Which temperature matters? Effects of origin, rearing and test conditions on the chemical sensitivity of Pardosa amentata

Spiders may be adversely affected by pesticides, yet they are not included in regulatory risk assessment and a related standard guideline to test their sensitivity to chemicals is lacking. Different laboratory setups, including test temperature and relative humidity, have been shown to influence the sensitivity of spiders. The climate from which spiders originate and the rearing conditions in the laboratory prior to ecotoxicological testing may also alter their sensitivity to chemicals, potentially in interaction with test conditions. We investigated the influence of population origin, rearing and test temperature on the chemical sensitivity of the spider Pardosa amentata towards lambda‐cyhalothrin. We collected female P. amentata carrying egg sacs from two climates, i.e., boreal and cool temperate. Spiders were kept in the laboratory and their offspring were reared and tested at 15, 20 and 25°C. Hatching of egg sacs largely failed at 15°C, while a moderate spiderling mortality (40%) was recorded at 20°C. At 25°C, mortality increased (63%) and a faster developmental rate was observed. Rearing and test temperature had no significant effects on spider chemical sensitivity. However, spider chemical sensitivity differed between populations, with spiders from boreal climate being 38% more sensitive than spiders from cool temperate climate. A higher sensitivity towards lambda‐cyhalothrin increases the risk of population reduction in treated areas, with potential alterations of ecosystem functions such as biological control. Our results suggest that the climatic origin of test organisms deserves stronger attention in ecotoxicological research.

As a consequence, areas treated with pyrethroids exhibited reduced abundance and diversity of spiders and other natural enemies (Fritz et al., 2013;Rodrigues et al., 2013).This may translate into a reduction of their capacity for biological control (e.g., Hanna & Hanna, 2013;Tahir et al., 2019), an important ecological function provided by spiders (Michalko et al., 2019).
Despite the known adverse effects of pesticides, spiders have received little attention in ecotoxicology compared to other nontarget arthropods (EFSA Panel on Plant Protection Products and their Residues, 2015;Pekár, 2012).Moreover, spiders are not routinely included as test organisms for the risk assessment of pesticides in Europe (European Commission, 2013a, 2013b).Although methods for testing pesticides on spiders from the family Linyphiidae (Aukema et al., 1990) and the genus Pardosa (Wehling et al., 1998) have been proposed, so far, no standard protocol applicable to all spider taxa has been accepted for regulatory risk assessment.Hence, the available spider ecotoxicological data have been derived from many different test designs (Pekár, 2012), mainly focusing on the lethal and sublethal effects of field doses or pesticide residues.Spider pesticide sensitivity varies between different test designs, and this variation has been mainly attributed to the abiotic parameters from the test setup (Jagers Op Akkerhuis et al., 1997).In particular, higher spider mortality is expected at low test temperatures and low relative humidity (Everts et al., 1991).We hope that the current research will provide valuable information for the potential development of a standardised protocol to test the chemical sensitivity of spiders, which could improve the reproducibility and comparability of ecotoxicological studies.
Apart from test temperature, the climatic origin and rearing conditions may also influence the pesticide sensitivity of spiders.For example, climatic factors influence the cuticular structure and composition of arthropods.Spiders from warm and dry ecosystems are expected to have a stronger cuticle to resist desiccation (Sprenger et al., 2018), which could translate to a reduced uptake and higher pesticide tolerance.In a multi-species comparison, spiders from boreal climates were indeed more sensitive to lambda-cyhalothrin than spiders from warmer climates (Duque et al., 2023).Moreover, when comparing the chemical sensitivity between spiders of different origins, differences may arise because standard test or rearing temperatures are differently amenable for different species.
For example, a test or rearing temperature of 25°C could represent heat stress for a spider adapted to cold environments, while it may represent the optimum temperature for spiders from a warm temperate climate.Although the relationship between spider pesticide sensitivity and the test temperature has been previously evaluated (Everts et al., 1991;Michalko & Košulič, 2016), information about potential interactions of test temperature, rearing temperature and climatic background on spider sensitivity is lacking.
The aim of the present study was to investigate the influence of climatic origin, rearing and test temperature on the chemical sensitivity of a spider species.We used the wolf spider P. amentata (Clerck, 1757) as a test organism, which is sensitive to pyrethroids (Baatrup & Bayley, 1993;Shaw et al., 2006).P. amentata is common and widely distributed in Europe, preferring damp habitats in open areas (Nentwig et al., 2023).It is common in agricultural field margins where pesticide application may directly affect it.We collected 81 females of P. amentata carrying egg sacs in two European climates: 36 in boreal and 45 in cool temperate, respectively.Spiders were reared in the laboratory at three different temperatures (rearing; 15, 20 and 25°C) and their spiderlings were used for ecotoxicological assessment using the pyrethroid insecticide lambda-cyhalothrin.
To derive median lethal concentrations (LC 50 ), we conducted 24-h acute tests in a crossed-treatment design with test temperatures of 15, 20 and 25°C.We addressed the following research questions: (1) Are spiders originating from boreal climate more sensitive to lambda-cyhalothrin than spiders from cool temperate climate?(2) or Switzerland (Milano et al., 2021).Collected spiders were preidentified visually in the field (Roberts, 1995) and placed individually into a glass jar (35 mL, 44 mm Ø × 42.5 mm height) with a layer of moistened plaster for transport to the laboratory (iES Landau, Germany).

