Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus.

The intensification of agriculture leads to increased pesticide use and significant transformation from small fields towards large-scale monocultures. This may significantly affect populations of non-target arthropods (NTA). We aimed to assess whether the multigenerational exposure to plant protection products has resulted in the evolution of resistance to insecticides in the ground beetle Poecilus cupreus originating from different agricultural landscapes. Two contrasting landscapes were selected for the study, one dominated by small and another by large fields. Within each landscape the beetles were collected at nine sites representing range of canola coverage and a variety of habitat types. Part of the collected beetles, after acclimation to laboratory conditions, were tested for sensitivity to Proteus 110 OD-the most commonly used insecticide in the studied landscapes. The rest were bred in the laboratory for two consecutive generations, and part of the beetles from each generation were also tested for sensitivity to selected insecticide. We showed that the beetles inhabiting areas with medium and large share of canola located in the landscape dominated by large fields were less sensitive to the studied insecticide. The persistence of reduced sensitivity to Proteus 110 OD for two consecutive generations indicates that either the beetles have developed resistance to the insecticide or the chronic exposure to pesticides has led to the selection of more resistant individuals naturally present in the studied populations. No increased resistance was found in the beetles from more heterogeneous landscape dominated by small fields, in which spatio-temporal diversity of crops and abundance of small, linear off-crop landscape elements may provide shelter that allows NTAs to survive without developing any, presumably costly, resistance mechanisms.

Introduction

Agriculture is the most important type of land-use in Europe, covering almost half of the terrestrial area of European Union (EU), and the major pressure on biodiversity and provision of ecosystem services [1]. In agricultural areas important roles are played by all kinds of non-cropped habitats, serving as a reservoir for many plant and animal species [2]. Natural or semi-natural habitats provide not only shelter, reproduction and hibernation sites but also serve as starting points for cyclic recolonization of fields after mowing, fertilization, tillage or pesticide applications and harvest [3,4]. Unfortunately, the intensive development of agriculture promotes large-scale monoculture farming leading to a decrease in spatio-temporal heterogeneity of agricultural landscapes. Additionally, the ever-increasing demand for agricultural products makes it impossible to stop using plant protection products. Although it is difficult to disentangle the impacts of intensified management of local fields from changes in land use at the landscape level–as both occur simultaneously in most agricultural landscapes [5]–one of the most important components of agricultural intensification behind the undesired loses of non-target arthropods (NTA) in agricultural landscapes [6] is the widespread use of pesticides [7]. Pesticides are a quick, highly effective, easy to access, consistent and easy to use tool of choice for farmers for controlling weeds and pests in agriculture landscapes. The worldwide use of agrochemicals has led, however, to contamination of almost every part of our environment in even the most remote places [8]. Because of the similarity of basic biochemical processes among insects and many other invertebrates, insecticides are not specific enough to affect only pest species, and have harmful effects also on beneficial organisms inhabiting agricultural landscapes [7]. The eventual effect of insecticides on NTA depends on which specific products are used (some are more toxic and/or persistent than others), how they are used (e.g., repetitive use may lead to the accumulation of a chemical and/or its effects but also to the development of resistance in chronically exposed populations), and what is the environmental context (e.g., landscape structure).

Carabids, as a taxonomically and ecologically diverse group, have different habitat requirements and may respond differently to landscape structure and its management [9-11]. Both larvae and adults of most carabid species are carnivorous and are known to be predators of many invertebrate pests, but can also consume seeds of different weeds [12,13]. Virtually all agriculture practices result in direct or indirect effects on carabid communities, either through mortality and emigration or changes in conditions in occupied microhabitats [14,15]. Carabids are sensitive to many insecticides, including all the most commonly used groups: organophosphates, organochlorines, carbamates, pyrethroids, and neonicotinoids [16]. During and/or soon after a spraying, adult beetles, which spend most of the time at the soil surface when not hibernating, may easily be exposed topically to pesticide droplets from a sprayer and/or falling from sprayed plants. Other exposure routes include consumption of contaminated prey and seeds, contact with contaminated soil, water, and plant surfaces.

In the field, apart from direct mortality caused by insecticides, NTAs, including carabids, are frequently exposed to sub-lethal doses that can lead to more subtle effects, for example on learning performance, behavior and neurophysiology [17]. Organisms chronically exposed to low doses of pesticides may not show signs of acute toxicity (e.g., in the form of increased mortality), but may have reduced tolerance to other stress factors such as other toxic chemicals and/or various types of natural environmental factors, e.g., low or high temperatures, shortage of food, etc. [18]. To survive in environments with a long history of intensive agriculture, where insecticides have been used regularly for many years, populations of any species may be pushed towards evolution of resistance to the most commonly used plant protection products. Although this phenomenon has been proved for many pest populations [19-21], it is much less recognized in NTAs [22-25]. The evolution of resistance, although temporarily beneficial to organisms living in an insecticide -treated area, may be metabolically costly [26], making insecticide-resistant individuals less fit in uncontaminated environments and/or more sensitive to other stressors [18]. Hence, even if increased resistance among NTAs may seem beneficial, it may bring undesired long-term effects. On the other hand, the presence of resistance in pest insects may lead to increased use of insecticides [27]. This potentially may start a cascade of unwanted effects, jeopardizing numerous NTA populations and even bringing human health hazard. Maintaining NTAs biodiversity, abundance and stable populations is, thus, important also from an economic and human health perspective.

It may be expected that changes caused by chronic exposure to pesticides in the field are expressed to a different degree in populations inhabiting differently transformed agricultural areas. Therefore, understanding changes in NTA communities along gradients of agricultural landscape complexity will help us to not only sustain vital habitat conditions to prevent beneficial species from extinction, but also to save billions of dollars which are spent on plant protection products and practices [28]. In the end, understanding insect responses to pesticide pressures in their local ecological context poses a key challenge in developing balanced pest control strategies.

The objective of this study was to investigate the importance of agricultural landscape structure to the sensitivity of the carabid beetle Poecilus cupreus, representing an important group of NTAs–the pest control agents, to insecticides and the evolution of resistance in its populations. We located our study area in the agricultural landscapes of the Wielkopolska [Greater Poland] province in Poland where, within a relatively small area, hugely different landscapes can be found, ranging from large-scale agriculture to more traditional small-scale farming. We hypothesized that populations inhabiting agricultural landscape dominated by large fields with high percentage of canola coverage exhibit increased resistance to the insecticide most commonly used in the area (Proteus 110 OD), while small-fields landscape, with its high diversity of crops and, especially, off-crop habitats serving as refuge areas (e.g., field margins), prevents development of resistance in beetles. We focused in our study on the canola (winter variety) because it is an important crop in the European Union (10% of the EU’s arable land). Although the production of canola in EU is slowly decreasing, in Poland we can still see an increase in the production [29]. Canola plantations usually receive one to four insecticide applications in a season, but in some cases there can be even five or more treatments [30]. We assumed, thus, that the gradient of canola coverage represents a gradient of pesticide pressure. Most treatments in canola plantations are carried out in spring and early summer because this is when the risk of pest outbreaks is the highest. Adult P. cupreus, as spring breeders, are present in the fields during spraying, and therefore can be particularly exposed to plant protection products via direct spray or residues on plants, food, water and soil, possibly favoring the evolution of resistance towards insecticides. To sort out possible temporary effects through direct selection of the most resistant individuals collected from the field right after the spraying from possible genetically fixed adaptation to the used pesticides, the sensitivity of beetles towards Proteus 110 OD was tested on field collected beetles (P) and two consecutive laboratory cultured generations (F1 and F2).

Methods

Study area and site selection

The study area lies in the southwest part of the Wielkopolska province (western Poland, Fig 1) and represents a typical farmland, where arable fields occupy nearly 65–70% of the area. The growing season is one of the longest in Poland, beginning around the end of March and lasting for approximately 220 days. Mean annual temperature is ca. 8°C and mean annual precipitation over 550 mm [31,32]. Within the study area, two distinct landscapes, 12 x 16 km each, were selected: one dominated by large fields (hereafter ‘large-fields landscape’, L) and the second one with prevailing small-fields family farming (hereafter ‘small-fields landscape’, S) (Fig 1). Both landscapes are similar in terms of land cover (i.e., percentage of arable areas, woodlands, water bodies) and pedo-climatic conditions, but differ in the farmland structure (Table 1).

Fig 1

Location of the study landscapes and sites.

