Biomarker responses and lethal dietary doses of tau-fluvalinate and coumaphos in honey bees: Implications for chronic acaricide toxicity.

Evidence suggests that acaricide residues, such as tau-fluvalinate and coumaphos, are very prevalent in honey bee colonies worldwide. However, the endpoints and effects of chronic oral exposure to these compounds remain poorly understood. In this study, we calculated LC50 and LDD50 endpoints for coumaphos and tau-fluvalinate, and then evaluated in vivo and in vitro effects on honey bees using different biomarkers. The LDD50 values for coumaphos were 0.539, and for tau-fluvalinate, they were 12.742 in the spring trial and 8.844 in the autumn trial. Chronic exposure to tau-fluvalinate and coumaphos resulted in significant changes in key biomarkers, indicating potential neurotoxicity, xenobiotic biotransformation, and oxidative stress. The Integrated Biomarker Response was stronger for coumaphos than for tau-fluvalinate, supporting their relative lethality. This study highlights the chronic toxicity of these acaricides and presents the first LDD50 values for tau-fluvalinate and coumaphos in honey bees, providing insights into the risks faced by colonies.

Toxicity of Coumaphos Residues in Beeswax Foundation to the Honey Bee Brood.

Coumaphos is one of the most frequently detected pesticides in recycled beeswax. The objective was to assess the maximal level of coumaphos in foundation sheets that could exist without lethal effects on the honey bee larvae. Brood development was followed in cells drawn on foundation squares containing coumaphos ranging from 0 to 132 mg/kg. Furthermore, larval exposure was determined by measuring the coumaphos level in the drawn cells. Coumaphos levels in the initial foundation sheets up to 62 mg/kg did not increase brood mortality because the emergence rates of bees raised on these foundation squares were similar to controls (median of 51%). After a single brood cycle, coumaphos levels in the drawn cells were up to three times lower than the initial levels in foundation sheets. Hence, coumaphos levels of 62 mg/kg in the initial foundation sheets, almost the highest exposures, resulted in levels of 21 mg/kg in drawn cells. A significantly reduced emergence rate (median of 14%) was observed for bees raised on foundation sheets with initial coumaphos levels of 132 mg/kg, indicating increased brood mortality. Such levels resulted in coumaphos concentrations of 51 mg/kg in drawn cells, which is close to the median lethal concentration (LC50) as determined in previous in vitro experiments. In conclusion, brood mortality was increased on wax foundation sheets with initial coumaphos levels of 132 mg/kg, while no elevated mortality was observed for levels up to 62 mg/kg. Environ Toxicol Chem 2023;42:1816-1822. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evaluating the chronic effect of two varroacides using multiple biomarkers and an integrated biological response index.

There is mounting evidence that acaricides are among the most prevalent medicinal compounds in honey bee hive matrices worldwide. According to OCDE guideline No. 245 chronic lethal concentration of tau-fluvalinate (at concentrations ranging from 77.5 to 523.18 ppm), coumaphos (59.8 ppm) and dimethoate (0.7 ppm) were determined. The activity of the biomarkers acetylcholinesterase (AChE), carboxylesterase (CbE), glutathione S-transferase (GST), catalase (CAT) and malondialdehyde (MDA) was analysed and as they are implicated in neurotoxicity, biotransformation and antioxidant defences, these values were combined into an integrated biomarker response (IBR). There was enhanced AChE, CAT and GST activity in honey bees exposed to tau-fluvalinate, while dimethoate inhibited AChE activity. Both dimethoate and coumaphos inhibited CbE activity but they enhanced CAT activity and MDA formation. Our results highlight how these biomarkers may serve to reveal honey bee exposure to commonly used acaricides.

An in vitro model for assessing the toxicity of pesticides in beeswax on honey bee larvae.

