Differential responses of Orthoptera in vineyards to organic farming, pesticide reduction, and landscape heterogeneity

Conservation measures such as those under the European Green Deal aim to counteract the biodiversity loss by increasing the share of organic farming and reducing pesticide use, as well as increasing the proportion of semi-natural habitats (SNH) in agricultural landscapes. Given the large environmental impacts of agriculture, it is important to thoroughly understand effects of such measures on organisms to provide evidence-based and effective implications for conservation. In this study, we analysed how vineyard management, pesticide reduction, and landscape composition affect Orthoptera densities and species composition. Therefore, we sampled herb- and vine-dwelling orthopterans in a paired design of classic and fungus-resistant grape (FRG) varieties in conventionally and organically managed vineyards along a landscape heterogeneity gradient. Here, FRG varieties allowed us to study the effect of 44% reduced pesticide applications under real-world conditions. Total densities of herb-dwelling Orthoptera did not differ between grape varieties in conventional vineyards, but were 2.9 times higher in FRG varieties under organic management. In contrast, total densities of vine-dwelling Orthoptera, mainly driven by the dominant species Phaneroptera falcata, were similar between grape varieties in organic vineyards, but tended to be 1.4 times higher in classic grapes under conventional management. Furthermore, the management system and SNH in a radius of 500 m in the surrounding landscape influenced species composition. Our work shows that the cultivation of FRG varieties, at least in organic viticulture, clearly benefits some orthopteran species. It appears that the reduction of non-specific pesticides such as copper and sulfur is important to mitigate negative effects and promote Orthoptera in viticulture.