Upper right, administrative division of Poland into provinces. In green–Wielkopolska province. Upper left–administrative division of Wielkopolska with the capital of the region [Poznań] marked in yellow and the location of the two study landscapes (red squares; enlarged bellow) representing two contrasting landscape types: S–small-fields landscape, L–large-fields landscape. Within each landscape the locations of the study sites are marked (red circles– 500 m radius). The first character stands for landscape type (S vs. L); the second character stands for canola coverage: 0 –none, S–small (10–14%), M–medium (20–52%), L–large (80–98%). Land-cover maps were generated according to methodology described by Ziółkowska et al. [33], see Methods section for more details.

Table 1

Two study landscapes, each 12 x 16 km, with share of land of the main land cover types [%], share of arable land in given field size classes [%], field border length [km] and total number of arable fields.

		Large-fields landscape (L)	Small-fields landscape (S)
Share of land cover units [%] in a landscape	Arable	73.2	76.9
	Herbaceous	8.8	8.2
	Woodland	11.2	8.7
	Build up	5.3	4.7
	Water bodies	0.8	0.6
	Other	0.7	0.9
Share of arable land [%] in given field size classes	< 3 ha	23.5	41.7
	3–10 ha	25.7	40.1
	10–30 ha	21.7	11.5
	30–50 ha	16.8	4.8
	≥ 50 ha	12.3	1.9
field borders [km]		4443	6131
total number of arable fields		4072	6494

Raster land-cover maps (resolution of 1 m2) for the landscapes were generated in a step-by-step process according to the methodology described in [33] by combining data from: (1) the National Database of Topographic Objects (BDOT) providing the land cover and land use information at the scale of 1:10.000, and (2) the Land Parcel Identification System (LPIS) providing information on the type of cultivated crops. In Poland, LPIS, managed by the Polish Agency of Restructuring and Modernization of Agriculture, is based on the national land and building cadaster and therefore information on type of cultivated crop is provided at the level of cadastral parcels. From LPIS we used information for 2018 to allocate major crop types to individual cadastral parcels. The information for canola was further verified in the field, together with delineation of individual fields if the cadastral parcel was shared by more crops than canola only. Spatial data were handled and analyzed using ArcGIS 10.4 (ESRI, Inc., USA).

Based on the analysis of land-cover maps, three habitat types per landscape–each consisting of three study sites (therefore in total nine study sites per landscape)–were selected based on the canola coverage (CC) expressed as percent of total area within a 500 m radius around the midpoint where the beetle traps were located (Fig 1 and Table 2). The midpoints of the study sites with canola were located in canola fields and were separated from each other by at least 800 m to avoid situations were beetles from one population could be caught in different study sites. At the same time, the most distant study sites were located not more than 60 km apart to ascertain similar climatic and edaphic conditions. In the large-fields landscape (L) sites representing the following beetle habitat types were distinguished: with medium CC (28–33%, LM), with large CC (80–98%, LL), and without CC (L0). In the small-fields landscape (S), it was impossible to establish study sites with CC larger than 60% due to the lack of large canola fields and therefore the beetle habitat types were distinguished as follows: with small CC (10–14%, SS), with medium CC (20–52%, SM), and without CC (S0). Note that in both landscapes, sites with no CC and with medium CC were established and could serve for direct comparison between the landscapes. The midpoints (beetle traps) of study sites L0 and S0, serving as a control, were located on meadows subjected to agricultural practices with the exclusion of pesticide applications. The whole area, in both landscapes, is strongly dominated by conventional agriculture, and no fields managed as organic were present in our study areas.

Table 2

Geographic coordinates (decimal degrees) of the study sites.

Habitat type	Coordinates
	Longitude	Latitude
L0	52.1341	16.8568
L0	52.0627	16.8140
L0	52.0864	16.8559
LM	52.1462	16.8688
LM	52.1396	16.8478
LM	52.1148	16.8475
LL	52.1349	16.9091
LL	52.0563	16.7829
LL	52.0287	16.8044
S0	51.8162	17.3959
S0	51.7568	17.3524
S0	51.7249	17.2615
SS	51.8198	17.3252
SS	51.8125	17.3381
SS	51.7373	17.2597
SM	51.8069	17.3728
SM	51.7943	17.2919
SM	51.7292	17.2873

Study species

The ground beetle Poecilus cupreus was chosen for investigation because it was numerous enough at all the study sites, easy to identify in the field, and with a well-tested culturing procedure. It is one of the most common and dominant carabids found on arable land across Europe [34] and an example of a typical beneficial predator [35]. Adult beetles are strictly diurnal and disperse mainly by walking but can occasionally fly [36]. Usually individuals do not show a high level of movement throughout the active period that falls in spring–summer. It was demonstrated that beetles disperse over hundreds of meters depending on landscape composition and availability of resources [37]. Poecilus cupreus is associated with many different crops, with canola being the most favorable spring habitat [38]. It also inhabits different types of meadows with relatively high soil humidity [39]. Poecilus cupreus is a typical representative of so-called spring breeders, with the period from April to the end of July being the main reproductive time. New generation adults emerge in August, and in the late September the beetles start overwintering hibernation [40]. As P. cupreus can live up to 2–3 years, it is possible for adults to have two activity periods: the first, shortly after hatching just before winter diapause and the second, longer one, in the following spring and summer.

Beetle collection and laboratory culture

In the midpoint of each study site, 64 Barber traps without any preservative were set up in a grid of 8 x 8 m (64 m2). The beetles were collected in 2018 during their peak activity in April–May. The traps were emptied every 2–3 days, the beetles sorted in the field and individuals of P. cupreus were placed in plastic containers (23 x 17 x 11 cm) with moist peat, transported to the laboratory and kept in a climatic chamber (relativity humidity 70% ± 5%, temperature 20°C ± 2°C; day:night 16 h:8 h). The authorisation for capture, transport and keeping the beetles was granted by the Regional Directorate for Environmental Protection in Kraków, Poland, document no. OP-I.6401.128.2017.MMr, and by the Regional Directorate for Environmental Protection in Poznań, Poland, document no. WPN-II.6401.83.2017.AG.2.

To obtain sufficient numbers of beetles for each habitat type, the beetles from three sites representing particular CC within particular landscape type were pooled together. Part of the field collected beetles (P generation) were utilized for insecticide sensitivity test on parental generation and the remaining beetles were used to establish laboratory cultures to obtain the next two generations (F1 and F2). The beetles were cultured according to Bednarska and Laskowski [41] procedure with only one modification: the animals were fed ad libitum with artificial food made of frozen ground mealworms mixed with ground apple but without any preservative to eliminate contact with any potentially harmful chemicals. After overwintering, adult beetles from the laboratory cultures (F1 and F2 generations) underwent the same experimental procedure as the parental generation.

Experimental design

In total, 250–280 individuals were tested for sensitivity to the insecticide in each generation, with 15–40 beetles per habitat type per landscape, depending on availability. Proteus 110 OD (Bayer, Germany) was selected for the experiment as the most frequently used insecticides in the studied landscapes (according to the survey on pesticide usage conducted among local farmers and farmer advisors from regional agricultural advisory center), applied only in winter rape in the period of sampling the beetles (April-May). It has two active ingredients: thiacloprid (100 g L-1) and deltamethrin (10 g L-1) (see S1 Table in Supplementary materials for full product specification). Nevertheless for testing the resistance of beetles to insecticides other formulations might as well have been used, as long as their active ingredients belong to the main classes of insecticides (here, pyrethroids and neonicotinoids) commonly used in the studied landscapes. If some resistance mechanisms become established in a population, they are universal for all insecticides of a given class, not only for a specific chemical compound representing that class.

Twenty-four hours before insecticide application, the beetles were placed individually in plastic Petri dishes with a diameter of 35 mm (FL Medical, Italy) to acclimatize. The commercial formulation of Proteus 110 OD was dissolved in acetone to obtain the concentration equivalent to 0.2 recommended concentration for field use (for canola pests 0.6 L of the product in 300 L of water per hectare is recommended). The beetles from each habitat type were exposed individually to a single topical application of 1 μl droplet of the insecticide solution applied on the scutellum with a Hamilton syringe with a repeater (Hamilton Company, USA). For control habitats (S0 and L0), additional 30–40 individuals per habitat were treated with 1 μl of pure acetone only (these groups are denoted S0-A and L0-A, respectively) to confirm the effectiveness of the insecticide dose used (positive control). Because S0 and L0 populations did not differ in survival within treatment groups (i.e., either acetone- or Proteus-treated), the S0 and L0 beetles from a given treatment were pooled together and tested for the insecticide effect by comparing the survival curves.

