Influence of repeated pyrethroid applications on the sensitivity of pyrethroid-resistant pollen beetles (Meligethes aeneus F.) and their offspring

Einfluss wiederholter Pyrethroid-Behandlung auf die Sensitivität des Rapsglanzkäfers (Meligethes aeneus F.) und dessen Nachkommen

Materials and Methods

Fig. 1. Layout of the field trial in Hötzum; B1 and B2 = treated with Karate^® Zeon, K1 and K2 = untreated.

Skizze des Versuchsschlages in Hötzum; B1 und B2 = behandelt mit Karate^® Zeon, K1 und K2 = unbehandelt.

Living pollen beetles were sampled regularly for the sensitivity tests in the laboratory. They were collected along two tramlines in the plot centre until a sufficient high number (at least 300 beetles per plot) for the resistance tests was sampled by beating inflorescences into a funnel with a perforated plastic bag attached. By this it was ensured that sampling areas in treated and control plots were in at least 14 m distance to differently treated plots which is more than 2 m used in EPPO Standards (EPPO, 2004). Beetles of the overwintered generation were collected on eight occasions in treated and untreated plots a few days before and after each insecticide application (Tab. 1). Before testing the beetles were kept in perforated plastic bags in a climatic chamber at 10°C and 16:8-hours L:D for a maximum of one week. They were provided with water and oilseed rape inflorescences from the untreated headlands as food.

To catch the emerging new beetle generation eight photoeclectors (each covering 0.25 m² of ground) were established on 23 March in the sampling area of each plot (four near to each tramline). The digging of the soil rings of the photoeclectors into the soil was carried out very carefully not to disturb the oilseed rape plants within and outside the photoeclector area. They were set up at a distance of 35 m to each other and approximately 0.5 to 1 m apart from the tramline.

Additionally eight plastic containers (17 cm × 12.5 cm) filled with a 10% sodium benzoate water solution for conservation were placed on 24 April close to each photoeclector in all plots to collect the larvae dropping off the plants. The containers were emptied weekly from 3 May to 23 May and the larvae were counted. The mortality of larvae and pupae in the soil was inferred by relating the number of emerged beetles to the number of captured larvae.

The photoeclectors were closed by a fabric tent on 16 May (BBCH 69 (Weber and Bleiholder, 1990; Lanca­shire et al., 1991)), before emergence of the new beetle generation started. To catch the beetles alive, a perforated plastic bag was fixed to the opening on top of the photo­eclectors. The plastic bags were checked once a week in May and from the beginning of June every three to four days. First new generation beetles were collected on 14 June in the bags of the photoeclectors. After this the bags were emptied every two to three days. The beetles were maintained alive in a climatic chamber, similar to the overwintered adults. They were supplied with pollen as food. Beetles trapped during the first sampling period (14 June until 18 June) and the last sampling period (18 June until 25 June) were pooled within each variant to obtain sufficient beetles for the sensitivity test.

To compare calculated curve progressions of the dose-response relationships of two populations, the hypotheses of equality and parallelism of the curves were tested by using the program POLO PLUS 2.0. If the hypotheses are rejected, a significant difference between the sensitivity of the two populations compared exists. Further, if the confidence intervals do not overlap, the populations differ significantly.

Based on the mortality of overwintered pollen beetles in the Adult-Vial-Test, 81.25% of samples were classified as highly resistant (according to Heimbach and Müller, 2013). This classification means that more than 50% of beetles survived at 0.075 μg lambda-cyhalothrin cm^–2 or at higher doses. The remaining 18.75% of the samples were classified as resistant (11–50% survival at 0.075 μg lambda-cyhalothrin cm^–2 or at higher dose). Similarly, 75% of the new beetle generation was classified as highly resistant and 25% as resistant. These results were confirmed by the LD₅₀-values (Tab. 3).

Fig. 2. LD₅₀-values (μg lamb­da-cyhalothrin cm^–2) and 95% confidence in­tervals of overwintered pollen beetles collected at eight sampling dates in untreated plots K1 and K2 and lambda-cy­halothrin treated plots B1 and B2, assessment after 5 hours; # = calcu­lation of LD₅₀ not pos­sible.

LD₅₀-Werte (μg lamb­da-cyhalothrin cm^–2) und 95% Konfidenzin­tervalle der überwinter­ten Rapsglanzkäfer, die an acht Terminen in den unbehandelten Kon­trollparzellen K1 und K2 und den mit lambda-cyhalothrin behandelten Parzellen B1 und B2 ge­sammelt wurden, Boni­tur nach 5 Stunden; # = Berechnung des LD₅₀-Wertes nicht möglich.