| Rearing Pardosa amentata under laboratory conditions
In the laboratory, P. amentata females were transferred individually into polypropylene boxes (1 L, 18 cm length × 13.2 cm width × 6.8 cm height) with a layer (~1 cm) of moistened plaster.To maintain a polytypic diet (Uetz et al., 1992), spiders were fed ad libitum twice per week (Figure 1b) with a mixture of fruit flies (Drosophila hydei and D. melanogaster; b.t.b.e. Insektenzucht, Bad Wörishofen, Germany) and springtails (Coecobrya tenebricosa, MyAnts.de,Weiden, Germany).Spiders were kept avoiding direct light exposure in three climate chambers at constant relative humidity (100%), light/dark cycle (16/8 h), and illuminance (300 lux), but with different temperatures.One-third of the spiders (~12; Table 1) collected from each location were bred at 15°C (Treatment L; Figure 1c), the second third at 20°C (Treatment M; Figure 1c) and the remaining females at 25°C (Treatment H; Figure 1c) until the juveniles hatched.During the rearing period, we recorded female mortality, the number of egg sacs hatched, the number of hatched spiderlings and the survival of spiderlings (Table 1).
Once the spiderlings hatched, they were kept with their mother in the container and more springtails were offered as food.
Approximately 1 week after, when spiderlings became independent, their second instar after being independent (Table 1).The rearing at 15°C (Treatment L, Figure 1c) resulted in low hatching success, for boreal and cool temperate origin, and was omitted from further analysis.Each mother spider was preserved in 70% ethanol and identified to species level to confirm the field identification using Nentwig et al., 2023.

| Ecotoxicological assessment
To assess the influence of climatic origin, rearing and test temperatures on the sensitivity of P. amentata, we performed a fullfactorial experiment.For spiders from both climatic origins, boreal and cool, spiderlings reared at 20 and 25°C were tested at 15, 20 and 25°C (Table 2).This resulted in a full 2 × 2 × 3 factorial design with 12 temperature combinations.For each temperature combination, we performed a 24-h acute exposure toxicity test for spiders as in Aukema et al. (1990), with modifications to allow for dose-response calculations.Moreover, every test was performed with six different concentrations of lambda-cyhalothrin plus a blank control, each one with five replicates, i.e., five spiderlings.
Two days before the ecotoxicological assessment, at least 105 spiderlings reared at the same temperature and originating from the same climate, were transferred into glass jars with moistened plaster, but without food (Pekár, 1999).During this time, spiders were stored at the same temperature treatments in which they had developed.1e), and petri dishes were closed and placed in the respective climate chamber to have a crossed treatment (Figure 1f).P. amentata survival was visually assessed after 24 h of the application.Spiderlings were classified as alive, dead or paralysed (Baatrup & Bayley, 1993).

| Chemical sensitivity
Since rearing at 15°C largely failed (Table 1), only the tests for the rearing treatments at 20 and 25°C were performed (Table 2).Spider LC 50 s ranged from 7.06 to 16.81 ng a.i./cm 2 for spiders collected in the cool temperate climate (Table 2; Figures S1-S6), and from 4.61 to 8.77 ng a.i./cm 2 for the boreal climate (Table 2; Figures S7-S12).For both climatic zones, the treatment of spiderlings reared at 20°C with a test temperature of 25°C resulted in the most sensitive endpoints (Table 2).However, neither rearing temperatures nor test temperatures or any of their interactions had a significant effect on spider chemical sensitivity (Table 3, Figure 2b,c).P. amentata chemical sensitivity differed exclusively between the climatic zones (F (1,4) = 9.96, p = 0.03; Table 3).The LC 50 s of spiderlings from the boreal climate were on average 38% lower than those originating from the cool temperate climate (Figure 2a).