After the treatment, the beetles were placed back in the climatic chamber. The mortality and immobility were recorded after 0.5, 1, 2, 3, 6, 8, 10, 12, 24 h and daily afterwards. In the analysis of mortality, we assumed “dead” to be all individuals that either indeed died or were paralyzed (unable to walk/relocate). The beetles were not fed during the experiment. Each experiment (i.e., on generations P, F1 and F2) was terminated when all the individuals had died. The dead beetles were placed into 2 ml Eppendorf tubes (Sarstedt AG and Co. KG, Germany) and dried at 105°C for 24 h to obtain dry mass. We weighed all the beetles in order to exclude body mass as a factor for possible differences in susceptibility to the insecticide. Dry body mass was recorded to the nearest 0.00001 g using XA 110/2X scale (Radwag, Poland).

Statistical analysis

Survival curves of beetles originating from different habitat types were compared using survival analysis (Wilcoxon test), assuming p ≤ 0.05 as statistically significant. We choose Wilcoxon test because it is more suitable than log-rank test when the hazard is higher at early survival times than later [42] as is the case for insecticide-treated organisms [43]. The survival analyses were performed separately for each generation. First, we compared survival curves of beetles from habitat types with no CC, but separately for insecticide-treated (S0 and L0) and acetone-treated (S0-A and L0-A) ones. Subsequently, survival curves for the insecticide-treated beetles within a particular generation were analyzed for the effect of habitat type. As statistically significant differences were found, this was followed by pairwise comparisons of each habitat type against one another. Dry body mass of beetles was compared among all habitat types and generations using two-way analysis of variance with habitat type and generation as factors. Because both factors and the interaction were highly significant (p ≤ 0.0002), in the next step we performed one-way ANOVA with Tukey post hoc test separately for each generation. All statistical analyses were performed using Statgraphics Centurion 18 (Statgraphics Technologies, Inc., USA).

Results

Parental generation (P–Field collected beetles)

The survival curves for beetles from control habitats from the two analyzed landscapes did not differ, regardless if beetles were treated with insecticide (S0 vs. L0, p = 0.95) or acetone only (S0-A vs. L0-A, p = 0.52, Fig 2). The insecticide significantly increased the mortality rate of the meadow beetles (p < 0.0001 for all three generations), confirming that the insecticide dose used was effective. Thus, meadow populations of beetles from S and L landscapes were similar in terms of their background mortality (with no insecticide treatment) and in terms of their sensitivity to the insecticide. Wilcoxon test showed, however, a significant (p ≤ 0.0001) difference in survival rates of insecticide-treated beetles originating from different habitat types (Fig 3). Pairwise comparisons of the habitats with one another revealed that only insecticide-treated beetles from the LM and LL habitats survived significantly better than those from other habitat types (Fig 3, Table 3), and even from acetone-treated beetles from the control habitats (S0-A and L0-A). The LT50 values for beetles from the LM habitat type were almost three times higher than in beetles from the S0 and SM habitat and ca., 1.5 times higher than those found for beetles from the control habitats treated with acetone only (S0-A and L0-A) (Fig 4; Table 3). Although SM beetles originated from habitats similar to LM in terms of CC, they had the lowest LT50 recorded for P generation.

Fig 2

Survival curves of all three generations of Poecilus cupreus from control habitats after exposure to acetone and Proteus 110 OD pesticide treatment.

Solid lines stand for small-fields landscape; dashed lines stand for large-fields landscape. The first character stands for landscape type: S–small fields landscape, L–large fields landscape; the second character stands for canola coverage: 0 –none. Red uppercase letters indicate generations: P–parental, F1 & F2 –laboratory breed. In all three generations the insecticide treatment significantly increased the mortality rate (p < 0.0001); p values on the graphs indicate the significance level for comparisons between S0 and L0 beetles.

Fig 3

Survival curves of all three generations of Poecilus cupreus from different habitat types and landscapes after exposure to the insecticide Proteus 110 OD.

Dashed lines stand for small-scale landscape; solid lines stands for large-scale landscape. The first character stands for landscape type: S–small-fields landscape, L–large-fields landscape; the second character stands for canola coverage: 0 –none, S–small (10–14%), M–medium (20–52%), L–large (80–98%). Red uppercase letters indicate generations: P–parental, F1 & F2 –laboratory breed. Note the generally shorter survival times in the first laboratory generation (F1).

Table 3

Median lethal times (LT50, days) for all three generations (P–Parental, F1, F2 –Laboratory bred) from all habitat types.

Site	LT50 (days)
	P	F1	F2
S0-A	14.32b (40)	13.03d (30)	20.29d (40)
S0	7.31a (40)	0.42ab (40)	0.42abc (40)
SS	12.3a (30)	0.5ab (30)	0.25a (30)
SM	7.29a (15)	1bc (20)	0.5ab (30)
L0-A	15.28b (40)	13d (30)	20.26d (40)
L0	10.27a (40)	0.25a (40)	0.33a (40)
LM	23.26c (30)	10.97c (30)	15.24bc (30)
LL	21.25bc (30)	7.96c (30)	15.23c (30)

The sites are named as follows: First character stands for landscape type (S–small-fields agriculture, L–large-fields agriculture); second character stands for canola coverage (0 –none, S–small, 10–14%, M–medium, 20–52%, L–large, 80–98%). All groups except those marked with letter “A” (for acetone only) were exposed to the insecticide. Groups with the same lowercase letter do not differ significantly at p ≤ 0.05 in terms of survival (pairwise comparison of survival curves, Wilcoxon test); two groups showing resistance are typed in boldface; beetles originating from meadows and not treated with the insecticide are in italics. The numbers of individuals used in particular treatments are reported in brackets.

Fig 4

Median lethal times (LT50s) with standard errors for the three generations of Poecilus cupreus from all experimental groups, representing different habitat types, landscapes and treatments.

The first character stands for landscape type (S–small fields landscape, L–large fields landscape); the second character for canola coverage (0 –none, S–small, 10–14%, M–medium, 20–52%, L–large, 80–98%). All groups except those marked with letter “A” were exposed to Proteus 110 OD insecticide. Red uppercase letters indicate generations: P–parental, F1 & F2 –laboratory breed.

The one-way ANOVA revealed a statistically significant difference in beetle dry body mass among habitat types (p ≤ 0.0001). The dry mass of both insecticide-treated and acetone-treated beetles from the control habitats in large-fields landscape (L0 and L0-A) was the lowest, and significantly lower than in all other habitat types with the only exception that L0 did not differ from S0 (Fig 5A).

Fig 5

Dry body mass in the three generations of Poecilus cupreus from all experimental groups, representing different habitat types and treatments (A) and across all generations (B). The first character stands for landscape type: S–small fields landscape, L–large fields landscape; the second character stands for canola coverage: 0 –none, S–small (10–14%), M–medium (20–52%), L–large (80–98%). Within each generation all groups except those marked with letter “A” were exposed to Proteus 110 OD insecticide. Red uppercase letters indicate generations: P–parental, F1 & F2 –laboratory breed. Means of groups marked with the same letter above box-and-whisker plots do not differ significantly at p ≤ 0.05. The graphs indicate median (shorter horizontal line), average (plus sign), second and third quartile (wider horizontal lines), minimum and maximum (whiskers) except for outliers > 1.5 interquartile rage (asterisks); the notch indicates approximate 95% confidence interval for the median.

Laboratory bred generations (F1 and F2)

As in the case of P generation, the survival curves of F1 and F2 generations of beetles originating from control habitats showed no statistically significant difference either for acetone-treated beetles (S0-A vs. L0-A, p = 0.35 for F1, p = 0.85 for F2, Fig 2) or insecticide-treated beetles (S0 vs. L0, p = 0.12 for F1, p = 0.17 for F2, Fig 2). Also as in the P generation, the comparison among insecticide-treated beetles from different habitat types revealed statistically significant difference in survival rates of beetles in both the F1 and F2 generations (p < 0.001 for F1, p < 0.03 for F2, Fig 3). The increased tolerance to the insecticide found in beetles from the P generation from the LM and LL habitats was still present in both next generations F1 and F2, as insecticide-treated beetles from the LM and LL habitats survived significantly better than insecticide-treated beetles from all other habitat types (Fig 3, Table 3), but significantly worse than acetone-treated beetles from the control habitats (S0-A and L0-A; Table 3). The LT50s for beetles from the LM and LL habitats, although lower than in the P generation, also in the F1 and F2 were higher than for insecticide-treated beetles from other habitat types (Fig 4).

Although one-way ANOVA on beetle dry body mass revealed statistically significant differences among beetles from different habitat types in both the F1 and F2 generations (p ≤ 0.0001), the beetles from the LM and LL habitats–with confirmed increased tolerance to the insecticide–did not differ significantly in weight from the beetles from most other habitats. The only exception were the F2 beetles from the LM and LL habitats, which were significantly heavier than the F2 beetles from the control habitats, either insecticide- or acetone-treated (L0 and L0-A, Fig 5A). In general, the mean dry body mass of the insecticide-treated beetles from the control habitat in the large-fields landscape (L0) was the lowest in all generations (Fig 5A). Additionally, the dry mass of beetles decreased over the generations (p ≤ 0.0001, Fig 5B), showing that laboratory conditions could have an unforeseen impact on long-term experiments.