Introduction
Together with the impact of climate change, habitat destruction, and invasive species, intensified agriculture is considered one of the main drivers of the serious loss of biodiversity in recent decades, harming the environment through regular Marvin Kaczmarek marvin.kaczmarek@julius-kuehn.de non-target effects, e.g. on arthropods, can be expected (Pertot et al. 2017). While organic farming promotes biodiversity in many cropping systems compared to conventional farming (Bengtsson et al. 2005;Hole et al. 2005;Tuck et al. 2014), the effect seems to be less clear in viticulture and vary between and even within organism groups (Bruggisser et al. 2010;Döring et al. 2019;Ostandie et al. 2021;Paiola et al. 2020;Schirmel et al. 2022, Kaczmarek et al. 2023. Since the use of synthetic chemicals is prohibited in organic management, non-synthetic components such as copper and sulfur are used in high quantities in viticulture to control pests, which, however, can also negatively impact non-target organisms (Biondi et al. 2012;Nash et al. 2010;Vogelweith and Thiéry 2018). The copper accumulates in the soil and thus may have long-term risks on the environment (Komárek et al. 2010). Further, an increased number of pesticide applications in organic viticulture leads to more tractor passages, and may affect non-target organisms by higher disturbance of the ground vegetation and increased soil compaction (Bruggisser et al. 2010).
Regardless of conventional or organic vineyard management, the impact of pesticides on biodiversity can be reduced by cultivating fungus-resistant grape (FRG) varieties, which are characterized by resistance traits to the major fungal diseases (Töpfer et al. 2011). While the first resistant grape varieties were developed in the late 19th and early 20th centuries, the first cultivars with convincing wine qualities were only developed by the end of the 20th century and nowadays, over 38 cultivars are available for winegrowers (Töpfer and Trapp 2022). However, despite that the resistances can reduce the need for pesticides by up to 80% for some multi-resistant varieties and increase the sustainability of viticulture (Töpfer and Trapp 2022), only about 2.7% of the area under cultivation is planted with FRG varieties in our study region (Statistisches Bundesamt (Destatis) 2023). Positive effects of the cultivation of such varieties are recently reported to benefit non-target organisms, including predatory mites and some spider families (Pennington et al. 2017(Pennington et al. , 2019Reiff et al. 2021aReiff et al. , 2023, making the cultivation of FRG varieties a promising approach to promote biodiversity in viticulture. While this is of importance in the context of, e.g., the European efforts to reduce pesticides, evidence is lacking as there are only few recent research studies on the effects of the cultivation of FRG varieties on biodiversity. Although organisms are usually exposed to high levels of pesticides due to the intensive management in viticulture, vineyards can provide a suitable habitat for species through the vines themselves, but especially through the vegetation in the inter-rows. Improved ground vegetation management may promote biodiversity by higher vegetation cover and species-rich cover crops providing habitat especially for typical grassland species (Blaise et al. 2022;Ortis et al. 2021;Paiola et al. 2020;Winter et al. 2018). In addition, biodiversity is usually enhanced by heterogeneous landscapes with a high proportion of SNH (Martin et al. 2019;Ostandie et al. 2021;Paiola et al. 2020). Forests, hedges, shrubs, and grasslands can provide habitat for shelter and overwintering as well as food and breeding resources for species for which such resources are not available in vineyards (Holland et al. 2017).
Orthopterans are an important arthropod group in terrestrial food webs and provide a food source for various predatory species (Belovsky and Slade 1993). However, agricultural land use intensification, including the use of pesticides, is considered one of the major threats to Orthoptera species (Zuna-Kratky et al. 2016). They may be highly exposed to pesticides through surface contact, their feeding behavior, and the egg-laying substrate (Bundschuh et al. 2012;Ingrisch and Köhler 1998), making them sensitive to environmental changes caused by management practices. In viticulture, the orthopteran fauna is further determined by tillage, cover crop management, the presence of habitat structures in the surrounding area, and the location of the vineyard in the landscape context (Detzel 1998). In Central Europe, vineyards can mainly be found in climatically favorable regions (Bruggisser et al. 2010) and can provide habitat for several Orthoptera species including both shrubdwelling orthopterans (mainly species of the order Ensifera) in the vines and herb-dwelling orthopterans (mainly of the order Caelifera) in the inter-row vegetation (Detzel 1998). Hence, Orthoptera are appropriate indicators of the effects of pest, soil, and cover crop management practices and landscape heterogeneity in viticulture as they meet many of the criteria for effective ecological indicators (Noss 1990). In addition, density and species composition can be easily measured (Gardiner et al. 2005), which is why orthopterans are widely used as indicator species in ecological studies (Alignan et al. 2018;Bazelet and Samways 2011;Dvořák et al. 2022).
To assess how organic vineyard management, pesticide reduction, and landscape heterogeneity affect orthopteran densities and species composition, we sampled herb-and vine-dwelling orthopterans using box quadrats and transect walks with song detection, respectively. For this purpose, we used a paired design with classic and FRG varieties in either conventionally or organically managed vineyards along a gradient of landscape heterogeneity. In particular, we expected that (1) orthopterans are promoted by organic compared to conventional farming and (2) that they benefit from the cultivation of FRG varieties. Lastly, we investigated whether (3) SNH-rich compared to vineyard-dominated landscapes favor orthopterans.