| Chemical sensitivity
Our results suggest that the climatic origin of P. amentata influenced their sensitivity to lambda-cyhalothrin, independent of rearing or test temperatures.The higher sensitivity of the population from the boreal climate is in accordance with a generally higher sensitivity of multiple spider species from boreal and polar climates when compared to spiders from a temperate climate after exposure to the same insecticide (Duque et al., 2023).
Nevertheless, the high sensitivity of both populations of P. amen-  below the recommended application dose of lambda-cyhalothrin (i.e., 75 ng a.i./cm 2 ), indicates a high potential risk of Indeed, a reduction of spider abundance in fields treated with this insecticide has been reported by Rodrigues et al. (2013).As a consequence, ecosystem functions such as biocontrol may be reduced (Hanna & Hanna, 2013;Tahir et al., 2019), an important function provided by spiders, especially free hunters, such as P. amentata (Michalko et al., 2019).
The differences in chemical sensitivity between spiders from different climates may be explained by differences in biological traits related to climate adaptation, such as metabolism or desiccation resistance.To prevent desiccation, arthropods living in dry or warm habitats can adapt the chemical composition of their cuticular hydrocarbons (Sprenger et al., 2018).This adaptation may also reduce pesticide uptake when direct contact is the main exposure route.
Moreover, a waterproofing cuticle may reduce pesticide effects, as water depletion appears to contribute to the mortality of spiders exposed to pyrethroids (Jagers Op Akkerhuis et al., 1997).This mechanism could explain sensitivity differences in the direction observed in the current study.In addition, reduced chemical sensitivity in warmer climates may reflect higher tolerance to natural plant secondary metabolites that are ingested e.g., with herbivore prey.
While relationships of metabolite concentrations with temperature are variable, higher concentrations at warmer temperatures seem to prevail (Pant et al., 2021;Yang et al., 2018).This could lead to a higher natural exposure of spiders in warmer climates to plant secondary compounds.
Lastly, the adaptation of spiders to lower environmental temperatures in a boreal climate may influence their internal response to lambda-cyhalothrin.Such adaptation can be related to a higher nerve sensitivity and changes in the receptor binding, as has been documented in other arthropods (Ahn et al., 1987).Information on the internal effect of pesticides on populations from different habitats may help to understand the climate effect observed in our study.
This would require toxicogenomic (Hamadeh et al., 2002) studies analysing other ecotoxicological endpoints, such as the activity of detoxification enzymes (Zhou et al., 2019), the binding of active ingredients to target and non-target receptors (Narahashi et al., 2007), and gene expression (Giambò et al., 2021).
As each of the two climatic origins was only represented by one location, we cannot exclude that other factors than climate contributed to the observed differences.For example, it could be expected that pesticides were more common in the temperate climate, since no crops are grown at the altitude where the boreal population was sampled (Ding et al., 2023).We tried to avoid confounding effects of background pollution and possible adaptation by sampling the temperate climate population in a location with no agriculture within at least a 3 km radius.Furthermore, a study comparing the chemical sensitivities of 28 spider species found no indication that species from agricultural areas (e.g., Pardosa agrestis) were more sensitive than related species from more natural areas (Duque et al., 2023).Also, in this multi-species comparison that comprised 25 sampling locations, spiders from boreal to arctic climates were more sensitive to lambda-cyhalothrin than spiders from warmer climates.Thus, we assume that climate is the most likely explanation for the higher sensitivity of spiders from colder climate also in the current experiment.In contrast to our results, linyphiid spider mortality from pyrethroids increased from 30°C towards 10°C test temperature at high air humidity (Everts et al., 1991).In addition, the mortality of Philodromus when exposed to lambda-cyhalothrin was higher at 31°C in comparison with lower test temperatures (Michalko & Košulič, 2016).Previous research suggests that the range of temperatures that we tested (15-25°C) may have been too small to detect test temperature effects on spider sensitivity.The elevated spider mortality at high temperatures, i.e., >30°C, observed by Everts et al. (1991) and Michalko and Košulič (2016) was beyond the conditions tested in our study.Nevertheless, our results indicate that rearing and test conditions have no major effect on chemical sensitivity, as long as the conditions used are favourable for spider reproduction.