Discussion

The goal of this study was to contribute to the knowledge on the evolution/selection towards elevated insecticide resistance in carabids by investigating the sensitivity of the predatory P. cupreus to Proteus 110 OD over three generations. Because we assumed that the development of resistance may depend on the environmental context, we used beetles originating from different habitat types, defined based on canola coverage within the presumed beetle home range, located within two landscapes with distinct farmland heterogeneity. Our results showed that beetles from the landscape dominated by large fields and living in habitats with medium and large coverage of canola (LM and LL habitats) exhibited significantly lower mortality after exposure to Proteus 110 OD than all other populations, and that the difference was maintained in two consecutive laboratory-bred generations. Proteus 110 OD is the plant protection product containing two active ingredients, thiacloprid which belongs to neonicotinoids and affects insect nervous system by stimulating nicotinic acetylcholine receptors, and deltamethrin which is a pyrethroid preventing the closure of the voltage-gated sodium channels in the axonal membranes resulting in dysfunction of spiracles–insect death is caused by overdrying. However, the ultimate effect of commercial formulations depends not only on the active ingredients but also adjuvants/co-formulants, which may affect the efficacy of penetration of the active ingredients into insect bodies, the persistence of an insecticide in field conditions [44]. Hence, other formulations with exactly same active ingredients may have somewhat different effects than those reported herein.

As all beetles in the experiment received an identical dose of the insecticide (except those treated with acetone only), theoretically the observed higher survival of the beetles from the LM and LL habitats could result from higher body mass of the beetles from these two populations rather than from increased resistance. This hypothesis can be, however, rejected as the dry mass of the beetles from the LM and LL habitats did not stand out in any way. We can thus conclude that body mass was not the factor differentiating the beetles in terms of survival after the insecticide treatment, and that the increased resistance found in populations from the LM and LL habitats resulted from the few co-varying factors occurring at local and landscape scales: the presence of large monocultures, homogenization of landscape by disappearance of non-cultivated elements and/or repeated episodes of pesticide sprays; possibly an increased abundance of pesticide adapted prey can also be a factor [25].

One of the main mechanisms of developing resistance to an insecticide is detoxification through the overexpression of metabolic genes. Increased detoxification usually results in energy reallocation at the expense of metabolic and developmental processes. Because resistance to pesticides seems to be costly and in pesticide-free environments the selection acts against it [45,46], the increased resistance has low chances to be fixed in populations with low or sporadic pesticide exposure. Hence, a genetically fixed resistance may be expected in populations that are chronically and repetitively exposed to insecticides, being thus under continuous strong selective pressure. The exposure to insecticides not only depends on management of fields at a local scale (the type and number of product applications on a given field) but also on the context of agriculture and land-use at the landscape level [6]. The necessity to analyze these mechanisms at various spatial scales simultaneously is clearly visible in our results, as the increased resistance to Proteus 110 OD was found exclusively in populations from habitats with medium and large coverage of canola (local-scale measure of stressor’s intensity) only if they were located in landscapes dominated by large fields (landscape-scale measure of stressor’s intensity).

Large coverage of canola within a local habitat increases the probability of beetles being directly exposed to repeated episodes of insecticide sprays. It has been shown by Schoonees and Giliomee [47] that insects receiving more sprays per year exhibited higher resistance to the used pesticides than those which experienced only one application. However, we showed that the proportion of canola coverage within a local habitat is not the sole deciding factor for the evolution of resistance. Beetles originating from SM habitat type did not develop resistance, although the canola coverage in the SM habitat type was similar to the LM habitat type, indicating the importance of factors acting at a landscape-scale. In landscapes dominated by large fields, carabids have less possibilities to migrate to non-crop habitats or to fields with other crops where other management practices are used. This not only increases the probability of beetles being exposed to direct spraying but also to contaminated prey, which has been indicated as an important factor contributing to the development of resistance in beneficial organisms [25]. In contrast to the beetles inhabiting landscapes dominated by large fields, those living in more diverse landscapes–like the beetles from SS and SM habitats–can more easily emigrate during spraying or right after to adjacent fields or non-crop habitats and part of the population is always present in unsprayed non-crop areas [48]. Even if pesticide application in canola fields in S landscape is similar to that in L landscape and the temporal selection pressure on beetle populations is similar across all canola fields, the presence of other habitats within the beetle home range in S landscape acts against fixing the resistance genes in local populations.

The necessity of taking into account not only local but rather various spatial scales simultaneously when investigating the impacts of stressors on species in agroecosystems is generally well recognized [6]. Riggi et al. [49] investigated the importance of the proportion of canola coverage and crop heterogeneity at two spatial scales (defined either as a 3 km radius around the sampled canola field or Sweden’s administrative region) on the occurrence of resistance to lambda-cyhalothrin insecticide in the pollen beetle–one of the major canola pests. They found a positive effect of the canola coverage on insecticide resistance in pollen beetle populations, but only at the regional spatial scale and suggested an effect of regional landscape history on the current pest resistance. To the best of our knowledge, similar studies on carabid beetles, being natural pest enemies, do not exist. Our results, by showing that heterogeneity at the landscape level can play a major role in the development of increased insecticide resistance, confirm the importance of maintaining or increasing landscape (spatio-temporal) heterogeneity as a mechanism of supporting beneficial organisms in agroecosystems. The presence of extensively managed semi-natural habitats–even if only as narrow, linear elements–plays a key role for NTAs in reducing the negative impacts of insecticides by providing shelter and access to uncontaminated food [48], but also as overwintering refugia or source habitats for the recolonization of fields [7,50]. With landscape simplification and land consolidation, the abundance of semi-natural habitats is decreasing together with the multiple ecosystem services they are providing [51].

The results for dry mass of beetles showed that with each generation the beetles became lighter. One potential explanation may be the inbreeding. Limited gene flow and accumulation of specific mutations in a laboratory culture can result in many changes in insect life history [52] such as slower pre-adult development, reduced adult survival [53], decreased larval competitive success and adult fecundity [54] and decreases in body size [55], as shown for Drosophila melanogaster, but similar processes may occur also in beetles. Another possible explanation can be the use of artificial diet. Carabid beetles, like many invertebrates, derive energy from a variety of compounds, and a partially and/or fully artificial diet can significantly affect the insect development, in particular its mass and growth rate [56]. We cannot exclude that the artificial diet used in our culture lacks some elements or compounds necessary to achieve high body mass. Nevertheless, it did not prevent the successful culturing of the beetles through three generations, enabling us to separate out the actual genetically fixed adaptation from possible direct or maternal effects [57]. It should be stressed that despite the overall decrease in body mass in consecutive generations, the survival of the LM and LL beetles exposed to the insecticide was significantly and similarly higher than all other populations in all three generations.

Conclusions

We confirmed that the effect of insecticides on the evolution of resistance in populations of the beneficial insect, the ground beetle P. cupreus, strongly depends on large-scale landscape characteristics. The small-fields landscape, with its higher spatio-temporal heterogeneity, is able to provide refuges that allow the beetles (and possibly other NTAs) to survive without developing any, presumably costly, resistance mechanisms. Understanding the significance of landscape structure for insect responses to pesticide pressures poses a key challenge in developing balanced pest control strategies and can be fundamental for forecasting the consequences of agricultural practices. NTAs, especially beneficial insects serving as natural pest enemies or pollinators, should be able not only to survive insecticide sprays, but also to provide ecosystem services. This requires maintenance of vital habitat conditions to prevent beneficial species from extinction or impairment. While increased resistance to pesticides among beneficial species can be considered a desired phenomenon, it has to be kept in mind that such adaptations are usually costly and may negatively affect the performance of resistant individuals, when other stress factors appear. Although the evolution of resistance is a complex process, influenced by many factors, we believe that our results emphasize the importance of landscape‐scale management and pesticide use in agricultural management practices.

Proteus 110 OD (Bayer, Germany) specification.

(DOCX)

Click here for additional data file.

20 Oct 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ramzi Mansour

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional location information, including geographic coordinates of your field collection site if available.

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

5. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-21-27829

Title: Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

Brief.

The manuscript was well written, and the methodology approach was appropriate for this study. Nonetheless, references were missed in the introduction section. In addition, part of the materials and methods sections is long. Although it is interesting, part of the information provided in the materials and methods sections should be moved to the discussion section. Suggestions and corrections were left in the general comments to improve the manuscript.

