Methoxyfenozide is an insect growth regulator (IGR) commonly used in agriculture to simultaneously control pests and preserve beneficial insect populations; however, its impact on honey bees in not fully understood. We conducted field and laboratory experiments to investigate bee health in response to field-relevant concentrations of this pesticide. Significant effects were observed in honey bee colony flight activity and thermoregulation after being exposed over 9 weeks to supplemental protein patty containing methoxyfenozide. Compared to bee colonies in the control group, colonies fed pollen patty with 200 ppb methoxyfenozide (as measured by residue analysis) had: 1) a significantly reduced rate of weight loss due to forager departure in the morning; and 2) higher temperature variability during the winter. Colonies in the 100 ppb (as measured by residue analysis) treatment group had values between the 200 ppb group and control for both response variables. The dusk break point, which is the time associated with the end of forager return, differed among all treatment groups but may have been confounded with direction the hives were facing. Bee colony metrics of adult bee mass and brood surface area, and measurements of bee head weight, newly-emerged bee weight, and hypopharyngeal gland size were not significantly affected by methoxyfenozide exposure, suggesting that there may be significant effects on honey bee colony behavior and health in the field that are difficult to detect using standard methods for assessing bee colonies and individuals. The second experiment was continued into the following spring, using the same treatment groups as in the fall. Fewer differences were observed among groups in the spring than the fall, possibly because of abundant spring forage and consequent reduced treatment patty consumption. Residue analyses showed that: 1) observed methoxyfenozide concentrations in treatment patty were about 18–60% lower than the calculated concentrations; 2) no residues were observed in wax in any treatment; and 3) methoxyfenozide was detected in bee bread only in the 200 ppb treatment group, at about 1–2.5% of the observed patty concentration.

Colony brood levels, frames of bees (adult bee mass) and internal hive temperature were monitored for 60 colonies for each of two years as they were moved from agricultural, tree crop and mountain landscapes in southern California to blueberry and almond pollination sites. Hive weight was also continuously monitored for 20 of those hives for 6 weeks for both years, during commercial pollination. Pesticide residues in wax, honey and beebread samples were analyzed by composite apiary samples. While colonies in mountain sites had more adult bees and brood than those in agricultural sites in August, by October brood levels were higher in colonies from agricultural sites. Though hives from different original landscapes differed in size in October, hive assessments revealed no differences between the groups after co-wintering when graded for commercial almond pollination. Beebread from hives in agricultural sites had greater agrochemical diversity and in general higher pesticide hazard quotients than those from mountain sites, but those hives also had higher and more constant temperatures from September until January than hives from mountain sites. Hives placed in commercial almond pollination gained on average 287 g per d, compared to an average loss of 68 g per d for colonies in commercial blueberry pollination, although weight data indicated greater foraging effort by colonies in blueberries, possibly due to the proximity and abundance of almond pollen during bloom. Temperature monitoring was effective at distinguishing hive groups and had the best overall value in terms of equipment, installation, colony disturbance and information yield.

The effects of agricultural pesticide exposure upon honey bee colonies is of increasing interest to beekeepers and researchers, and the impact of neonicotinoid pesticides in particular has come under intense scrutiny. To explore potential colony-level effects of a neonicotinoid pesticide at field-relevant concentrations, honey bee colonies were fed 5- and 20-ppb concentrations of clothianidin in sugar syrup while control colonies were fed unadulterated syrup. Two experiments were conducted in successive years at the same site in southern Arizona, and one in the high rainfall environment of Mississippi. Across all three experiments, adult bee masses were about 21% lower among colonies fed 20-ppb clothianidin than the untreated control group, but no effects of treatment on brood production were observed. Average daily hive weight losses per day in the 5-ppb clothianidin colonies were about 39% lower post-treatment than in the 20-ppb clothianidin colonies, indicating lower consumption and/or better foraging, but the dry weights of newly-emerged adult bees were on average 6–7% lower in the 5-ppb group compared to the other groups, suggesting a nutritional problem in the 5-ppb group. Internal hive CO2 concentration was higher on average in colonies fed 20-ppb clothianidin, which could have resulted from greater CO2 production and/or reduced ventilating activity. Hive temperature average and daily variability were not affected by clothianidin exposure but did differ significantly among trials. Clothianidin was found to be, like imidacloprid, highly stable in honey in the hive environment over several months.

15Honey bee colonies were exposed to sublethal concentrations (5 and 20 ppb) of clothianidin in sugar 16 syrup, while control colonies were fed syrup with no pesticide. In addition to standard colony 17 assessments of adult bees and brood, hive weight and internal temperature were monitored on a 18 continuous basis at all sites. Experiments were conducted twice in Arizona, in successive years at the 19 same site, and once in Mississippi, to examine the concomitant effects of weather and landscape. Adult 20 bee masses at the Arizona site were significantly affected by clothianidin concentration. Newly-emerged 21 bee dry weights, measured only at the Arizona site, were significantly lower for colonies fed 5 ppb 22 clothianidin compared to the other groups. CO 2 concentration, also only measured at the Arizona site, 23 was higher in colonies fed 20 ppb clothianidin. Neither daily hive weight change nor colony 24 thermoregulation were affected by clothianidin exposure. The Mississippi site had higher rainfall, more 25 diverse land use, and a different temperature regime, and bee colonies there did not show any effects of 26 clothianidin. These results suggest that bee colonies in more stressful environments, such as the 27 Sonoran desert of southern Arizona, are affected more by clothianidin exposure than colonies at sites 28 with higher rainfall and more forage. Clothianidin was also found to be, like imidacloprid, highly stable in 29 honey in the hive environment at least over several months. These results also showed that CO 2 30 concentration within the hive is potentially valuable in measuring the effects of stressors on bee health. 31 32 Key words: continuous hive weight, continuous hive temperature, hive CO 2 concentration, newly-33 emerged bees, neonicotinoid, pesticide residues. 34 35 36 3 37

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