While many studies have examined residue levels in beeswax, little is known about the levels that pose a risk for honey bee development. In an in vitro study, we aimed to assess the toxicity of pesticides in wax for worker larvae using coumaphos as a model substance. First, we reared larvae in beeswax with the aim to correlate the larval toxicity to the corresponding levels of coumaphos in beeswax. In a second step, we tested to which extent coumaphos migrates from the beeswax into the larval diet and if such dietary levels are toxic to larvae. We observed dose-related toxicity when larvae were exposed to coumaphos concentrations in beeswax from 30 to 100 mg/kg. The lethal concentration in 50% of the individuals (LC50) was calculated to be 55.9 mg/kg, while the no observed effect concentration (NOEC) for exposure of larvae to coumaphos in wax was 20 mg/kg. Additional test series without larvae allowed to assess the migration of coumaphos from the beeswax into the diet. The resulting dietary coumaphos concentrations were four to five times lower than the initial concentrations in wax. In accordance, the LC50 for chronic exposure of larvae to coumaphos in the diet was 12.5 mg/kg, which was 4.5 times lower than the LC50 obtained for wax exposure. Finally, a coumaphos level of 20 mg/kg in wax led to a dietary concentration of 3.9 mg/kg that was close to the NOEC of 3 mg/kg obtained in the diet. In conclusion, both experimental approaches suggest that coumaphos concentrations of up to 20 mg/kg in wax are non-lethal.

Organophosphate intoxication in 2 dogs from ingestion of cattle ear tags.

OBJECTIVE: To describe 2 cases of organophosphate intoxication through a previously unreported method of exposure. CASE SERIES: A 2-year-old intact male Australian Cattle Dog (case 1) presented with progressive muscarinic and nicotinic clinical signs, and a 3-year-old neutered male mixed breed dog (case 2) presented after known ingestion of cattle ear tags. The dog in case 1 was discovered to have ingested cattle ear tags after abdominal radiographs. Organophosphate testing of gastric contents confirmed diazinon toxicosis. The dog in case 2 was found to be eating ear tags by the owner. The tags in case 2 contained diazinon and coumaphos. The dog in case 1 was treated with gastric lavage, gastroprotectants, prokinetics, antiemetics, pralidoxime chloride, and atropine. The dog in case 2 was treated with pralidoxime chloride. Both patients received standard supportive and nursing care and recovered completely with no further concerns. NEW OR UNIQUE INFORMATION PROVIDED: This is a novel exposure to organophosphates that has not been reported in small animals. In dogs with relevant clinical signs and potential environmental exposure, cattle ear tag ingestion is an important differential diagnosis to consider.

Exposure to pesticides during development negatively affects honey bee (Apis mellifera) drone sperm viability.

Honey bee (Apis mellifera) colonies invest a substantial amount of colony resources in the production of drones during the reproductive season to enable mating with virgin queens from nearby colonies. Recent studies have shown significant differences in the production of sperm cells that are viable (i.e., sperm viability) and can fertilize an ovule among sexually mature drones that are exposed to different environmental conditions during development or as adults. In particular, sperm viability may be negatively affected during drone development from exposure to pesticides in contaminated beeswax. To assess whether sperm viability is negatively affected during drone development from exposure to beeswax contaminated with in-hive pesticides, we compared the viability of sperm collected from drones reared in pesticide-free beeswax with that of drones reared in beeswax contaminated with field-relevant concentrations of the pesticides most commonly found in wax from commercial beekeeping operations in the United States. These pesticides include the miticides fluvalinate, coumaphos and amitraz, and the agro-chemicals chlorothalonil and chlorpyrifos. Sperm from drones collected at 10 and 18 days post emergence were classified as viable or non-viable to calculate sperm viability. For all pesticide treatment groups, drones that were reared in pesticide-laden beeswax had lower sperm viability compared to those reared in pesticide-free beeswax. This difference was especially pronounced among drones reared in miticide-laden wax. Our results reinforce the notion that pesticide contamination of beeswax negatively affects the reproductive quality of drones, which can affect the queens they mate with, ultimately compromising colony health.

Lethality of synthetic and natural acaricides to worker honey bees (Apis mellifera) and their impact on the expression of health and detoxification-related genes.