General comments

L27 – Please, ‘inheritable’ should be deleted from the sentence. Even though the sensitivity of P. cupreus was lower in the subsequent generation the inheritability of resistance of P. cupreus to Thiacloprid + Deltamethrin is not proved through these experiments and results.

L80 – Please, see the references Rodrigues et al. 2013 Response of different populations of seven lady beetle species to lambda-cyhalothrin with a record of resistance https://doi.org/10.1016/j.ecoenv.2013.06.014 and Torres et al. 2015 Lambda-Cyhalothrin Resistance in the Lady Beetle Eriopis connexa (Coleoptera: Coccinellidae) Confers Tolerance to Other Pyrethroids https://doi.org/10.1093/jee/tou035

L181, 183, … – Please, the scientific name of species starts the sentence with full name. This should be corrected in the entire manuscript.

L173 to 188 – The subsection is interesting from the point of view of details on the species studied. However, this subsection contains a lot of information that would not be appropriate for the materials and methods sections. Some of this information can be moved to the discussion section. Information about the species in previous studies detailed here will be important to explain the results found in this research.

L208 – Please, the commercial name of insecticides should be avoided in the manuscript because the commercial name of insecticide could be different in other countries. Instead, the name of insecticide active ingredients would be indicated to use in the manuscript.

L212 to 215 – The mode of action of each active ingredient (Thiacloprid + Deltamethrin) of insecticide (Proteus 110) should not be described in the materials and methods section. This type of information should be suppressed or moved to the introduction or discussion sections.

L215 to L220 – The reasons about the application history of specific insecticides presented here are interesting, but they are not suitable for the materials and methods section. Please, the sentences should be moved to the discussion section.

L236 to 239 – The sentence should be deleted. The criteria for the assessment of beetles have already been defined in the previous sentence.

L247 to 249 – Please, the sentence should be deleted from the statistical analysis subsection. Perhaps, the reference should be left in the sentence. However, the explanation why one was chosen than the other analysis is not appropriate in the materials and methods section. Although this information clarifies why one analysis is appropriate than the other it became a long manuscript with an excess of information.

Figure 4 – The legend of the x-axis was missed in figure 4. Please, the legend of the x-axis should be inserted in figure 4.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

26 Nov 2021

Response to Reviewer and Editor

We thank the Referee and Editor for the helpful comments. Below we provide point-by-point answers to the points raised by the Reviewer #1 and the Editor.

REVIEWER #1:

1. L27 – Please, ‘inheritable’ should be deleted from the sentence. Even though the sensitivity of P. cupreus was lower in the subsequent generation the inheritability of resistance of P. cupreus to Thiacloprid + Deltamethrin is not proved through these experiments and results.

We admit that we have not proven that any inheritable resistance mechanism has developed in the studied population, another possibility being the selection of more resistant individuals from the large populations inhabiting the studied areas. We have clarified this in the revised manuscript as follows: “The persistence of reduced sensitivity to Proteus 110 OD for two consecutive generations indicates that either the beetles have developed resistance to the insecticide or the chronic exposure to pesticides has led to the selection of more resistant individuals naturally present in the studied populations” (lines 23-26 in the revised paper without tracked changes).

2. L80 – Please, see the references Rodrigues et al. 2013 Response of different populations of seven lady beetle species to lambda-cyhalothrin with a record of resistance https://doi.org/10.1016/j.ecoenv.2013.06.014 and Torres et al. 2015 Lambda-Cyhalothrin Resistance in the Lady Beetle Eriopis connexa (Coleoptera: Coccinellidae) Confers Tolerance to Other Pyrethroids https://doi.org/10.1093/jee/tou035.

We are grateful to the Reviewer for pointing on these important publications, which are now cited in the revised manuscript (line 80 and 391) and have been added to the list of references (lines 549-555).

3. L181, 183, … – Please, the scientific name of species starts the sentence with full name. This should be corrected in the entire manuscript.

We have changed our wording across the manuscript as indicated by the Reviewer.

4. L173 to 188 – The subsection is interesting from the point of view of details on the species studied. However, this subsection contains a lot of information that would not be appropriate for the materials and methods sections. Some of this information can be moved to the discussion section. Information about the species in previous studies detailed here will be important to explain the results found in this research.

We thought through this comment carefully, but would prefer to leave this sub-section as it was in the original version of the manuscript. In this sub-section, only information which allowed us to select this particular species for our research, determine the time of beetles collecting and select specific research sites (and crop) has been included (e.g., species numerous enough at all studies sites, disperse mainly by walking, easy to identify in the field, with a well-tested culturing procedure, one of the most common and dominant carabids found on arable land across Europe, including oilseed rape). Moreover, as the Reviewer stressed information detailed here is important to explain some of our results. We agree, that description of experimental species, its special characteristics and criteria for selection, does not necessary need to be a separate sub-section in manuscripts; the general methodological approach, including species description, often is a part of Introduction. We are not aware of any strict rules for placing the criteria for selecting species in the manuscript, but we feel it is better to have one consistent paragraph instead of covering species selection criteria in several places in the manuscript. Of course, if the Editor decides that this sub-section must be changed, we see no problem to include some information about the species in other places in in the manuscript, although Introduction seems to be better place at least for some information than Discussion.

5. L208 – Please, the commercial name of insecticides should be avoided in the manuscript because the commercial name of insecticide could be different in other countries. Instead, the name of insecticide active ingredients would be indicated to use in the manuscript.

The reviewer is right that reporting the commercial name of an insecticide does not guarantee exactly same composition in different countries, but in this case we cannot refer to the active ingredients only because the purpose of the experiment was to test the actual product used by farmers (i.e., active ingredients + additives), not the specific active ingredients. Please note, however, that the exact contents of the active ingredients in the product are reported in the manuscript (line 218-219). Moreover, although exact concentrations of all product ingredients except the insecticides themselves may differ slightly, the commercial name usually means a mixture of specific chemicals. Hence, we prefer to keep in the manuscript the commercial name of the product used.

6. L212 to 215 – The mode of action of each active ingredient (Thiacloprid + Deltamethrin) of insecticide (Proteus 110) should not be described in the materials and methods section. This type of information should be suppressed or moved to the introduction or discussion sections.

As suggested by the Reviewer, the sentence describing the modes of action of both active ingredients has been moved to the Discussion. We also added a few sentences stressing the necessity of using actual formulations in pesticide testing. The section reads now: “Proteus 110 OD is the plant protection product containing two active ingredients, thiacloprid which belongs to neonicotinoids and affects insect nervous system by stimulating nicotinic acetylcholine receptors, and deltamethrin which is a pyrethroid preventing the closure of the voltage-gated sodium channels in the axonal membranes resulting in dysfunction of spiracles – insect death is caused by overdrying. However, the ultimate effect of commercial formulations depends not only on the active ingredients but also adjuvants/co-formulants, which may affect the efficacy of penetration of the active ingredients into insect bodies or the persistence of an insecticide in field conditions [45]. Hence, other formulations with exactly same active ingredients may have somewhat different effects than those reported herein. (lines 369-377).

7. L215 to L220 – The reasons about the application history of specific insecticides presented here are interesting, but they are not suitable for the materials and methods section. Please, the sentences should be moved to the discussion section.

The indicated sentence has been moved to the Discussion section (lines 377-380).

8. L236 to 239 – The sentence should be deleted. The criteria for the assessment of beetles have already been defined in the previous sentence.

Done.

9. L247 to 249 – Please, the sentence should be deleted from the statistical analysis subsection. Perhaps, the reference should be left in the sentence. However, the explanation why one was chosen than the other analysis is not appropriate in the materials and methods section. Although this information clarifies why one analysis is appropriate than the other it became a long manuscript with an excess of information.

Although indeed this sentence may appear not crucial as this can be considered a general knowledge of statistics, the Reviewer admitted himself that “this information clarifies why one analysis is [more] appropriate than the other”. Because we are convinced that in this particular case it is important to clarify why this specific test was used, we prefer to keep this sentence in the manuscript. These are just two lines of text so we don’t think that shortening the manuscript by 30 words is worth sacrificing this information. If, however, the Editor and the Reviewer decide otherwise, we can, of course, make the necessary correction.

FIGURES

Figure 4 – The legend of the x-axis was missed in figure 4. Please, the legend of the x-axis should be inserted in figure 4.

The x-axis legend has been added (“Habitat type”) and we have also unified with other figures the descriptions of the three plots relating to the three consecutive generations (red uppercase letters “P”, “F1”, “F2”).

Response to Editor

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

The manuscript has been revised to meet PLOS ONES’s style requirements as provided in the above mentioned templates.

2. In your Methods section, please provide additional location information, including geographic coordinates of your field collection site if available.

Information on the coordinates of all twelve study sites has been included as Table 2.

