by Clarence Collison
Pesticide exposure and pathogens may interact to have strong negative
effects on colony health.
Honey bees are constantly exposed to pesticides, particularly if their colonies are located in agricultural areas. While bees are non-target organisms for most field pesticide applications; nevertheless, they can be exposed to pesticides while collecting pollen and nectar from flowers, collecting resins from various plants, drinking water from numerous sources, breathing, and during flight (if the pesticides are airborne) (Mullin et al. 2010; Gregorc and Ellis 2011).
Pesticide exposure and pathogens/may interact to have strong negative effects on colony health (Pettis et al. 2013). Such findings are of great concern given the large numbers and high levels of pesticides found in colonies. These chemicals may affect the synthesis, transport, action or elimination of natural molecules, such as hormones or enzymes that are responsible for maintaining bee development, immune mechanisms and behavior (Chauzat et al. 2009).
Another area of concern is the sub-lethal effects of acaricides used within the hive for the control of Varroa mites (Johnson et al. 2010). Acaricide levels can build up in the wax comb of colonies (Mullin et al. 2010), and low level exposure to these products can impair a colony’s ability to rear queens (Collins et al. 2004), reduce sperm viability in drones (Burley et al. 2008), and impact the development and immune response of worker bees reared in contaminated comb (Desneux et al. 2007).
Numerous surveys have revealed high levels of pesticide residue contamination in honey bee comb. Wu et al. (2011) conducted studies to examine possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan. Worker bees were reared in brood comb containing high levels of known pesticide residues (treatment) or in relatively uncontaminated brood comb (control). Delayed development was observed in bees reared in treatment combs containing high levels of pesticides particularly in the early stages (day four and eight) of worker bee development. Adult longevity was reduced by four days in bees exposed to pesticide residues in contaminated brood comb during development. Pesticide residue migration from comb containing high pesticide residues caused contamination of control comb after multiple brood cycles and provided insight on how quickly residues move through wax. Higher brood mortality and delayed adult emergence occurred after multiple brood cycles in contaminated control combs. In contrast, survivability increased in bees reared in treatment comb after multiple brood cycles when pesticide residues had been reduced in treatment comb due to residue migration into uncontaminated control combs, supporting comb replacement efforts. These sub-lethal effects; delayed larval development and adult emergence or shortened adult longevity, from pesticide residue exposure can have indirect effects on the colony such as premature shifts in hive roles and foraging activity.
In a second study, honey bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with Nosema ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores + syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection (Wu et al. 2012).
Bees are particularly vulnerable to sublethal pesticide exposures because they gather nectar and pollen, concentrating environmental toxins in their nests in the process (James and Xu 2012). Pesticides do have effects on immunity. Organophosphates and some botanicals have been found to impact hemocyte number, differentiation, and thus affect phagocytosis. The phenoloxidase cascade and melanization have also been shown to be affected by several insecticides. Many synthetic insecticides increase oxidative stress, and this could have severe impacts on the production of some antimicrobial peptides. Pesticides can also affect grooming behaviors, rendering bees more susceptible to disease.
To further understand potential interactions between pesticides and bee pests/pathogens, Gregorc et al. (2012) determined physiological responses of bees to chemical and biological threats by measuring gene expression after exposure to the parasitic Varroa mite and a suite of pesticide threats. The tested pesticides (with pesticide class in parentheses) included two fungicides [myclobutanil (azole), chlorothalonil (substituted benzene)], two herbicides [simazine (triazine), glyphosate (phosphonoglycine)], and five insecticides/miticides [fluvalinate (pyrethroid), imidacloprid (nicotinoid), coumaphos (organophosphate), chlorpyrifos (organophosphate), amitraz (amidine)] and represent a range of modes-of-action and pesticide families. Three of these compounds (amitraz, fluvalinate, and coumaphos) are often used by beekeepers to control Varroa mites and/or small hive beetles in colonies. The other chemicals are used commonly in agricultural settings and, with the exception of glyphosate, have been found as residues in honey bee colonies (Mullin et al. 2010). Honey bee larvae reared in vitro (Latin: in glass; experiments done in a cell-free system) were exposed to one of these nine pesticides and/or were challenged with Varroa mites. Total RNA was extracted from individual larvae and first strand cDNAs were generated so that gene-expression changes in the larvae could be measured targeting transcripts (RNA copy of a gene sequence) for pathogens and genes involved in physiological processes, bee health, immunity, and/ xenobiotic (chemical substances that are foreign to the body) detoxification. Transcript levels for Peptidoglycan Recognition Protein, a pathogen recognition gene, increased in larvae exposed to Varroa mites and were not changed in pesticide treated larvae. As expected, Varroa-parasitized brood had higher transcripts of deformed wing virus than did control larvae. Varroa parasitism, arguably coupled with virus infection, resulted in significantly higher transcript abundances for the antimicrobial peptides abaecin, hymenoptaecin, and defensin 1. Transcript levels for Prophenoloxidase-activating enzyme, an immune end product, were elevated in larvae treated with myclobutanil and chlorothalonil (both are fungicides). Transcript levels for Hexameric storage protein (Hsp70) were significantly upregulated in imidacloprid, fluvalinate, coumaphos, myclyobutanil, and amitraz treated larvae. Definitive impacts of pesticides and Varroa parasitism on honey bee larval gene expression were demonstrated.