The highest LD₅₀-values of beetles were found in three of the four plots at the first sampling date. At this time favourable weather conditions with a maximum daily temperature of around 20°C encouraged the main immigration of beetles. This was confirmed by yellow water trap samples. The lowest LD₅₀-values were determined in the plots K1 and K2 on the second sampling date (Fig. 2). In plot K1, the sensitivity of beetles differed significantly between the second and the third sampling date, between the fifth and the sixth sampling date and between the sixth and the seventh sampling date. Similar results were obtained from plot K2. As an example, the dose-response relationships of pollen beetles collected on the fifth (24 April) and the sixth sampling date (30 April) were compared in plots K1 and K2 (Fig. 3). Based on the analysis of equality and parallelism of the curves the hypothesis of equality was rejected. Consequently, a significant difference between the sensitivity of the two populations compared exists (Tab. 4).

Fig. 3. Dose-response relationships of lambda-cyhalothrin for overwintered pollen beetles collected on 24 April and 30 April in plots K1 and K2 in Hötzum, assessment after 5 hours.

Dosis-Wirkungsbeziehung von lambda-cyhalothrin von überwinterten Rapsglanzkäfer, die am 24. April und 30. April in den Parzellen K1 und K2 in Hötzum gesammelt wurden, Bonitur nach 5 Stunden.

In contrast to the untreated plots K1 and K2, analyses of the LD₅₀-values of treated plots B1 and B2 showed no significant differences between individual sampling dates (Fig. 2).

Fig. 4. LD₅₀-values (μg lambda-cyhalothrin cm^–2) and 95% confidence intervals of overwintered pollen beetle samples collected on 24 April and of the new pollen beetle generation caught in photo­­eclectors from 21–25 June in a field trial in Hötz­um.

LD₅₀-Werte (μg lambda-cyhalothrin cm^–2) und 95% Konfidenzintervalle von überwinterten Raps­glanzkäfer, die am 24. April gesammelt wurden so­wie der Jungkäfer, die vom 21.–25. Juni mit Photoeklektoren im Feld­versuch in Hötzum gefan­gen wurden.

The first pollen beetles of the new generation were trapped in photoeclectors on 14 June. In plot K2 the higher number of larvae dropping was parallel to a higher number of emerging beetles (Fig. 5, Tab. 5). During the first sampling period from 14 to 18 June approximately 380 beetles per m² emerged from plot K2, while numbers of beetles emerging from the other plots varied between 270 and 280 beetles per m². During the sampling period from 19 to 25 June, the number of beetles caught in untreated plots was higher (355 and 365 beetles per m² in plot K1 and K2, respectively) than in treated plots (300 and 315 beetles per m² in plot B1 and B2, respectively). When the number of adults of the new beetle generation was related to the number of larvae, mean mortality of larvae and pupae in soil was 80.4% and 79.7% in the untreated and treated plots, respectively (Tab. 5). In the plots treated five times with lambda-cyhalothrin, the number of larvae dropping to the soil for pupation was about 18% lower than in the untreated plots. Similar differences were observed for the emerging new beetle generation (15% less in treated plots).

Fig. 5. Number of the new pollen beetle generation per m² emerging from treated and untreated plots from 14–18 June (white) and from 19–25 June (black); no standard deviation because pollen beetles emer­ging within each plot were pooled.

Anzahl der im Zeitraum vom 14.–18. Juni (weiß) und vom 19.–25. Juni (schwarz) in den behandel­ten und unbehandelten Parzellen geschlüpften Jungkäfer pro m²; keine Angabe der Standardab­weichung, da die in den Parzellen geschlüpften Rapsglanzkäfer gepoolt wurden.

A high within-season temporal variability in the sensitivity of overwintered pollen beetles to lambda-cyhalothrin was also detected in samples of beetles collected from another field trial located 10 km away from Hötzum in Wendhausen in 2012 (data not reported here). Further, Heimbach and Müller (2010) reported a temporal variability in the sensitivity of pollen beetles within one season in fields of oilseed rape near Schwerin. This variability over time might be a result of the continuous migration of beetles between differently treated plots within the experimental field as well as of immigration or emigration of beetles between fields.

In addition, sensitivity of individual pollen beetles may change with age, physiological condition and reproductive status. Egg formation and egg laying might have affected the fitness and sensitivity of females. Thus the low LD₅₀-value observed on the second sampling date in all plots at Hötzum may be connected with the oviposition period. The first eggs were observed in the Adult-Vial-Test from beetles collected on 10 April. Therefore, the initiation of oviposition in beetles collected on 2 April might have increased their sensitivity. Similarly, sensi­tivity of males might increase because of their mating activity. Some reports indicate a higher susceptibility of male insects to pesticides than of females, e.g. Pielou and Glasser (1952), Respicio and Forgash (1984). Unfortunately, the sensitivity of male and female beetles was not checked separately in our experiments.

In addition, the amount and quality of food ingested by the beetles has a strong influence on the physiological condition, and might have affected fitness and sensitivity. A higher sensitivity of worker honey bees to pesticides fed with qualitatively or quantitatively inadequate pollen has been reported by Wahl and Ulm (1983).