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

The explanation of the permits obtained has been added and the text now reads: “The authorisation for capture, transport and keeping the beetles was granted by the Regional Directorate for Environmental Protection in Kraków, Poland, document no. OP-I.6401.128.2017.MMr, and by the Regional Directorate for Environmental Protection in Poznań, Poland, document no. WPN-II.6401.83.2017.AG.2.” (lines 198-201).

4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Yes, we confirm that repository information for our data will be provided upon acceptance of our manuscript

5. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

The above mentioned figure is neither in whole nor in part a satellite image. The upper part of the figure showing location of the two study landscapes in the Wielkopolska province and the location of the Wielkopolska province in Poland was generated using data from the National Register of Boundaries in Poland which is provided free of charge (via the service www.geoportal.gov.pl) and can be used for any purpose according to the amendments to the Surveying and Cartographic Act from April 16 2020. The land-cover maps of our study landscapes shown at the bottom of the figure are simplified versions of very detailed land cover maps generated by us in a step-by step process using the methodology described in Ziółkowska et al. (2021) based on two types of source data: (1) the National Database of Topographic Objects (BDOT) providing the land cover and land use information at the scale of 1:10.000, and (2) the Land Parcel Identification System (LPIS) providing information on the type of cultivated crops (see Methods section). We are the copyright holders of these products. However, the source data (BDOT database and data on parcels - Land and building register) are also provided free of charge (via the service www.geoportal.gov.pl) and can be used for any purpose according to the amendments to the Surveying and Cartographic Act from April 16 2020. Therefore, the entire figure may be published under the Creative Commons Attribution License (CC BY 4.0).

In addition, to be more clear, we have now added the following explanation to the main text of the manuscript: “Raster land-cover maps (resolution of 1 m2) for the landscapes were generated in a step-by-step process according to the methodology described in [33] by combining data from: (1) the National Database of Topographic Objects (BDOT) providing the land cover and land use information at the scale of 1:10.000, and (2) the Land Parcel Identification System (LPIS) providing information on the type of cultivated crops.” [lines 145-149]

We also provided some additional explanation in the Figure 1 caption:

“Land-cover maps were generated according to methodology described by Ziółkowska et al. [33], see Methods section for more details.” [lines 139-140].

Submitted filename: Response to reviewers.docx

Click here for additional data file.

17 Dec 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ramzi Mansour

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: #PONE-D-21-27829R1

Title: Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

Brief.

The manuscript has been improved after revisions. However, some critical points of the manuscript should be a better-clarified regarding the results found in the experiments and terms used throughout the manuscript. Furthermore, a broad interval confidence interval found for the LT50 values of the LA and LL habitat beetles should be appropriately discussed in the manuscript. Suggestions and corrections were left in the general comments to improve the manuscript.

General comments

L19, 24, 105, 119, 214, 227, 235, 284, 292, 314, 326, 362, 367, 369, and 402 - The reason given in the section of the response to the reviewers by the author for keeping the insecticide trade name Proteus 110 OD in the manuscript is conflicting because the insecticide Proteus 110 OD is a mixture of ingredient actives thiacloprid + deltamethrin. Several products with ingredient actives mixture have been released in the last years. What were the additives tested in the experiment? Oil dispersion (nonpolar) allows break plant barriers such as the wax layer of leaves plants (nonpolar) to lead the ingredient active improving an insecticide’s uptake by the plant. Acetone is an adjuvant that should not have to act in the beetle. The suggestion to change the trade name of Proteus 110 OD to its active ingredient was based on making it internationally standardized to identify it other countries exclusively through the name of ingredient active. However, decisions can be made by consensus of the author and editor.

L81 to 82, 86, 95, 171, 236, 268, 269, 352, 395, 397, 421, 465 and 470 – Please, all references used in the sentence referred to insecticides. Thus, pesticides should be changed to insecticides. Pesticide is a broad term that includes various products to kill different types of organisms, insecticide is a term designated for products that act specifically on insects. Please, the correct term should be reviewed in the entire manuscript.

L345 to 348 – The broad confidence intervals to the LT50 values for insecticide-treated beetles from the LM and LL habitat types and acetone-treated beetles from the control habitats (S0-A and L0-A, Fig 4) should be discussed in the discussion section. Why were only beetles from these areas had a broad confidence interval in the different generations' studies (P, F1, and F2)?

L339 and 351 - How was it determined that the beetle was tolerant to insecticides? Would not this be an evolution of the beetle's resistance to insecticides? The term used to describe the results found in the experiment should be clearer.

L360 to 362 - The first sentence of the discussion section contradicts the results described in the materials and methods section, see comments above in lines 339 and 351. In materials and methods, the tolerance of the beetle to insecticides has been described. However, the discussion concerns the development of insecticide resistance in the beetle. Please, the terms used in the materials and methods section and the discussion should be clearer.

L377 to 380 - The sentence is confusing as a massive number of studies have used both commercial formulations and unique active ingredients. Please, the sentence should be rephrased or deleted from the discussion section.

L408 to 409 – There was no found tolerance as well as the use of pesticides in this study mentioned in the sentence. Schoonees and Giliomee (1982) found an increase of resistance of two strains of parasitoids to the insecticides in different localities where the application of insecticide was intensified through the years.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Jan 2022

Response to Reviewer

We thank the Referee for the helpful comments. Below we provide point-by-point answers to the points raised by the Reviewer #1.

REVIEWER #1:

1. L19, 24, 105, 119, 214, 227, 235, 284, 292, 314, 326, 362, 367, 369, and 402 - The reason given in the section of the response to the reviewers by the author for keeping the insecticide trade name Proteus 110 OD in the manuscript is conflicting because the insecticide Proteus 110 OD is a mixture of ingredient actives thiacloprid + deltamethrin. Several products with ingredient actives mixture have been released in the last years. What were the additives tested in the experiment? Oil dispersion (nonpolar) allows break plant barriers such as the wax layer of leaves plants (nonpolar) to lead the ingredient active improving an insecticide’s uptake by the plant. Acetone is an adjuvant that should not have to act in the beetle. The suggestion to change the trade name of Proteus 110 OD to its active ingredient was based on making it internationally standardized to identify it other countries exclusively through the name of ingredient active. However, decisions can be made by consensus of the author and editor.

We still agree with the Reviewer on the fact that when reporting the commercial name of an insecticide, one can expect different composition in different countries. However, we stand by our arguments that providing information only on active ingredients, without reporting commercial name, may be confusing. By giving the full name of the product, together with the exact contents of the active ingredients in the product and the country of purchase (lines 212-218 in the revised paper without tracked changes), we avoid any doubts whether the toxicity of the active ingredients alone could be potentially different from that of the mixture (i.e. the commercially available product). The purpose of the experiment was to test the actual product used by farmers (i.e., active ingredients + additives), not the specific active ingredients, as we wanted to reflect as closely as possible the real situation in the field. To make it unambiguously clear what the composition of the product used was, we have included, as Supporting information, a table with product specification as provided by the manufacturer (Bayer, Germany) (S1 Table). Additional text in lines 217-218 in the revised manuscript was also added: “(see Table S1 in Supplementary materials for full product specification)”.

2. L81 to 82, 86, 95, 171, 236, 268, 269, 352, 395, 397, 421, 465 and 470 – Please, all references used in the sentence referred to insecticides. Thus, pesticides should be changed to insecticides. Pesticide is a broad term that includes various products to kill different types of organisms, insecticide is a term designated for products that act specifically on insects. Please, the correct term should be reviewed in the entire manuscript.

Of course we agree with the Reviewer that pesticide is a broad term that includes various products (e.g. insecticides, herbicides, fungicides) to kill different types of organisms, whereas insecticides act specifically on insects. We have now changed the term “pesticide” into “insecticide” wherever we refer to our laboratory test results on Proteus 110 OD, which is the insecticide (lines 236, 268, 269, 352, now lines 234, 265, 266, 345). The changes were also done in lines 81-82 and 86 (now lines 79-80 and 84): although the references we used are not specific to insecticides (they relate to resistance to xenobiotics in general), we agree that for consistency with the earlier sentence, in which “insecticides” were mentioned, the same term should be used in the indicated sentences. In line 94, the term “pesticide” was mentioned because the use of other products then insecticides (e.g. herbicides, fungicides), even though they are not directly harmful to ground beetles, still may indirectly influence their survival, for example through the impact on their habitat and/or food source (e.g., disappearance of plant food). Therefore, the use of the term “pesticide” is appropriate in this context. A similar situation arises in line 171 (now line 169 in the revised paper without tracked changes) where the term “pesticide” is used to emphasize that the populations of the two habitats mentioned (S0 and L0) were not exposed to any potentially toxic substances in the field. The use of the term “insecticides” would imply that other types of treatments could have been applied there. A similar justification for using a term “pesticide” applies to line 421 (now line 411). Regarding lines 465 and 470 (now lines 455 and 460), we decided to keep the original terminology, as we draw general conclusions here on either significance of landscape for insect response (not resistance) to pressures from different pesticides used in that landscape (line 465, now line 455) or resistance of beneficial species (not specifically insects) to pesticides (line 470, now line 460).

