Good production practices minimize their impact on natural ecosystems and maximize sustainability (Tilman et al., 2002). Consumers are aware of this, so they are changing their habits and acquiring commodities that are produced through sustainable agricultural practices (Vermeir & Verbeke, 2006). However, a growing human population and modern lifestyle create demand for an ever-increasing volume and variety of agricultural goods. Modern intensive agricultural technology holds the promise to fulfill current and future humanity needs, even though it commonly involves the use of conventional chemical insecticides. Entire crops are sprayed to manage insect pests that reduce crop yield (Tilman et al., 2002), and though the use of insecticides reduces the damage caused by pests, the practice usually comes with deleterious side effects on non-target organisms, especially beneficial insects such as predators, parasitoids and pollinators (Carvalho, 2006). The honey bee,
Biological control, i.e. the use of organisms or their metabolites for the control of pests, is currently considered a better, more environmentally friendly alternative to conventional synthetic insecticides (Heimpel & Mills, 2017). Spinosyns, a group of molecules that is produced through the fermentation of the actinomicete
Because the “attract and kill” formulation lures the pest to the application points, it is unnecessary for the crop to be completely covered with the insecticide, and instead the formulation is applied either as large drops, bursts on the base of trees or placed inside bait stations (Gazit et al., 2013). Non-targets are not attracted to the bait, and the probability of contact between non-target insects and the insecticide formulation dramatically decreases. Therefore, the use of “attract and kill” formulations should be significantly more compatible with the management of honey bee colonies for crop pollination than conventional cover sprays of chemical pesticides for crop protection (Kirkland, 1999; Cabrera-Marín et al., 2016). GF-120 (Dow Agrosciences, CA) is a formulation that consists of NH3-releasing compounds, sugars and the bioinsecticide spinosad (a mixture of spinosyns A and D), aimed to control fruit flies in Mexico (Sánchez et al., 2012). Even though spinosad is considered to be a minor threat to wildlife including mammals, it has been shown to be toxic in contact and oral tests to pollinators such as the honey bee (Miles, 2003; Hardstone & Scott, 2010). However, given that the GF-120 formulation has shown to be mostly repellent to bee pollinators (Mangan & Moreno, 2009; Gómez-Escobar et al., 2014), actual oral toxicity to pollinators is substantially reduced. More importantly, application at an ultra low volume (ULV, 2 liters of diluted formulation per ha) with large size droplet completely eliminates the necessity of complete crop coverage, leaving most of the crop free of insecticides, which might provide refuge for pollinators (Cabrera-Marín et al., 2016).
The Spotted-Wing Drosophila,
A new product, Acttra SWD (ISCA Technologies Inc., Riverside, CA, US), aimed for the management of
The experimental work was carried out at a raspberry (
Acttra SWD® [A.I. sugars (invertose 33.1%, sucrose 3.0%), methionol 0.6% and spray adjuvants 63.1%, ISCA Technologies Inc., USA] is a feeding stimulant adjuvant registered as a commercial formulation for the SWD in USA. The pesticide Entrust® (A.I. spinosad 22.14%, Dow Agro-Sciences, Mexico S.A. de C.V.) was chosen as the source of spinosad because of their frequent use by berry farmers in Mexico. We added Entrust to Acttra SWD (from now on Acttra/Entrust) to obtain a final concentration of 110.7 ppm of spinosad, as used for the control of the SWD. Because Exalt® is commonly used against SWD, it was included in the experiment (A.I. spinetoram 5.87%, Dow Agro-Sciences, Mexico S.A. de C.V.) in order to evaluate its impact on bees. Spinetoram is a mixture of spinosyns J and L and shares many features with spinosad (Schutze et al., 2018). Exalt was diluted with water to a final concentration of 29.35 ppm of spinetoram according to directions provided by the manufacturer for SWD control.
Two honey bee colonies from a local apiary were used in the experiments. Both colonies were considered in good health by an experienced local beekeeper who visually inspected brood, food reserves, number of adults, and for the presence of parasites and disease. The colonies were located on the edge of the raspberry plantation to minimize disturbance by routine work in the farm (Fig. 1A). Experiments were carried out between 08:00 and 15:00, since at this time honey bees were commonly visiting floral resources and it was possible to train them to feeding stations.