The differences between the sensitivity of pollen beetles of treated and untreated plots probably may have been caused by repeated applications of lambda-cyhalothrin in the treated plots B1 and B2. Beetles were selected for low sensitivity to this insecticide in treated plots, whereas in control plots sensitive beetles survived resulting in lower LD₅₀-values. But it should be taken into account that the actively flying pollen beetles could easily move between adjacent treated and untreated plots, even at the large plot size of 1.5 ha used in our experiments. Stechmann and Schütte (1976) showed that pollen beetles are able to fly mean distances of 1 to 3 kilometers per day. Therefore beetles immigrating from surrounding fields might have mixed with beetles present in the plots and affected the sensitivity of the population as well.

Beetles of the new generation emerging in June turned out to be more sensitive to lambda-cyhalothrin than overwintered beetles. The LD₅₀-values of overwintered and new generation beetles differed significantly. The period from the oviposition of overwintered beetles to the hatching of new generation beetles depends on weather conditions and usually lasts seven to eight weeks (Friederichs, 1921); thus the overwintered beetle population tested by the Adult-Vial-Test might have served as parent population of the new generation.

After emergence the new beetle generation was not exposed to resistance selection by pyrethroid applications. Further, unlike the overwintered beetles, they had not been selected for high fitness over a period of several months by environmental toxins or other stress factors such as harsh winter conditions. Different sensitivity of honey bees in the age of two to eight days towards DDT has been reported by Graves and Mackensen (1965). In their experiments the age-dependent LD₅₀-values of young honey bees differed up to three times, with two day old bees being most sensitive. These findings were confirmed by Ladas (1972). In studies comparing the sensitivity of newly emerged adults and more than one year old adults of the Carabid beetle Harpalus aeneus to the organophosphorus pesticide thionazin, mature beetles were found to be less susceptible than newly emerged beetles (Critchley, 1972). These findings were explained by the higher migration activity of the younger individuals. As consequence of their high activity they may have taken up a higher dosage of the pesticide.

Another reason for the higher sensitivity of the new generation of pollen beetles might be that they were introduced into the Adult-Vial-Test shortly after emergence, after only one to five days of feeding possibilities before the test. Consequently they could accumulate only small fat reserves which might have increased the sensitivity to the insecticide. A similar effect has been reported by Frey (1950) who found that young pollen beetles were more resistant to the insecticide powder “Kümex” (a.i. rotenone) when they had developed a pronounced fat body. In our experiments the average fresh weight of overwintered beetles and new generation beetles showed only little difference (3% lower weight of the new beetle generation) but their fat bodies might have differed to a larger extend.

The extensive application of five pyrethroid sprays during the developmental period of pollen beetle eggs and larvae showed only a negligible effect on the number of new generation beetles in our experiment. In total the number of both the mature larvae dropping to the soil and the emerging beetles was only slightly higher in untreated plots than in treated plots. The reason for the lower number of new generation beetles in treated plots might be a slightly reduced abundance of overwintered beetles in treated plots due to the insecticide sprays, which was not exactly measured in the experiment, or might be caused by sublethal effects of the insecticide applications on oviposition and larval development. Insecticidal effects on the larvae seem unlikely because they showed a high level of resistance to lambda-cyhalothrin in our laboratory experiments performed with glass fiber filters coated with different dosages of this active. Three populations of larvae from two locations showed LD₅₀-values between 0.22 and 0.44 μg cm^–2 (details not reported here). In addition oviposition might have been reduced by a repellent effect of the applied pyrethroid. It is well known that pyrethroids have a repellent effect on aphids and can prevent probing or colonization on plants (Rieckmann and Wüstefeld, 2006; Thieme et al., 2009). Repellency might also have affected the behaviour of pollen beetles by avoiding the treated plots B1 and B2, resulting in fewer eggs and consequently in lower numbers of larvae and new generation beetles.

The mean mortality of larvae and pupae from dropping to the soil to hatching of new generation beetles in treated and untreated plots was on a high level (on average 80%). Parasitism of larvae was observed but not analyzed in detail at Hötzum. Parasitoids such as Tersilochus heterocerus (Thomson), Phradis morionellus (Holmgren) and P. interstitialis (Thomson) are known as important mortality factors of larvae (Nilsson, 1988b; Nilsson and Andreasson, 1994; Ulber et al., 2010). The application of insecticides during flowering of oilseed rape can also affect para­sitization (Nitzsche, 1998). Consequently, the insecticide application in treated plots may have reduced parasitoid numbers, leading to a higher number of surviving larvae and new generation beetles. However, in this study the number of larvae and emerging new generation beetles was higher in untreated plots compared to treated plots indicating no negative effects of pyrethroid application on parasitoids, which might similar to pollen beetles have also developed resistance to this insecticidal group. Some studies have shown that some parasitoids can develop resistance to insecticides and subsequently survive field application rates of insecticides (e.g. Baker and Weaver, 1993; Liu et al., 2003).