In this study, honey bees (Apis mellifera L.) were exposed to LD05 and LD50 doses of five commonly used acaricides for controlling the parasitic mite, Varroa destructor. LD50 values at 48 h post-treatment showed that tau-fluvalinate was the most toxic, followed by amitraz, coumaphos, thymol, and formic acid. However, the hazard ratios, which estimate the hive risk level based on a ratio of a standard dose of acaricide per hive to the LD50 of the acaricide, revealed that tau-fluvalinate was the most hazardous followed by formic acid, coumaphos, amitraz, and thymol. The expression of the honey bee acetylcholinesterase gene increased after treatment with the LD05 and LD50 acaricide doses and could distinguish three patterns in the timing and level of increased expression between acaricides: one for amitraz, one for tau-fluvalinate and formic acid, and one for coumaphos and thymol. Conversely, changes in cytochrome P450 gene expression could also be detected in response to all five acaricides, but there were no significant differences between them. Changes in vitellogenin gene expression could only detect the effects of tau-fluvalinate, amitraz, or coumaphos treatment, which were not significantly different from each other. Among the acaricides tested, coumaphos, amitraz, and thymol appear to be the safest acaricides based on their hazard ratios, and a good marker to detect differences between the effects of sub-lethal doses of acaricides is monitoring changes in acetylcholinesterase gene expression.

Chronic toxicity of amitraz, coumaphos and fluvalinate to Apis mellifera L. larvae reared in vitro.

The effects of chronic exposure to common acaricides on Apis mellifera survival, developmental rate and larval weight were tested in the laboratory. Larvae were reared in vitro and fed a diet containing amitraz: 1.5, 11, 25 and 46 mg/L; coumaphos: 1.8, 6, 8 and 25 mg/L; or fluvalinate: 0.1, 1, 2.4 and 6 mg/L. The dependent variables were compared for groups feeding on treated diets and control diets: positive control, 45 mg/L dimethoate; solvent control; and negative control. Bee survival decreased in the 46 mg/L amitraz and 25 mg/L coumaphos treatments but not in any fluvalinate treatment. Furthermore, the developmental rate decreased in individuals treated with 46 mg/L amitraz. In our study, larvae exposed to acaricides at concentrations similar to maximum residue in pollen and honey/nectar had no detectable change in survival or developmental rate. Given that pollen and honey/nectar represent only a small part of larval diet, we suggest that residues of amitraz, coumaphos and fluvalinate at the levels we tested are unlikely to impact immature worker bee survival in the field, though our data do not preclude any sublethal effects that may result from bee exposure to these compounds or possible synergisms when they co-occur in bee colonies.

Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos.

Honey bee population declines are of global concern. Numerous factors appear to cause these declines including parasites, pathogens, malnutrition and pesticides. Residues of the organophosphate acaricide coumaphos and the neonicotinoid insecticide imidacloprid, widely used to combat Varroa mites and for crop protection in agriculture, respectively, have been detected in wax, pollen and comb samples. Here, we assess the effects of these compounds at different doses on the viability of sperm stored in the honey bee queens' spermatheca. Our results demonstrate that sub-lethal doses of imidacloprid (0.02ppm) decreased sperm viability by 50%, 7days after treatment. Sperm viability was a downward trend (about 33%) in queens treated with high doses of coumaphos (100ppm), but there was not significant difference. The expression of genes that are involved in development, immune responses and detoxification in honey bee queens and workers exposed to chemicals was measured by qPCR analysis. The data showed that expression levels of specific genes were triggered 1day after treatment. The expression levels of P450 subfamily genes, CYP306A1, CYP4G11 and CYP6AS14 were decreased in honey bee queens treated with low doses of coumaphos (5ppm) and imidacloprid (0.02ppm). Moreover, these two compounds suppressed the expression of genes related to antioxidation, immunity and development in queens at day 1. Up-regulation of antioxidants by these compounds in worker bees was observed at day 1. Coumaphos also caused a repression of CYP306A1 and CYP4G11 in workers. Antioxidants appear to prevent chemical damage to honey bees. We also found that DWV replication increased in workers treated with imidacloprid. This research clearly demonstrates that chemical exposure can affect sperm viability in queen honey bees.