Study area and site selection
We conducted the study in the district Südliche Weinstraße and the district-free city Landau in der Pfalz (49.273280 °N, 8.020602 °E / 49.147516 °N, 8.175736 °E). The area is located in the wine-growing region Palatinate in the south of Rhineland-Palatinate in southwestern Germany with a temperate climate with an average annual temperature of 11.1 °C and a total annual precipitation of 687.5 mm (Agrarmeteorologie Rheinland-Pfalz 2022; Beck et al. 2018).
We chose 16 different landscapes along a gradient of landscape heterogeneity in the study region that differed in their proportion of SNH in the surrounding landscape ( Fig. 1). In each landscape, we sampled in two vineyards planted with a FRG and a classic grape variety, respectively, while the management system was organic in half of the pairs of vineyards and conventional in the other half. Of the vineyards studied, those managed conventionally had 10 pesticide applications in classic grape varieties (SD = ± 2) Fig. 1 Locations of the 16 landscapes in the south of Rhineland-Palatinate, Germany, with organic (red) and conventional (blue) management and fungus-resistant (FRG, brighter) and classic grape varieties (darker). Basic map data by © GeoBasis- DE/LVermGeoRP (2022) We used an information-theoretic approach to multimodel inference (Burnham and Anderson 2002) to analyze the effects of vineyard management and vegetation and landscape parameters on Orthoptera densities. We conducted linear mixed models (R command 'lmer' in the R package lme4, Bates et al. 2015) and used total density of herb-dwelling Orthoptera, total density of vine-dwelling Orthoptera, and densities of the three most frequent herband vine-dwelling Orthoptera, respectively, as dependent variables. In order to meet model assumptions, densities were log (x + 1) transformed. We standardized the regression predictors using the 'standardize' function (R package arm, Gelman and Su 2016). As explanatory variables we included vineyard management (factor with the two levels 'organic' and 'conventional'), grape variety (factor with the two levels 'FRG' and 'classic'), vegetation cover (continuous), vegetation height (continuous), and the amount of SNH in the surrounding landscape (continuous) in the full models. To assess whether the effect of grape variety differs among organic and conventional vineyards, we further included their interaction in the models. Due to our paired design, we included the site ID as a random effect in the models. For automated model selection, we used the 'dredge' function (R package MuMln, Bartoń 2020) and selected those top-ranked models within Δ AICc < 4. We used the AICc for small sample sizes. Conditional averaged parameter estimates from this top set of models were then produced using the 'model.avg' function. To check for correlations among the explanatory variables we calculated the variation inflation factors (VIF). In cases where an explanatory variable had a VIF > 2 (which was either vegetation height or vegetation cover), we excluded this variable in the full model which resulted in VIF < 2 of all remaining variables.
The species composition of Orthoptera was analyzed using redundancy analysis (command 'rda' in package vegan, Oksanen et al. 2020). For the multivariate analysis, we used binary data (presence / absence) because densities of herb-and vine-dwelling Orthoptera are not comparable due to the differently used sampling methods. We used the same explanatory variables as in the univariate models.

Sampling of herb-dwelling Orthoptera
In mid-August 2021, during dry and warm weather with temperatures ranging from 20 to 30 °C, a 40 m section was sampled in two randomly selected inter-rows in the center of each vineyard using a 1.96 m 2 box quadrat to assess the density of herb-dwelling Orthoptera. We sampled in two adjacent, differently tilled inter-rows and placed the isolation square on the ground seven times per inter-row. We identified orthopterans within the box quadrat (according to Fischer et al. 2020) and released individuals afterwards. Only adult individuals were analyzed.

Sampling of vine-dwelling Orthoptera
In early September 2021, after sunset and during dry weather with temperatures above 10 °C, we walked through two randomly selected inter-rows in the center of each vineyard to assess the density of vine-dwelling Orthoptera in the adjacent rows of grape vines. We detected individuals by their species-specific songs (according to Orthoptera.ch 2021) and additionally used a bat detector (Observer 2 HD², CIEL-electronique) to make calls in the high frequency range audible. We calculated the number of individuals per 100 m inter-row.

Vegetation and landscape parameters
We measured the vegetation height in two randomly selected inter-rows per vineyard by using a cardboard-disc with a diameter of 30 cm and a measuring stick. Further, we visually assessed the proportion of ground covered by vegetation in the two whole inter-rows. A mean value for vegetation height and cover was calculated out of all measurements for each vineyard (Table S1). For each landscape, we calculated the mean proportion of SNH, which we defined as forests, hedges, shrubs, and grassland, within a radius of 500 m of each vineyard using ATKIS data (Basis-DLM by ©GeoBasis-DE/BKG (2013)) with intersection of spatial data in an Oracle database 12c (Oracle 2017).

Data analysis
We used R 4.0.5 (R Core Team 2021) for statistical analyses and the R package ggplot2 (Wickham 2016) for creating figures.

