesidiocoris tenuis in nine different treatments. See Table 1 for treatment descriptions. Treatment

AUC for T. absoluta

AUC for N. tenuis

Control

358.63 ± 41.83 a

-

P1+Tb10

371.25 ± 42.20 a

-

P1+Tb30

376.00 ± 48.47 a

-

P1+Nt

314.50 ± 31.88 a

189.38 ± 7.31 a

P1+Nt+Tb10

268.13 ± 35.73 ab

158.25 ± 3.62 b

P1+Nt+Tb30

235.75 ± 19.51 ab

155.75 ± 3.56 b

Nt1+P2

110.75 ± 8.37 b

193.63 ± 2.68 a

Nt1+P2+Tb10

98.75 ± 14.58 b

158.25 ± 3.84 b

Nt1+P2+Tb30

96.63 ± 3.16 b

154.00 ± 0.98 b

F

14.35

16.31

df

8, 27

5, 18

P

< 0.001

< 0.001

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Table 3. Mixed linear regression of weekly Tuta absoluta population growth rate on its own population density (no. adult moths per m2). Treatments not significantly different from one another ( > 0.05) were pooled. See Table 1 for treatment descriptions.

Treatment

Intercept

Slope

t

P

0.446 ± 0.070

-0.026 ± 0.004

6.73

< 0.001

0.452 ± 0.070

-0.037 ± 0.005

7.51

< 0.001

0.796 ± 0.082

-0.171 ± 0.016

10.85

< 0.001

Control, P1+Tb10 and P1+Tb30 P1+Nt, P1+Nt+Tb10 and P1+Nt+Tb30 Nt1+P2, Nt1+P2+Tb10 and Nt1+P2+Tb30 22

Table 4. Mixed linear regression of weekly Nesidiocoris tenuis population growth rate on its own population density (no. adult bugs per m2). Treatments not significantly different from one another ( > 0.05) were pooled. See Table 1 for treatment descriptions. 23

Treatment P1+Nt and Nt1+P2

Intercept

Slope

0.318 ± 0.080

-0.032 ± 0.006

0.351 ± 0.058

-0.042 ± 0.006

t 5.07

P < 0.001

P1+Nt+Tb10, P1+Nt+Tb30, Nt1+P2+Tb10 and Nt1+P2+Tb30

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7.16

< 0.001

Table 5. Mixed linear regression of weekly Tuta absoluta and Nesidiocoris tenuis population growth rates on both pest and predator densities (no. insects per m2) and parasitoid presence, in treatments that included N. tenuis. Non-significant terms were eliminated from the model. See Table 1 for treatment descriptions. Species

T. absoluta

N. tenuis

Regression term

Value ± SE

t

P

Intercept

0.649 ± 0.120

5.42

< 0.001

Pest density

-0.061 ± 0.012

5.16

< 0.001

Predator density

-0.049 ± 0.017

2.93

0.004

Pest*predator

0.004 ± 0.002

2.40

0.017

Intercept

1.169 ± 0.057

20.65

< 0.001

Pest density

-0.016 ± 0.004

3.92

< 0.001

Predator density

-0.150 ± 0.008

19.32

< 0.001

Pest*predator

0.003 ± 0.001

4.85

< 0.001

Parasitoid present

-0.171 ± 0.023

7.47

< 0.001

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Graphical abstract

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Highlights 

N. tenuis and T. brassicae were augmented against T. absoluta in a greenhouse cage study



The egg parasitoid reduced the impact of the zoophytophagous bug on tomato leafminer density



Highest impact was obtained when predators were released prior to pest infestation, and without parasitoids



Pest density was reduced by 40%, indicating N. tenuis augmentation will require integration with other tactics

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Contribution of authors MAM, YF and MS conceived and designed the research, MAM conducted the experiments and MAM, NH and JPM analyzed the data, JPM edited the final version. All authors read and approved the manuscript.

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