Genomic analysis of the interaction between pesticide exposure and nutrition in honey bees (Apis mellifera).

Populations of pollinators are in decline worldwide. These declines are best documented in honey bees and are due to a combination of stressors. In particular, pesticides have been linked to decreased longevity and performance in honey bees; however, the molecular and physiological pathways mediating sensitivity and resistance to pesticides are not well characterized. We explored the impact of coumaphos and fluvalinate, the two most abundant and frequently detected pesticides in the hive, on genome-wide gene expression patterns of honey bee workers. We found significant changes in 1118 transcripts, including genes involved in detoxification, behavioral maturation, immunity, and nutrition. Since behavioral maturation is regulated by juvenile hormone III (JH), we examined effects of these miticides on hormone titers; while JH titers were unaffected, titers of methyl farnesoate (MF), the precursor to JH, were decreased. We further explored the association between nutrition- and pesticide-regulated gene expression patterns and demonstrated that bees fed a pollen-based diet exhibit reduced sensitivity to a third pesticide, chlorpyrifos. Finally, we demonstrated that expression levels of several of the putative pesticide detoxification genes identified in our study and previous studies are also upregulated in response to pollen feeding, suggesting that these pesticides and components in pollen modulate similar molecular response pathways. Our results demonstrate that pesticide exposure can substantially impact expression of genes involved in several core physiological pathways in honey bee workers. Additionally, there is substantial overlap in responses to pesticides and pollen-containing diets at the transcriptional level, and subsequent analyses demonstrated that pollen-based diets reduce workers' pesticide sensitivity. Thus, providing honey bees and other pollinators with high quality nutrition may improve resistance to pesticides.

Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae.

Recently, the widespread distribution of pesticides detected in the hive has raised serious concerns about pesticide exposure on honey bee (Apis mellifera L.) health. A larval rearing method was adapted to assess the chronic oral toxicity to honey bee larvae of the four most common pesticides detected in pollen and wax--fluvalinate, coumaphos, chlorothalonil, and chloropyrifos--tested alone and in all combinations. All pesticides at hive-residue levels triggered a significant increase in larval mortality compared to untreated larvae by over two fold, with a strong increase after 3 days of exposure. Among these four pesticides, honey bee larvae were most sensitive to chlorothalonil compared to adults. Synergistic toxicity was observed in the binary mixture of chlorothalonil with fluvalinate at the concentrations of 34 mg/L and 3 mg/L, respectively; whereas, when diluted by 10 fold, the interaction switched to antagonism. Chlorothalonil at 34 mg/L was also found to synergize the miticide coumaphos at 8 mg/L. The addition of coumaphos significantly reduced the toxicity of the fluvalinate and chlorothalonil mixture, the only significant non-additive effect in all tested ternary mixtures. We also tested the common 'inert' ingredient N-methyl-2-pyrrolidone at seven concentrations, and documented its high toxicity to larval bees. We have shown that chronic dietary exposure to a fungicide, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrants further investigation. We suggest that pesticide mixtures in pollen be evaluated by adding their toxicities together, until complete data on interactions can be accumulated.

Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera.

As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses.

Exposure to multiple cholinergic pesticides impairs olfactory learning and memory in honeybees.