Effects of vineyard management, grape variety, and local and landscape parameters
Total density of herb-dwelling Orthoptera was significantly affected by grape variety and the interaction of management system and grape variety (Table 1). This indicates that total densities were not different between classic and FRG varieties in conventional vineyards, but were almost 3 times higher in FRG varieties under organic management ( Fig. 2A). Management, vegetation cover, and the amount of SNH in the surrounding were included in the final model of total density of herb-dwelling Orthoptera but had no significant effects ( Table 1). The two most common herb-dwelling species C. brunneus and C. biguttulus showed similar responses: Both species had higher densities in FRG varieties under organic management, while no such differences were observed in conventional vineyards. Furthermore, C. brunneus densities significantly increased with increasing vegetation height ( Fig. 2B and C). Management, grape variety, and the amount of SNH in the surrounding were all not significantly related to densities of C. brunneus and C. biguttulus (Table 1). For P. parallelus we found a significant influence of the grape variety while all other parameters had no significant effect ( Table 1). Densities were on average about two times higher in FRG compared to classic varieties (organic vineyards: three times higher, conventional vineyards: 1.7 times higher; Fig. 2D).
As a trend, total density of vine-dwelling Orthoptera was affected by the interaction of management and grape variety ( Table 2). Densities were similar between classic and FRG varieties in organic vineyards, but 1.4 times higher in classic grapes under conventional management (Fig. 3A). Further, their density tended to increase with increasing vegetation cover. All other parameters included in the final model of total density of vine-dwelling Orthoptera had no significant effects ( Table 2). For the density of the most frequent vine-dwelling Orthoptera P. falcata, we found no significant effects of the explanatory variables ( Table 2). P. falcata densities were higher in conventional than in organic vineyards, but this pattern was statistically not significant and driven by two extreme values in classic and FRG varieties, respectively ( Fig. 3B; Table 2). Organic management had Table 1 Model-averaging results of the top-ranked models for total densities of herb-dwelling Orthoptera and the three most common species in vineyards with different management (conventional, organic) and grape varieties (classic, fungus-resistant). Indicated are the standardized coefficient (Coeff.), adjusted standard error (SE), z-score (z), and P-value (p) for management, grape variety, their interaction (Man:Var), semi-natural habitats (SNH), and vegetation cover and height.  (Table 3). However, it was not affected by the grape variety. Species related to conventional vineyards were T. viridissima and C. biguttulus, while L. punctatissima were more common in organic vineyards (Fig. 4). L. punctatissima was also related to vineyards with a higher amount of SNH in the surrounding landscape, while P. falcata was more common in vineyards with less SNH (Fig. 4).
a significant negative effect on T. viridissima and densities were on average three times higher in conventional than in organic vineyards (Table 2; Fig. 3C). The amount of SNH in the surrounding landscape, grape variety, and its interaction with management as well as vegetation height had all no significant influence on the density of T. viridissima (Table 2). L. punctatissima densities increased significantly with increasing amount of SNH while no other explanatory variable had a significant effect ( Table 2; Fig. 3D). The Orthoptera species composition, based on presenceabsence data, was significantly influenced by vineyard management and the amount of SNH in the surrounding of the

Discussion
We assessed how organic and conventional viticulture, reduced pesticide application by using FRG varieties, and the proportion of SNH in the landscape around vineyards affected the density and species composition of herb-and vine-dwelling Orthoptera. Our main findings were that total densities of herb-dwelling Orthoptera were greatly enhanced in FRG varieties under organic management, while total densities of vine-dwelling Orthoptera tended to be higher in classic grapes under conventional management. Further, the management system and SNH in the surrounding landscape influenced species composition.
In contrast to our first hypothesis, we did not find any general effects of organic viticulture on total densities of Orthoptera. The only species where we found an effect was T. viridissima, being even three times less abundant in organically managed vineyards than in conventional ones. Thus, a conversion from conventional to organic viticulture, such as that aimed for by the European Green Deal to counteract the loss of biodiversity (European Commission 2019), does not seem to increase Orthoptera densities in our study. However, it is important to note that the diversity and conservation value of orthopterans that we found was rather low compared to some areas with different soil and relief, such as in the Middle Rhine valley (Wersebeckmann et al. 2023). Similarly, other studies showed that general effects of organic farming in viticulture on biodiversity are less clear then in other cropping systems, where both positive and negative effects were reported (Bengtsson et al. 2005;Bruggisser et al. 2010;Ostandie et al. 2021;Paiola et al. 2020). In organic viticulture, non-synthetic compounds (mainly copper and sulfur) are used instead of synthetic chemicals, but they can have strong effects on non-target organisms, too (Biondi et al. 2012;Nash et al. 2010;Vogelweith and Thiéry 2018). Möth et al. (2021) found higher toxicity levels in organic vineyards, where high concentrations of copper accumulate in the soil (Mackie et al. 2012). The effects of management and fungicide reduction observed in our study could have been weakened by pesticide drift from neighboring fields (Druart et al. 2011), because vineyards of our region are often small and adjacent vineyards are frequently managed differently. However, the observed differences in species abundances and species composition confirm that our study design was suitable to detect local management effects on Orthoptera. Vineyard management influenced species and taxonomic groups differently, and thus, may affect species interactions (Caprio et al. 2015;Ostandie et al. 2021;Pedneault and Provost 2016;Vogelweith and Thiéry 2018). Further research is needed to determine the extent to which species relevant to nature conservation or Table 2 Model-averaging results of the top-ranked models for total densities of vine-dwelling Orthoptera and the three most common species in vineyards with different management (conventional, organic) and grape varieties (classic, fungus-resistant). Indicated are the standardized coefficient (Coeff.), adjusted standard error (SE), z-score (z), and P-value (p) for management, grape variety, their interaction (Man:Var), semi-natural habitats (SNH), and vegetation cover and height. Significant P-values are in bold, P-values with a trend are in italics Predictor