3. L345 to 348 – The broad confidence intervals to the LT50 values for insecticide-treated beetles from the LM and LL habitat types and acetone-treated beetles from the control habitats (S0-A and L0-A, Fig 4) should be discussed in the discussion section. Why were only beetles from these areas had a broad confidence interval in the different generations' studies (P, F1, and F2)?

The confidence intervals to the LT50 were calculated fusing Litchfield’s method (lines 256 – 258 in the previous version of the manuscript) which is a method for rapid graphic solution of time-per cent effect curves. We have now checked that confidence intervals are much narrower when they are calculated for medians (LT50 is the median lethal time). To give an example, for insecticide-treated beetles from LL habitat types in F2 generation, with LT50 of 15.23 days, the confidence interval calculated using Litchfield’s method is between 5.67 and 40.92 days, whereas the confidence interval for the median of 15.23 days is between 12.23 and 16.23 days (based on equations on https://www.statology.org/confidence-interval-for-median/).

However, following the Reviewer’s remark about the broad confidence intervals to the LT50 values we concluded that we unnecessarily confused the reader suggesting that confidence intervals were used to compare the mortality rates between the habitat types. In fact, as stressed in table 3, we compared the whole survival curves using Wilcoxon test (“Groups with the same lowercase letter do not differ significantly at p �  �  0.05 in terms of survival (pairwise comparison of survival curves, Wilcoxon test)”, now lines 299). Because of that, we decided to replace the Figure 4 with a similar figure, but instead confidence intervals, standard errors for LT50 values are now presented. Thus, all sentences refereeing to confidence intervals in Statistical analysis and results describing differences comparison of LT50s between habitat types based on confidence intervals overlap have been removed from the revised version of the manuscript.

4. L339 and 351 - How was it determined that the beetle was tolerant to insecticides? Would not this be an evolution of the beetle's resistance to insecticides? The term used to describe the results found in the experiment should be clearer.

Each individual beetle from each habitat type had its own survival time after insecticide treatment (i.e., its own insecticide sensitivity = insecticide tolerance) and we showed that: “as in the P generation, the comparison among insecticide-treated beetles from different habitat types revealed statistically significant difference in survival rates of beetles in both the F1 and F2 generations (p < 0.001 for F1, p < 0.03 for F2, Fig 3)” (now lines 332 - 335) and “The increased tolerance to the insecticide found in beetles from the P generation from the LM and LL habitats was still present in both next generations F1 and F2, as insecticide-treated beetles from the LM and LL habitats survived significantly better than insecticide-treated beetles from all other habitat types (Fig 3, Table 3), but significantly worse than acetone-treated beetles from the control habitats (S0-A and L0-A; Table 3)”. Thus, because similar results of increased tolerance to the insecticide were found in beetles from the LM and LL habitats over three generations (P, F1 and F2), the results suggest the evolution of resistance. Results and Discussion sections are, however, separated in the manuscript, so we do not interpret our results in Results section. We assumed that the Discussion section is designed to tell what the results mean. Thus, we would prefer to keep our original writing and first present the results on increased tolerance to the insecticide in beetles from the LM and LL habitats over three generations (P, F1 and F2) and then conclude that such results suggest the evolution of resistance. Nevertheless, if the Editor decides otherwise, we don't see a problem with adding following summary/conclusion sentence to the Results section: “Thus, the results suggest that the effect of insecticides on the evolution of resistance in populations of P. cupreus strongly depends on large-scale landscape characteristics.”.

5. L360 to 362 - The first sentence of the discussion section contradicts the results described in the materials and methods section, see comments above in lines 339 and 351. In materials and methods, the tolerance of the beetle to insecticides has been described. However, the discussion concerns the development of insecticide resistance in the beetle. Please, the terms used in the materials and methods section and the discussion should be clearer.

Yes, in the Material and Methods section we described the methods used to study the tolerance of the beetles to insecticide (in other words, we studied susceptibility of beetles to pesticide). This tolerance was studied for three generations to sort out possible temporary effects through direct selection of the most resistant individuals collected from the field right after the spraying from parental effect and/or possible genetically fixed resistance to insecticides (as stressed already in the Introduction, lines 114 -118 in the revised paper without tracked changes). As we stressed in the Discussion section, the successful culturing of the beetles through three generations enabled us to separate the actual genetically fixed adaptation from possible direct or maternal effects (lines 443 - 445). Thus, we don’t see any contradiction between Methods (testing tolerance of beetles to insecticides in generation P, F1 and F2) and Discussion sections (drawing conclusions from obtaining similar results on tolerance of beetles to insecticide in all three generations). Please see also our answer to the previous comment as well as to the last comment.

6. L377 to 380 - The sentence is confusing as a massive number of studies have used both commercial formulations and unique active ingredients. Please, the sentence should be rephrased or deleted from the discussion section.

As suggested the mentioned sentence has been deleted form the discussion section.

7. L408 to 409 – There was no found tolerance as well as the use of pesticides in this study mentioned in the sentence. Schoonees and Giliomee (1982) found an increase of resistance of two strains of parasitoids to the insecticides in different localities where the application of insecticide was intensified through the years.

Schoonees and Giliomee (1982) evaluated the toxicity of methidathion (organophosphorus insecticide) to two strains of Aphytis africanus and Comperiella bifasciata, both parasitoids of Aonidiella aurantii in South Africa. They found that different geographic strains varied; one of them, which used to receive three to four sprays of organophosphorus insecticides per year over many years, was much more tolerant to methidathion than the one, which received only one spray per year. To be precise, the authors used the word “susceptibility” to check difference of the studied species between localities, but being more susceptible to something means less tolerance to it. In the Methods they described how this “susceptibility” was measured and expressed (survival was checked in contact toxicity test and LC50 was calculated). In the Results they described differences in susceptibility between the two strains (collected from different localities) and calculated “ resistance factor”, which is LC50 of one strain divided by LC50 of the second strain. In the Discussion section, the authors concluded about “the greater resistance to methidathion in A. africanus and C. bifasciata from Letsitele as compared with those from Mooinooi and Zebediela” which simply means that A. africanus and C. bifasciata from Letsitele had higher LC50 (were less susceptible to methidathion = were more tolerant to methidathion) as compared with those from Mooinooi and Zebediela.

The paper by Schoonees and Giliomee (1982) proves that organisms subjected to higher number of insecticide sprays are able to develop tolerance to the most commonly used product (=are more resistant, and the word “tolerance” has been now replaced by the “resistance”). We agree that the paper does not directly answer the question of whether landscape structure is one of the causes of resistance (=being more tolerant to methidathion). However, the described situation (higher number of sprays) may occur more often in homogenised landscapes where the insects inhabiting them have limited or no access to a refuges. With this in mind, we consider the work by Schoonees and Giliomee (1982) as a good example that more frequent contact with insecticides promotes the development of resistance.

ADDITIONAL COMMENT

In Table 3, we made a typo error for the LT50 values of beetles from P generation and LM habitat. Instead of 21.25 it should be 23.26.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

20 Jan 2022

PONE-D-21-27829R2

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

PLOS ONE

Dear Dr. Sowa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

For the second consecutive time, the authors clearly failed to address the expert Reviewer's comments and suggestions and provided confusing responses. Additionally, as stated by the expert Reviewer, there is a confusion between (wrong use and interpretation) the important scientific terms "resistance" and "tolerance" in the manuscript and wrong reedits from previous scientific articles. Accordingly, this manuscript could no longer be considered for publication in PLOS ONE. All comments of the expert Reviewer are stated below.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Ramzi Mansour

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: # PONE-D-21-27829R2

Title: Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

Brief.

The manuscript was partially revised following suggestions from the reviewers. Furthermore, even after revising the terms used in the manuscript, the author chose to keep the wrong term and justify the error by reediting the term used in another manuscript.

General comments

The comments about lines 339 and 351 and 360 to 362 is not suggesting any discuss. The terms “tolerance” and “resistance” in results and discussion section was not clear and it remain unclear. Briefly, resistance may be defined as “a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species” (IRAC, 2022 [https://irac-online.org/about/resistance/]). In contrast to resistance, insecticide tolerance is a natural tendency and not a result of selection pressure and following Tabashnik (1991) insecticide resistance can be viewed as a threshold trait, with tolerance as the underlying continuous variable (Finney 1971, Falconer 1981, Via 1986, Tabashnik & Cushing 1989). Thus, the term should be rethought, because if beetles have been exposed to active ingredients at the collect site and exposed in the generations P, F1 and F2 generations, would they be resistant or tolerant? There is an expectation supported by results found that beetles are not tolerant, but individuals resistant. This would all change the way approach this term in the sections. Again, a discussion of this in any section is not recommended.