Pesticides are important agricultural tools often used in combination to avoid resistance in target pest species, but there is growing concern that their widespread use contributes to the decline of pollinator populations. Pollinators perform sophisticated behaviours while foraging that require them to learn and remember floral traits associated with food, but we know relatively little about the way that combined exposure to multiple pesticides affects neural function and behaviour. The experiments reported here show that prolonged exposure to field-realistic concentrations of the neonicotinoid imidacloprid and the organophosphate acetylcholinesterase inhibitor coumaphos and their combination impairs olfactory learning and memory formation in the honeybee. Using a method for classical conditioning of proboscis extension, honeybees were trained in either a massed or spaced conditioning protocol to examine how these pesticides affected performance during learning and short- and long-term memory tasks. We found that bees exposed to imidacloprid, coumaphos, or a combination of these compounds, were less likely to express conditioned proboscis extension towards an odor associated with reward. Bees exposed to imidacloprid were less likely to form a long-term memory, whereas bees exposed to coumaphos were only less likely to respond during the short-term memory test after massed conditioning. Imidacloprid, coumaphos and a combination of the two compounds impaired the bees' ability to differentiate the conditioned odour from a novel odour during the memory test. Our results demonstrate that exposure to sublethal doses of combined cholinergic pesticides significantly impairs important behaviours involved in foraging, implying that pollinator population decline could be the result of a failure of neural function of bees exposed to pesticides in agricultural landscapes.

The organophosphate insecticide Coumaphos induces oxidative stress and increases antioxidant and detoxification defences in the green macroalgae Ulva pertusa.

It is well established that many pesticides used in the farming and horticultural industries are harmful to not only the target species they were developed for, but also other organisms. Organophosphates were introduced as a replacement for the organochlorines and are generally considered non-toxic to plants and algae. This study investigated the impact of Coumaphos, a commonly used organophosphate, on the estuarine macrophyte Ulva pertusa. In a seven-day experiment U. pertusa cultures were exposed to four environmentally relevant concentrations of Coumaphos (0.01 mg/L, 0.05 mg/L, 0.1mg/L, 0.5 mg/L), well below the aqueous solubility maximum of the insecticide. The impact of Coumaphos was determined at a cellular level by assessing oxidative damage in the form of protein carbonyl and lipid hydroperoxide levels. Furthermore, non-enzymatic antioxidant levels and changes in the levels of enzymatic antioxidants and the enzyme GST were measured. Concentrations of Coumaphos above 0.01 mg/L caused rapid increases in the levels of protein carbonyls and lipid hydroperoxides peaking after 2-3 days of exposure, followed by a rapid decline in both markers of oxidative stress. Glutathione levels and the activities SOD, CAT, GR, APOX and GST all increased in response to the higher concentrations of Coumaphos tested and remained elevated for the duration of the experiment. These results demonstrate that environmentally relevant levels of the insecticide Coumaphos can cause oxidative damage and increase the antioxidant scavenging capacity, and GST activity in U. pertusa. This could potentially alter resource allocation within this alga, impacting algal growth and development, with possible indirect ecological consequences.

The antioxidant effect of wheat germ oil on subchronic coumaphos exposure in mice.

Forty-eight male Balb/C mice, allocated to 4 equal groups, constituted the material of the study. The first group was maintained as the control group and was administered solely with a vehicle, which was used to dissolve coumaphos in the third and fourth groups. The second group was administered with 1.5 ml/kg.bw/day (∼1400 mg/kg.bw/day) of wheat germ oil. The third group received 5.5mg/kg.bw/day (1/10 LD50(oral)) of coumaphos. Finally, the fourth group was given both coumaphos and wheat germ oil at the doses indicated above. In all groups, the compounds were given directly into the stomach using a gastric tube, and treatment was continued for a period of 45 days. At the end of the 45th day, the liver, lungs, kidneys, brain, heart and spleen were extirpated in all of the animals. Tissue homogenates prepared from the tissue specimens were analysed for malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. In conclusion, it was determined that coumaphos led to adverse alterations in the majority of the oxidative stress markers investigated. The administration of wheat germ oil alleviated the coumpahos-induced adverse effects detected in the tissues examined.

Weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis.

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.

Monitoring for resistance to organophosphorus and pyrethroid insecticides in Varroa mite populations.

