Two widely used neonicotinoids—a class of insecticide—appear to significantly harm honey bee colonies over the winter, particularly during colder winters, according to a new study from Harvard School of Public Health (HSPH). The study replicated a 2012 finding from the same research group that found a link between low doses of imidacloprid and Colony Collapse Disorder (CCD), in which bees abandon their hives over the winter and eventually die. The new study also found that low doses of a second neonicotinoid, clothianidin, had the same negative effect.
Further, although other studies have suggested that CCD-related mortality in honey bee colonies may come from bees’ reduced resistance to mites or parasites as a result of exposure to pesticides, the new study found that bees in the hives exhibiting CCD had almost identical levels of pathogen infestation as a group of control hives, most of which survived the winter. This finding suggests that the neonicotinoids are causing some other kind of biological mechanism in bees that in turn leads to CCD.
“We demonstrated again in this study that neonicotinoids are highly likely to be responsible for triggering CCD in honey bee hives that were healthy prior to the arrival of winter,” said lead author Chensheng (Alex) Lu, associate professor of environmental exposure biology at HSPH, and affiliated faculty member of the Center for Health and the Global Environment.
The study, titled "Sub-lethal Exposure to Neonicotinoids Impaired Honey Bees Winterization before Proceeding to Colony Collapse Disorder," appeared online May 9, 2017 in the Bulletin of Insectology.
The Study
Introduction
Since its emergence in 2005/2006, the continuing significant losses of honey bees (Apis mellifera L.) colonies resulting from the symptomatic disease of colony collapse disorder (CCD) has demonstrated our inability to identify and eradicate the responsible cause(s) of CCD (BBC News, 2013; The New York Times, 2013; vanEngelsdorp et al., 2008). While the prevailing opinions suggest the linkage of CCD to multi-factorial causes including pathogen infestation, beekeeping practices (including malnutrition), and pesticide exposure in general (Cox-Foster et al., 2007; Blanchard et al., 2008; Higes et al., 2008; vanEngelsdorp et al., 2009; Alaux et al., 2010; de Miranda et al., 2010; Williams et al., 2010; Di Prisco et al., 2011; Vidau et al., 2011; USDA, 2013), this notion ignores the differential mortality symptoms, in particular hive abandonment in CCD vs. diseased colonies. However, recent scientific findings linking CCD with exposure to neonicotinoids, a group of systemic insecticides, appear to be gaining traction (Maini et al., 2010; Pareja et al., 2011; Lu et al., 2012; Farooqui, 2013; Matsumoto, 2013) and have led to new regulatory control (Erickson, 2012). In this study, we extend our previous study (Lu et al., 2012) showing that sub-lethal exposure of imidacloprid and clothianidin affected the winterization of healthy honey bee colonies that subsequently leads to CCD.
Materials and Methods
In order to investigate the detrimental effects of sublethal neonicotinoid exposure in healthy honey bee colonies, we utilized the split-plot lifecycle study design in which honey bees are fed with pre-determined known amounts of neonicotinoids and allowed to freely forage in the environment. We then assessed their hive growth and strength, as well as their mortality and morbidity, throughout the lifecycle including multiple worker bee generations.
The setup and management of eighteen study colonies (using 10-frame Langstroth pine hive) in three apiaries in central Massachusetts was identical to that previously described (Lu et al., 2012). At each apiary, we separated six colonies into two groups in which honey bees were fed with either sucrose water or high-fructose corn syrup (HFCS) over the study period. Each sugar group consisted of two neonicotinoid-treated and one control colonies replicated in each of the three apiaries.
We purchased sucrose from a local food store and HFCS from a beverage company. Both sugar waters made of sucrose and HFCS were analyzed prior to being used in the experiment and found non-detectable residues of neonicotinoids using a published method (Chen et al., 2013).
Starting from July 2nd, 2012, we administered 258 µg of imidacloprid (1-(6-chloro-3-pyridinyl) methyl)-N-nitro-2-imidazolidinimine, CAS# 138261-41-3) or clothianidin (1-(2-chloro-1,3-thiazole-5-ylmethyl)-3-methyl-2-nitroguanidine, CAS# 210880-92-5) in 1.9 liters (0.5 gallon) of sucrose water and HFCS to the treated colonies each week, respectively, for thirteen consecutive weeks ending on September 17th, 2012. Assuming each colony consisted of 50,000 bees at any given day in spring and summer, we administered 0.74 ng/bee/day of either imidacloprid or clothianidin to treated hives for 13 consecutive weeks. This dosage is far below the oral LD50 of 3.4 and 118.7 ng/bee for clothianidin and imidacloprid, respectively (Laurino et al., 2013). Control colonies were given neonicotinoid-free sucrose or HFCS throughout the experimental period. Sugar water (both types) was completely consumed by each colony at the end of each week during the 13-week neonicotinoid administration.