| Rearing success
Surprisingly, the rearing temperature of 15°C was too low for hatching, even for spiders from boreal climate, which are expected to be better adapted to lower temperatures.At 15°C, the majority of the spiders lost their egg sacs and spiderlings did not hatch (Table 1).
In the field, P. amentata are known to reproduce at mean air temperatures during the day between 17 and 23°C (Nyffeler, 2000).
However, we observed the highest hatching and developmental success at 25°C, indicating a shift of optimal rearing conditions to higher temperatures compared to those observed in the field.Given their diurnal activity and occurrence in open habitats, it is likely that P. amentata females can heat their bodies and egg sacs above air temperatures in their natural environment using microhabitat selection, e.g., through sun basking or by staying on warmed-up surfaces.Thus, the poor reproduction at 15°C in the lab may be due to low light intensities and a lack of microclimatic heterogeneity available to the spiders in the climate chambers.Body temperature has a strong influence on arthropod metabolism and development (Mirhosseini et al., 2017).Specifically for spiders, a fast development at higher temperatures is an indicator of warm season adaptation (Li & Jackson, 1996), wherein P. amentata reproduction takes place (Vlijm et al., 1963).
The spiderling mortality observed during the rearing period (Table 1) can be partially explained by the monotypic diet of springtails offered during the rearing.P. amentata is a generalist predator, i.e., left their mother's back, they were transferred for individual rearing into 35 mL glass jars (44 mm Ø × 42.5 mm height) with moistened plaster and fed ad libitum with springtails twice per week.The temperature treatment of the mothers was maintained for rearing F I G U R E 1 Scheme of experimental procedure for one climate (cool temperate or boreal).(a) Sampling of Pardosa amentata females with egg sac.(b) Feeding of spiders in the laboratory and (c) temperature treatment in the climate chambers at 25°C (H), 20°C (M) or 15°C (L) until hatching of the spiderlings.(d) Separation of spiderlings and rearing at the same temperatures.(e) Pesticide application and (f) ecotoxicological assessment.[Colour figure can be viewed at wileyonlinelibrary.com] the spiderlings (Treatment L, M or H; Figure until they reached

F I G U R E 2
Chemical sensitivity of spiders towards lambdacyhalothrin expressed as median lethal concentration: Effects of (a) climate of origin, (b) rearing temperature and (c) test temperature.Different letters show significant differences (p < 0.05).LC 50 = median lethal concentration; a.i.= active ingredient.

2 | MATERIAL S AND ME THODS 2.1 | Source of test organisms
Development of Pardosa amentata under laboratory conditions (mean values with ± standard deviation).

captured Climate of origin Rearing temperature (°C) Female mortality (%) Egg sacs hatched (%) Spiderlings hatched a Spiderlings mortality (%) Time to reach 2nd instar (days)
a Average spiderlings hatched per egg sac.collection at cool temperate climate was done in late May 2022 at the Eußerthal Ecosystem Research Station in the Palatinate Forest in Germany (EERES; 49°15′16.4″N, 7°57′42.4″E).P. amentata is not a threatened species or under special protection in Germany (Rodrigues et al., 2013)previously been performed around the concentration of 75 ng a.i./cm 2(Duque et al., 2023), which has been reported to decrease spider abundance in fields(Rodrigues et al., 2013).On the test day, spiderlings were weighed to the nearest 0.1 mg (PA214® 210 g/0.0001 g, OHAUS, New Jersey, USA) and only spiders with a similar weight (1 ± 0.3 mg; Table2) were used in the test.Thirty-five spiderlings were used for each test The insecticide lambda-cyhalothrin (Hunter® EG, CERTIS Europe, Hamburg, Germany, 5% active ingredient [a.i.]) was used for acute exposure testing.The product was weighed to the nearest 0.01 mg (AT261 DeltaRange® 205 g/0.01 mg, Metler Toledo, Columbus, Ohio, USA), diluted in ultra-pure water, and stock solutions were created using serial dilutions.Stock solutions were homogenized with magnetic stirrers for 10 min at room temperature.
total, 81 spiders with egg sacs were collected, 36 in cool temperate and 45 in boreal climate.At the rearing temperature of 15°C, no hatching was observed for spiders collected in cool temperate climate, whereas two out of fifteen egg sacs hatched for the boreal spiders (Table1).All egg sacs hatched at 25°C, while 58 and 73% of egg sacs from spiders collected in cool temperate and boreal cli- mate, respectively, hatched at 20°C.A moderate spiderling mortality (~40%) was recorded at 20°C, while mortality increased to ~63% at 25°C.Spiderlings reached their 2nd instar 26 days after hatching when reared at 20°C, and 22 days after hatching when reared at 25°C (Table Effects of climatic origin, rearing and test temperature on spider chemical sensitivity. to lambda-cyhalothrin which is reflected in LC 50 values far TA B L E 3 Note: Statistically significant effects (p < 0.05) are printed in bold.a Interaction term for factors are represented by ":".