The occurrence of resistance in Varroa mite populations is a serious threat to the beekeeping industry and to crops that rely on the honey bee for pollination. Integrated pest management strategies for control of this pest include the judicious use of insecticides. To monitor field populations of Varroa mite for insecticide resistance, a glass vial bioassay procedure was developed to use in the development of a resistance management strategy. Diagnostic concentrations needed to separate susceptible genotypes from resistant individuals were determined for cypermethrin (0.1 microg per vial), fluvalinate (5.0 microg per vial), malathion (0.01 microg per vial), coumaphos (10.0 microg per vial), diazinon (5.0 microg per vial), methomyl (0.5 microg per vial), propoxur (0.1 microg per vial), and endosulfan (2.5 microg per vial). Resistance to organophosphorus insecticides (malathion, coumaphos) and pyrethroids (cypermetrhrin, fluvalinate) was widespread in both La Media Ranch, TX, and Wewahitchka, FL, from 2007 to 2009. There was no resistance to endosulfan, diazinon, methomyl, and propoxur in field populations of Varroa mite in the two locations where resistance was monitored. The seasonal patterns of resistance in Wewahitchka were different from those of La Media Ranch. In the former location, the frequency of resistance to all insecticides tested decreased significantly from 2007 to 2009, whereas it increased in the latter location. Resistance levels were unstable, suggesting that resistance could be successfully managed. The results validate use of the glass vial bioassay to monitor for resistance in Varroa mite and provide the basis for the development of a resistance management strategy designed to extend the efficacy of all classes of insecticides used for control of Varroa mite.

Coumaphos exposure and incident cancer among male participants in the Agricultural Health Study (AHS).

BACKGROUND: Coumaphos is an organophosphate livestock insecticide. Previous research in the Agricultural Health Study (AHS) cohort observed a positive association between coumaphos and prostate cancer in men with a family history of prostate cancer. OBJECTIVES: This study was performed to determine the association between coumaphos and other major cancer sites and to explore the consistency of the association with prostate cancer early (1993-1999) and later (2000-2005) in AHS follow-up. METHODS: This study included 47,822 male licensed pesticide applicators. Incident cases were ascertained by linkage to state cancer registries, and exposure data were collected by enrollment questionnaire. Poisson regression was used to estimate rate ratio (RR) and 95% confidence interval (CI) of cancer for coumaphos exposure controlling for potentially confounding variables. RESULTS: Approximately 8% of applicators reported use of coumaphos; 8.5% reported a family history of prostate cancer. Cumulative exposure to coumaphos was not associated with cancer risk overall or with any major cancer site including prostate. In men with a family history of prostate cancer, we observed a positive association between ever use of coumaphos and prostate cancer in both early (RR = 2.07; 95% CI, 1.19-3.62, p-interaction = 0.005) and later (RR = 1.46; 95% CI, 0.89-2.40; p-interaction = 0.11) periods of follow-up. Across all years, this association was statistically significant (RR = 1.65; 95% CI, 1.13-2.38; p-interaction = 0.004). CONCLUSION: Coumaphos was not associated with any cancer evaluated here. In men with a family history of disease, there was evidence of an association between coumaphos and prostate cancer, possibly due to genetic susceptibility; however, other explanations, including chance, are plausible.

First detection of Varroa destructor resistance to coumaphos in Argentina.

In Argentina, studies on Varroa destructor resistance to coumaphos are still unknown. At present, high infestation levels of V. destructor are being detected in colonies of Apis mellifera after treatment with this acaricide. The aim of the present study was to determine the LC50 of coumaphos in V. destructor from four apiaries with high mite density after treatment with coumaphos. The LC50's were 112, 319, 127 and 133 microg/Petri dish for mites from the four apiaries. Significant LC50 differences were detected between resistant and susceptible mites. LC50 increased 197-559-fold when compared to the corresponding baseline, suggesting the development of resistance. These results are the first report of resistance to coumaphos in V. destructor in Argentina.

Microarray analysis of acaricide-inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus.

Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13 000 probes, having been derived from a previously described R. microplus gene index (BmiGI Version 2; Wang et al., 2007). Relative quantitative reverse transcriptase-PCR, real time PCR, and serial analysis of gene expression data was used to verify microarray data. Among the differentially expressed genes with informative annotation were legumain, glutathione S-transferase, and a putative salivary gland-associated protein.