In this experiment we wanted to know if foragers would abandon a rich food source polluted with the Acttra/Entrust mixture, and if these foragers would move to nearby pesticide-free resources exploited by nestmates. Following the methodology of Cabrera-Marín et al. (2015), we trained five to ten foragers from one colony to collect a 2.0 M sucrose solution from a feeding station (FS1, a circular plastic plate containing 100 ml of sucrose solution atop a camera tripod), located 100 m away the colony. Next, a second group of foragers was trained to a different feeding station (FS2) located 130 m from the FS1, but at 100 m from the colony as shown in Fig. 1A. We differentiated trained foragers visiting the two feeders by painting them on the thorax with water-based paints of different colors. Newcomer foragers were painted with another set of colors (blue paint for FS1 foragers and red paint for FS2 foragers) to distinguish the feeder they were visiting and from the trained foragers (Sánchez et al., 2009). Observers were positioned at each feeder, so that any forager painted with a color not corresponding to the feeder they were visiting was trapped with a suction tube and maintained inside a plastic jar with sucrose solution until the end of the experiment. We carried out the previous procedure in order to maintain a stable and faithful set of foragers for each feeder. Since there were honey bee colonies other than ours in the surroundings, we marked the foragers from our colonies at the entrance with a painting device as carried out by Mikery-Pacheco et al. (2013). Over the paint marks deposited by this device, a different dot was painted as pointed out lines above to know the feeder station they were trained to.
Once we observed that 50–100 foragers were consistently visiting each feeder, and that no foragers were switching between feeders, we initiated the
Groups of 8–10 foragers, which were collected from feeding stations with the use of suction tubes, were transferred into plastic jars (24.5 cm height × 13.5 cm diameter) covered with a nylon cap. In the laboratory, ten groups were exposed to each of the following treatments: (a) control 1 [honey/water solution 50% with 10 ml of vegetable food dye per liter of solution (Rojo 122; McCormick Company Inc., Spain)]; (b) control 2 (10 ml of vegetable food dye per liter of water); (c) Acttra/Entrust at 110 ppm spinosad (added with 10 ml of vegetable food dye per liter of solution), and (d) Acttra/Entrust/Honey (Acttra/Entrust mixed in equal proportion with honey at 110 ppm spinosad, added with 10 ml of vegetable food dye per liter of solution). Exposure consisted of placing twenty 50 ml drops of each treatment on a 5 cm x 5 cm strip of Parafilm® (Heathrow Scientific, Vernon Hills, IL, USA) inside each container for 30 min. The dye was used to determine if the bees consumed the drops as described by Silva et al. (2013) for
Bee foragers were trained to FS2 between 09:00 and 11:00 h as described above. However, in this case the plate consisted of a 1 m2 square piece of Styrofoam. When approximately 100 foragers were observed feeding, the treatments were applied at the recommended dose to control
For experiment 1 we summed up the number of foragers observed in each phase resulting in three time-correlated data points per replicate and per feeding station. We analyzed these data following a Linear Mixed Model approach with the package lme4 v 1.1 (Bates et al., 2015), in which a replicate was considered a random variable and colony, treatment and phase as fixed variables. Percent mortality from experiment 2 and experiment 3 was analyzed with the use of the ANOVA approach. Statistics were run in R environment v4.0.3 (R Development Core Team, 2020) under Mac OS High Sierra v 10.13.6.
Data from both colonies followed similar patterns at α=0.01 (t=−2.36; df=37; p=0.02), so we pooled the data. The mean number of foragers (± standard error) visiting the FS2 during the exposure phase, when the formulation was offered, was extremely low (8.65±1.00), compared to the basal phase (385.65±27.80) and to the returning phase (227.05±23.08). This resulted in a significant difference between the basal and the exposure phases (t=5.69, df=78, p<0.001) and the exposure and the returning phases (t=3.91, df=78, p<0.001). On the other hand, the mean number of foragers visiting the FS1 during basal phase (396.60±36.37), exposure phase (453.7±38.94) and returning phase (455.05±36.50) remained high. The mean number of foragers in the basal and the returning phases was not significantly different (t=−1.78, df=78, p=0.08).
Interestingly enough, we observed many foragers switching from FS2 to FS1 during the exposure phase. We found significant differences (t<−3.40, df=218, p<0.001) in the mean number of foragers between the exposure phase (16.2±2.07) and the basal phase (6.18±2.31) and returning phase (7.12±1.12). No significant difference was found between basal and returning phases (t=0.367, df=218, p=0.72).