Leptophyes punctatissima
Coeff.  Table 3 Effect of vineyard management (conventional, organic), grape varieties (classic, fungus-resistant), vegetation height and cover, and the proportion of semi-natural habitats in the surrounding landscape on the species composition of Orthoptera in vineyards analyzed using redundancy analysis on presence-absence data. Indicated are the F-value (F) and P-value (p  . 3 Densities (mean ± SE and raw data points) of vine-dwelling Orthoptera (total and the three most common species) for classic and fungusresistant grape (FRG) varieties under conventional and organic management in the soil (Ingrisch and Köhler 1998), the eggs and hatching larvae, respectively, may be comparatively affected by accumulating copper in the topsoil (Karimi et al. 2021), which could explain the stronger negative effect. Furthermore, orthopterans can be exposed to copper through surface contact and their feeding behavior (Ingrisch and Köhler 1998). In a microcosm experiment, however, Karimi et al. (2021) have determined that the effects of copper on soil biodiversity are only measurable at annual concentrations far above those authorized by the European commission, while in toxicity tests, Duque et al. (2023) found that beneficial insects are harmed or benefited by conversion to organic viticulture. Herb-dwelling orthopterans were, in accordance with our second hypothesis, more common in FRG varieties with fewer pesticide applications compared to classic grape varieties. However, a positive effect of reduced pesticide application, with the exception of P. parallelus, was only present in organically managed vineyards, where copper and sulfur are used for plant protection. Since both the herb-dwelling Orthoptera and T. viridissima, which was affected by organic management as we discussed before, lay their eggs  Table 3 could benefit from increased pesticide use if their predators or competitors are affected negatively. Further research would be needed to substantiate such possible indirect effects. Regardless of the exact mechanisms, our results highlight how the use of pesticides can affect species abundance differently and how these differential effects can alter species composition.
While a strong positive effect of landscape heterogeneity on biodiversity has been reported in various studies (Barbaro et al. 2021;Kolb et al. 2020;Martin et al. 2019;Schmidt et al. 2005), the influence of landscape is comparatively low in our study. One reason for this rather low positive effect on Orthoptera could be that orthopterans tend to be sedentary species with a small range of action and the entire life cycle of the observed herb-and vine-dwelling species can take place in the vineyards or the immediate surroundings (Detzel 1998;Ingrisch and Köhler 1998). Only the species L. punctatissima occurs more frequently with a higher proportion of SNH. L. punctatissima prefers forest edges and is dependent on woody structures with adjacent grass and herbaceous areas (Detzel 1998;Schlumprecht 2003), so a higher abundance is expected here, as a higher proportion of SNH in our study is often associated with a higher proportion of forest in the surrounding area and thus a shorter distance to forest edges (Fig. S1).
Biodiversity is usually influenced by local practices besides pesticide use, such as tillage and cover crop management (Blaise et al. 2022;Ostandie et al. 2021;Reiff et al. 2021b). In line with this, we found that vegetation positively affected C. brunneus, which became more abundant with increasing vegetation height. Additionally, the total density of vine-dwelling Orthoptera tended to increase with higher ground vegetation cover, possibly being influenced by the inter-row vegetation during larval stages that live in the herb layer (Detzel 1998). Increased densities of Orthoptera with increasing vegetation height and vegetation cover might be linked to higher food supply, suitable microclimate, and the provision of hiding places. Further, more intensive tillage may harm egg pods development (Detzel 1998). Adapted management of vineyard interrows, such as grazing or less intensive mowing and tillage, can thus be another effective measure to promote the biodiversity of herb-dwelling species, in addition to the measures previously discussed (Blaise et al. 2022;Bosco et al. 2022;Detzel 1998).