From June 29th to September 24th, 2012, we assessed the brood rearing production of all colonies on a biweekly basis using a modified brood assessment method as previously described (Lu et al., 2012). In brief, the 20-frames in each hive were scored cumulatively for the area covered by “sealed brood” which is the pupal stage of honey bee development. Brood was estimated by dividing the face of each side of the frame into 32 squares (each square containing approximately 100 cells). All 20 frames in each hive were scored by visually estimating the number of squares of capped brood per frame face.
All colonies were treated with Miteaway Quick strips for controlling Varroa mite on August 13th, 2012, followed by Apistan strips from October 1st to November 15th, 2012. The Varroa mite counts were assessed twice using the common alcohol wash method on August 13th (pre-Miteaway application) and August 22nd (post-Miteaway application). In addition, colonies were treated with Fumagillan-B [9.1 g dissolved in 7.6 liters (two gallons) of sucrose or HFCS] in early October 2012 to control N. apis and N. ceranae, two common intestinal parasites. Entrance reducers were installed before the hives were ready for winterization.
All colonies were monitored weekly beginning on late October 2012. Notes were taken on the size of the clusters observed by counting the numbers of frames containing honey bees from the top of the hive in which it generally took no more than 10 seconds. Starting from November 2012, hives were supplemented either with crystallized HFCS or with granular sucrose mixed into a thick water paste. The food was placed on waxed paper on top of the frames inside the inner covers. Data were analyzed using SPSS Statistics (version 20.0).
Results
We found that honey bees in both control and neonicotinoid-treated groups progressed almost identically through the summer and fall seasons, and we observed no acute morbidity or mortality in either group until the end of winter. However, bees from six of the twelve neonicotinoid-treated colonies had abandoned their hives, and were eventually dead with symptoms resembling CCD. Only one of the six control colonies was lost due to Nosema-like infection. We observed a complete opposite phenomenon in the control colonies, in which instead of abandonment, they were re-populated quickly with new emerging bees.
Discussion
The findings of this study support the notion that sub-lethal exposure to neonicotinoids can lead to colony collapse disorder. We found that the sub-lethal exposure to neonicotinoids, imidacloprid and clothianidin, affected the winterization of healthy colonies that subsequently led to CCD. The observed symptoms in the neonicotinoid-treated colonies were consistent with those associated with CCD, including the abandonment of hives and eventual death of the colony. In contrast, control colonies remained healthy throughout the study period, with only one colony lost due to Nosema-like infection.
The fact that the neonicotinoid-treated colonies progressed almost identically to control colonies until the end of winter, and that no acute morbidity or mortality was observed, suggests that the effects of neonicotinoid exposure on honey bee colonies may not be immediately apparent. This may explain why the link between neonicotinoids and CCD has been difficult to establish.
Our findings are consistent with previous studies that have found a link between neonicotinoid exposure and CCD (Maini et al., 2010; Pareja et al., 2011; Lu et al., 2012; Farooqui, 2013; Matsumoto, 2013). Neonicotinoids are systemic insecticides that are commonly used in agriculture, and they can be found in pollen and nectar that honey bees collect from treated crops. The low doses of neonicotinoids used in this study were below the LD50 for honey bees, indicating that even low levels of exposure to these insecticides can have a significant impact on honey bee health.
The exact mechanism by which neonicotinoid exposure leads to CCD is not yet fully understood, but it is thought to involve immune suppression, impaired foraging behavior, and disruption of the colony's social dynamics (Bonmatin et al., 2015). Neonicotinoids are known to affect the nervous system of insects, and it is possible that exposure to these insecticides may affect the behavior and communication of honey bees, leading to a breakdown in the social structure of the colony.
Our study provides further evidence of the link between neonicotinoid exposure and CCD, and highlights the need for continued research into the effects of these insecticides on honey bees and other pollinators. The use of neonicotinoids in agriculture should be carefully regulated to minimize the exposure of honey bees and other beneficial insects, and alternative pest control methods should be explored to reduce the reliance on these systemic insecticides.
Conclusion
In conclusion, our study shows that sub-lethal exposure to neonicotinoids, imidacloprid and clothianidin, can affect the winterization of healthy honey bee colonies and subsequently lead to colony collapse disorder. The observed symptoms in the neonicotinoid-treated colonies were consistent with those associated with CCD, including the abandonment of hives and eventual death of the colony. Our findings support the notion that neonicotinoid exposure is a significant contributing factor to CCD, and highlight the need for continued research into the effects of these insecticides on honey bees and other pollinators.