Chauzat et al. (2009) conducted a three-year field survey in France from 2002 to 2005, to study colony health in relation to pesticide residues found in the colonies. Pesticide residue levels were determined in honey, pollen collected by bees, beeswax and bees. When all samples were pooled together, the number of pesticide residues detected per sampling period (four sampling periods per year) and per apiary ranged from 0 to 9, with the most frequent being two (29.6%). No pesticide residues were detected during 12.7% of the sampling periods. Residues of imidacloprid and 6-chloronicotinic acid were the most frequently detected in pollen loads, honey and bee samples. Several pairs of active ingredients were present concurrently within honey bees and in pollen loads but not in beeswax and honey samples. No statistical relationship was found between colony mortality and pesticide residues.
Chauzat and Faucon (2007) analyzed beeswax for pesticide residues in their French apiary survey. Beeswax samples were collected once a year over two years from a total of 125 honey bee colonies. Multi-residue analyses were performed on these samples in order to identify residues of 16 insecticides and acaricides and two fungicides. Residues of 14 of the searched-for compounds were found in samples. Tau-fluvalinate, coumaphos, and endosulfan residues were the most frequently occurring residues (61.9, 52.2 and 23.4% of samples, respectively). Coumaphos was found in the highest average quantities (792.6 µg/kg). Residues of cypermethrin, lindane and deltamethrin were found in 21.9, 4.3 and 2.4% of samples, respectively. Statistical tests showed no difference between years of sampling, with the exception of the frequency of pyrethroid residues. Beeswax contamination was the result of both in-hive acaricide treatments and, to a much lesser extent, environmental pollution.
Chauzat et al. (2006) also tested pollen from these 125 colonies. For three years, the colonies were sampled four times per year. Pollen loads from traps were collected at each visit. Multi-residue analyses were conducted to search for residues of 36 different molecules. Specific analyses were conducted to search fipronil and metabolites and also imidacloprid and metabolites. Residues of 19 searched compounds were found in samples. Contamination by pesticides ranged from 0 to 50%. Coumaphos and tau-fluvalinate residues were the most concentrated of all residues (mean concentrations were 925.0 and 487.2 µg/kg, respectively). Fipronil and metabolite contents were superior to the limit of detection in 16 samples. Residues of fipronil were found in 10 samples. Nine samples contained the sulfone compound, and three samples contained the desulfinyl compound. Residues of imidacloprid and 6-chloronicotinic acid were found in 69% of samples. Imidacloprid contents were quantified in 11 samples with values ranging from 1.1 to 5.7 µg/kg.
6-Chloronicotinic acid content was superior to the limit of quantification in 28 samples with values ranging from 0.6 to 9.3 µg/kg.
During 2007 to 2008, Mullin et al. (2010) actively sampled beebread, trapped pollen, broodnest wax, beeswax foundation, and adult bees and brood for pesticide residues. These samples were drawn largely from commercial beekeepers from 23 states and one Canadian province, and included samples from apparently healthy colonies as well as from operations that were diagnosed as having colony collapse disorder. Included in this survey were dead bees collected from local or community applications of insecticides. A total of 121 different pesticides and metabolites within 887 samples of wax, pollen, bee and associated hive samples were found. Almost 60% of the 259 wax and 350 pollen samples contained at least one systemic pesticide, and over 47% had both in-hive acaricides fluvalinate and coumaphos, and chlorothalonil, a widely-used fungicide. In bee pollen, chlorothalonil was found at levels up to 99 ppm and the insecticides aldicarb, carbaryl, chlorpyrifos and imidacloprid, fungicides boscalid, captan and myclobutanil, and herbicide pendimethalin at one ppm levels. Almost all comb and foundation wax samples (98 %) were contaminated with up to 204 and 94 ppm, respectively, of fluvalinate and coumaphos, and lower amounts of amitraz degradates and chorothalonil, with an average of six pesticide detections per sample and a high of 39. There were fewer pesticides found in adults and brood except for those linked with bee kills by permethrin (20 ppm) and fipronil (3.1 ppm).
Honey bees can be exposed to multiple chemical agents simultaneously (Mullin et al. 2010), synergistic or antagonistic interactions among these pesticides or between pesticides and bee pests/pathogens could also play a role in the bee and colony health (Johnson et al. 2009). Alaux et al. (2010) published physiological evidence that the insecticide imidacloprid and the fungal pathogen Nosema can interact synergistically to affect bee health negatively, including physiological changes initiated by pesticide exposure that decreased bee tolerance toward Nosema infection. Similarly, Pettis et al. (2012) showed an increase in Nosema spore loads in colonies treated with imidacloprid. Research has also shown that bees consuming pollen with high fungicide loads have an increased probability of Nosema infection (Pettis et al. 2013). In the past we have normally considered fungicides to be fairly safe for honey bees.
To address beekeeper concerns about pesticide residues in overwintered honey, paired samples were obtained from the extracting honey supers and brood chamber of the same colony (Ostiguy and Eitzer 2014). Only eight residues were detected: coumaphos, fluvalinate, boscalid, dimethoate, atrazine, bentazon, dichlorobenzene and thymol. Honey from extracting supers was significantly less likely to contain pesticide residues than in honey from brood comb. Fluvalinate was detected only in overwintered brood comb honey, and coumaphos was found at significantly higher levels in the overwintered samples from the brood comb-honey super pairs. Pesticide residues in honey, while low in comparison to other substrates in the hive, contribute to the overall pesticide exposure of honey bees, with overwintered brood comb honey contributing more than honey stored in other locations in the hive.
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Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.