Conclusion
According to our results, organic viticulture had no general positive effect on Orthoptera. Rather, reducing the number of pesticide applications and associated tractor passages, at least in organic viticulture, is important and shows a clear benefit to some orthopterans. Effects of the proportion of semi-natural habitats in the surrounding landscape on Orthoptera in concentrations also found in regional vineyards could have lethal effects on earthworms. However, Karimi et al. (2021) also point out that there is still a lack of field experiments that provide information on community dynamics under in situ conditions. In addition to the negative effects of copper, the use of dusting sulfur, which is used particularly in organic viticulture as fungicide but acts as a broad-spectrum pesticide, may also negatively affect non-target organisms in the inter-rows, potentially leading to a positive effect of reduced plant protection on herb-dwelling orthopterans. A negative effect of sulfur on non-target organisms such as parasitoids, predatory thrips and mites, and grapevine moths has been shown in earlier studies (Hanna et al. 1997;Jepsen et al. 2007;Tacoli et al. 2020). In addition to negative effects of pesticides in organic and conventional viticulture, the fact that P. parallelus is similarly affected by the cultivation of FRG varieties in both organic and conventional management may be also due to a lower overall workload in those vineyards. A reduced number of pesticide applications leads to less tractor traffic, resulting in less disturbance of the ground vegetation and reduced soil compaction likely enhancing orthopterans (Bruggisser et al. 2010). This could be particularly relevant in flightless species such as P. parallelus. Less intensive tillage may also be beneficial for egg pods development (Detzel 1998).
Vine-dwelling Orthoptera had similar densities in organic managed vineyards, but tended to be 1.4 times more abundant in classic than in FRG varieties in conventional managed vineyards. However, the total density of vine-dwelling Orthoptera was driven by high densities of P. falcata. T. viridissima seems to be more affected by pesticides used under organic management, where it occurs generally less common compared to conventional management. This may be because the larval stage lives in the herb layer (Detzel 1998), where it may come into contact with copper more intensively. Although copper also accumulates in the leaves of grapevines throughout the season (Angelova et al. 1999), species appear to be less affected here. One reason for this could be that copper on leaves is washed off by precipitation (Angelova et al. 1999). Furthermore, the use of sulfur as a widely used pesticide in organic viticulture may play a role here, too. Thus, reducing high toxicity levels in organic viticulture by cultivating FRG varieties can have a particularly large effect on promoting such affected species and may be a promising approach to reduce the pressure of intensified agriculture on biodiversity, as it was also reported to have positive effects on mites, spiders, and certain insects (Pennington et al. 2017(Pennington et al. , 2019Reiff et al. 2021aReiff et al. , 2023Kaczmarek et al. 2023). Furthermore, the trend for higher densities of vine-dwelling Orthoptera in classic grape varieties compared to FRG varieties in conventionally managed vineyards, driven by P. falcata, could possibly be explained by changes in species composition. For example, vine-dwelling Orthoptera vineyards were weak, while other studies showed strong positive effects for other taxonomic groups of insects as well as for spiders and birds. We conclude that under organic viticulture, reducing the use of non-specific pesticides is important to promote biodiversity and that the cultivation of fungus-resistant grape varieties can have a significant part to this progress.