One group from the Acttra-Entrust treatment was removed because half the bees escaped from the container during manipulation. All bees from the control 1 treatment had their abdomen colored red, while some had it red in control 2. As expected, no bee from the Acttra/Entrust and Acttra/Entrust/Honey treatments had their abdomen colored red and even rejected it if mixed with honey. Mortality among the four treatments was low and not significantly different (F=0.83, df=3, 35, p=0.48), even though a slightly higher mortality was observed in the Acttra/Entrust treatment (Fig. 3).
The results of this experiment showed that toxicity by contact is high. All treatments, except for control, showed mortalities of or close to 100% (Fig. 4; F=3645, df=3, 16, p<0.001).
This study revealed three important aspects about the use of Acttra SWD/Entrust in the field. First, we showed that the formulation repelled bees since most of the foragers moved from the Acttra/Entrust feeder to the feeder without it, and moreover the remaining foragers that continued visiting the Acttra/Entrust feeder did not collect it. Our data suggest that Acttra SWD was clearly discouraging foragers from collecting it, since the number of foragers in the Acttra/Entrust feeder was reduced by over 95% during the exposure phase. The GF-120 formulation elicited a similar behavior in honey bees and stingless bees (Gómez-Escobar et al., 2014; Mangan & Moreno, 2009). In this case, the protein contents in this formulation are assumed to keep bees from consuming it, despite the sugar in it. Honey bees should be able to taste in Acttra SWD invertose, sucrose, methionol and the inerts. We hypothesize that methionol discouraged our bees from collecting the formulation upon contact, since all foragers landed on the platform, but did not collect it and instead were repelled from a distance. Because this formulation constitutes proprietary technology, the composition of the vehicle is unknown to us and could also be a source of repellence, but this has to be tested. Many foragers that abandoned the Acttra/Entrust feeder during the exposure phase showed up at the sucrose feeder, which suggests that, as long as there are food alternatives available, some bees will prefer to move to these resources.
A similar result was obtained by Cabrera-Marín et al. (2015) with honey bees exposed to GF-120, when foragers abandoned the feeder that changed from offering sucrose to show the pesticide. Thus, some spaces must be left in the field untreated, for example through the application of the formulation at evenly spaced locations and that the application does not cause too much aerosolization in order to reach floral structures. In any case, our bees landed on the platform, and some had to make contact with the formulation to reject it, and some foragers still tried to collect it. Further evaluations need to be conducted in order to determine the presence of residues of spinosad in honey from honey bee colonies located nearby treated farms and to estimate actual risks.
Secondly, the low toxicity observed in Experiment 2 is in agreement with the lack of attraction of honey bees towards Acttra/Entrust observed in Experiment 1. No foragers exposed to droplets with Acttra SWD had their abdomens colored red, and they preferred to starve than to feed upon any of the Acttra treatments, which can be considered a no-choice test. This result gives further evidence on the safety of Acttra/Entrust to honey bees. However, during application it would be necessary to provide food for the bee colonies, since they could stop foraging completely and become weaker if no other food source were available. Although it is likely that a mixture Acttra/Exalt should also be repellent to honey bees, further studies must be done to confirm this.
The third important finding of our study is that the acute toxicity of spray applications aimed to completely cover the plantation with Acttra/Entrust can be as high as that observed for Entrust and Exalt formulations at the recommended field dose. Given that several studies have shown spinosyns to be highly toxic to
The environmental fate and half-life of spinosad and spinetoram is similar. They rapidly break-down on plant surfaces exposed to sunlight (2–16 d), water and soil (<1 d), but it takes longer in attenuated light conditions (9–17 d) and even longer in anaerobic conditions in water (161–250 d). It is strongly adsorbed by soil particles, so the probability of leaching is low (Cleveland, 2007). Thus, even though other routes of exposure do not seem to represent a threat, more research is needed, to discard any potential chronic intoxication by Acttra/Entrust, or that its effects (at field realistic concentrations) are not detrimental in the long term.
In conclusion, in this work we found that if Acttra/Entrust, which is formulated under the principle to “attract and kill” the pest (Klick et al., 2019), is applied according to manufacturer's instructions the probability of causing acute toxicity to honey bees is very low, making it compatible with commercial pollination in the conditions of the study site.