Bruce E. Tabashnik, 1991. Deterrnining the Mode of Inheritance of Pesticide Resistance with Backcross Experiments. Journal of Economic Entomology. V. 84, no. 3, p.703-712.

The explanation provide by authors of manuscript about lines 408 to 409 in the response to reviewers is completely wrong and they are reediting the terms “susceptibility and resistance” used correctly by Schoonees and Giliomee (1982).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

- - - - -

For journal use only: PONEDEC3

8 Feb 2022

Response to the Reviewer

We acknowledge receiving the decision rejecting our manuscript based on the review, but we fundamentally do not agree with the opinion expressed by the Reviewer and, consequently, the decision made by the Academic Editor. The Reviewer had only one comment, relating to the use of terms “tolerance” and “resistance” in our manuscript. Below please find the comment itself (in blue italics, indented) followed by our rebuttal.

Reviewer #1: # PONE-D-21-27829R2

Title: Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

Brief.

The manuscript was partially revised following suggestions from the reviewers. Furthermore, even after revising the terms used in the manuscript, the author chose to keep the wrong term and justify the error by reediting the term used in another

manuscript.

General comments

The comments about lines 339 and 351 and 360 to 362 is not suggesting any discuss. The terms “tolerance” and “resistance” in results and discussion section was not clear and it remain unclear. Briefly, resistance may be defined as “a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species” (IRAC, 2022 [https://iraconline.org/about/resistance/]). In contrast to resistance, insecticide tolerance is a natural tendency and not a result of selection pressure and following Tabashnik (1991) insecticide resistance can be viewed as a threshold trait, with tolerance as the underlying continuous variable (Finney 1971, Falconer 1981, Via 1986, Tabashnik & Cushing 1989). Thus, the term should be rethought, because if beetles have been exposed to active ingredients at the collect site and exposed in the generations P, F1 and F2 generations, would they be resistant or tolerant? There is an expectation supported by results found that beetles are not tolerant, but individuals resistant. This would all change the way approach this term in the sections. Again, a discussion of this in any section is not recommended.

Bruce E. Tabashnik, 1991. Deterrnining the Mode of Inheritance of Pesticide Resistance with Backcross Experiments. Journal of Economic Entomology. V. 84, no.

3, p.703-712.

The explanation provide by authors of manuscript about lines 408 to 409 in the response to reviewers is completely wrong and they are reediting the terms

“susceptibility and resistance” used correctly by Schoonees and Giliomee (1982).

We do not agree with the opinion expressed by the Reviewer and with the decision of the Academic Editor rejecting our manuscript. First of all, it should be noted that the Reviewer did not indicate any flaws in our study, data analysis or the manuscript itself except of contesting the use of just two terms: “tolerance” and “resistance”. In our opinion this might be, at the very best, the subject of discussion on the semantics but does not justify rejecting the manuscript. The questioned terms do not have such a strict scientific meaning as the Reviewer tries to imply. The meaning of the terms is defined in different ways, depending on the need, as shown in the examples below. Although some authors can define them in a way indicated by the Reviewer, this does not mean that such an understanding is the only one and obligatory. More importantly, in the revised version and in our previous Response to the Reviewer we explained very clearly how both terms are understood (defined) in our manuscript and why we decided to use both. Briefly, by testing tolerance to a pesticide (by estimating LC50 and comparing survival curves) throughout three generations we could conclude that most probably some of the tested populations exhibited genetically fixed resistance. This is exactly in accordance with the definition proposed by Tabashnik and Johnson in the “Handbook of Biological Control” (by the way, one of the authors, B. E. Tabashnik, is the very same author as the one cited by the Reviewer!):

Examples of the use of terms “resistance” and “tolerance”

“Pesticide resistance is a genetically based, statistically significant increase in the ability of a population to tolerate one or more pesticides. In most cases, resistance is documented with laboratory bioassays showing that a population with a history of extensive exposure to pesticides has a significantly greater LC50 or LD50 […] compared with a conspecific population that has had less exposure to pesticides. One can also document resistance by showing that treatment with a fixed concentration or dose causes significant differences in mortality among conspecific populations […]. Evidence of significant increase through time within a population in LC50, LD50, or survival in response to a fixed concentration or dose provides more direct documentation of resistance.” (from “Evolution of Pesticide Resistance in Natural Enemies” by B. E. Tabashnik, M. W. Johnson, in “Handbook of Biological Control”, 1999).

In turn, EPA defines tolerance in a very precise way for a very specific purpose: “A tolerance is the EPA established maximum residue level of a specific pesticide chemical that is permitted in or on a specific human or animal food in the United States”.

In pharmacology “Tolerance is a decrease in response to a drug that is used repeatedly. Resistance is development of the ability to withstand the previously destructive effect of a drug by microorganisms or tumor cells”.

Referring to pesticide resistance and tolerance: “The chemical arsenal we have developed in an attempt to rid our homes of rodents and our crops of insects is losing its power. We have simply caused pest populations to evolve, unintentionally applying artificial selection in the form of pesticides. Individuals with a higher tolerance for our poisons survive and breed, and soon resistant individuals outnumber the ones we can control.”

(https://www.pbs.org/wgbh/evolution/library/10/1/l_101_02.html)

Please note also that the English language thesaurus reports “resistance” and “resilience”

(together with “strength” and “toughness”) as the closest synonyms of “tolerance”.

Moreover, the Reviewer’s statement about the definition of resistance (“resistance may be defined as “a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species”) is completely irrelevant to our studies as we did not study effectiveness of any product against pests but rather the combined effect of infield pesticide applications and landscape structure on the resistance of a beneficial carabid. In our earlier response to the Reviewer, we provided a detailed answer to the Reviewer’s comments and explained that only “because similar results of increased tolerance to the insecticide were found in beetles from the LM and LL habitats over three generations (P, F1 and F2), the results suggest the evolution of resistance” (in terms of a heritable change in the sensitivity), as by doing the study on three generations of beetles we could “separate the actual genetically fixed adaptation (=heritable resistance) from possible direct or maternal effects”. We also explained that the increased tolerance to an insecticide means the decreased sensitivity to that insecticide. Even if this is not exactly the same as the Reviewer's reasoning, we are convinced that this is, at the best, a field for polemics rather than a basis for rejecting the manuscript. Indeed, if the Editor prefers to have the term “less sensitive” instead of “tolerant” (although tolerant means simply less sensitive), we do not see a problem with such a rewording.

We completely do not agree with the Reviewer’s statement that “The explanation provide by authors of manuscript about lines 408 to 409 in the response to reviewers is completely wrong and they are reediting the terms “susceptibility and resistance” used correctly by Schoonees and Giliomee (1982).” We read the paper carefully and would like to know where exactly we distorted the meaning of the words.

It must be stressed that the decision to reject our manuscript was based on the opinion of just one reviewer who apparently did not find any methodological flaws in our study or data analysis and simply has different opinion on the meaning of just two terms used in the manuscript. Also, in the first review no problem with the definition of the terms was mentioned by the Reviewer; nevertheless, the terms have been precisely defined in the revised version and should not bring any doubts about their meaning in the study.

Submitted filename: Response_to_Reviewer_PONE-D-21-27829_R2.pdf

Click here for additional data file.

22 Mar 2022

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

PONE-D-21-27829R3

Dear Dr. Sowa

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tuneera Bhadauria, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I believe they are extremely obvious in the text after reading the amended manuscript and the authors' responses to the reviewers' questions about the use of the phrases tolerance and resistance. I can explain it in the following context, as stated by the authors. I believe that the small-field environment, with its enhanced spatiotemporal variety, enables additional niches for beetles (and presumably other NTAs) to exist in while avoiding the development of insect resistance.In landscapes dominated by wide fields and ecosystem homogenization through mono cropping and recurrent periods of insecticide spraying, beetles are unable to move into fields with heterogenous cropping practices. This raises the chances of beetles being exposed to the insecticidal spray on a regular basis, developing tolerance to it over time, and finally becoming resistant to it.

I propose that the paper be approved for publishing in the journal because the authors have responded well to all of the additional clarifications, comments, and suggestions made by reviewers, incorporating them into the text as and where needed.

Reviewers' comments:

18 Apr 2022

PONE-D-21-27829R3

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus

Dear Dr. Sowa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tunira Bhadauria

Academic Editor

PLOS ONE