Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Nosema ceranae Escapes Fumagillin Control in Honey Bees

Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.     

Effects of abamectin and deltamethrin to the foragers honeybee workers of Apis mellifera jemenatica (Hymenoptera: Apidae) under laboratory conditions

This study aimed at evaluating the toxicity of some insecticides (abamectin and deltamethrin) on the lethal time (LT₅₀) and midgut of foragers honeybee workers of Apis mellifera jemenatica were studied under laboratory conditions. The bees were provided with water, food, natural protein and sugar solution with insecticide (concentration: 2.50 ppm deltamethrin and 0.1 ppm abamectin). The control group was not treated with any kind of insecticides. The mortality was assessed at 1, 2, 4, 6, 12, 24, 48, and 72 hour (h) after insecticides treatment and period to calculate the value of lethal time (LT₅₀). But the samples the histology study of midgut collected after 24 h were conducted by Scanning Electron Microscope. The results showed the effects of insecticides on the current results show that abamectin has an adverse effect on honeybees, there is a clear impact on the lethal time (LT₅₀) was the abamectin faster in the death of honeybee workers compared to deltamethrin. Where have reached to abamectin (LT₅₀ = 21.026) h, deltamethrin (LT₅₀ = 72.011) h. However, abamectin also effects on cytotoxic midgut cells that may cause digestive disorders in the midgut, epithelial tissue is formed during morphological alterations when digestive cells die. The extends into the internal cavity, and at the top, there is epithelial cell striated border that has many holes and curves, abamectin seems to have crushed the layers of muscle. Through the current results can say abamectin most toxicity on honeybees colony health and vitality, especially foragers honeybee workers.     

Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (<Emphasis Type="Italic">Apis mellifera L.</Emphasis>) larvae

Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 μg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.     

Long‐term risk assessment on noneffective and effective toxic doses of imidacloprid to honeybee workers

Imidacloprid is the most widely used insecticide in agriculture. Its impact on honeybees has received worldwide attention. Foliar sprays are commonly and frequently used for piercing insect control, particularly on cotton in southern USA. To simulate field exposures of formulated imidacloprid (Advise^® 2Fl), we used a modified spray tower to treat honeybee workers and monitored five enzyme activities and survival for up to 52 days. Results indicated that spray treatments twice a week for 52 days with 0.001 and 1 mg/L and once a week for three weeks with 4.3 mg/L Advise showed no adverse effect on bee survival, where imidacloprid‐treated bees could live as long as untreated bees. Concentration ≥80 mg/L significantly reduced bee survival, and substantial number of bees continued to die after 48‐hr of post‐treatment period which was commonly used for measuring insecticide toxicity. The body weight of imidacloprid‐treated bees (at LC₂₀ and LC₅₀) was also significantly reduced. Enzymatic data showed that activities of detoxification enzymes esterase and glutathione S‐transferase (GST), insecticide‐target enzyme acetylcholinesterase (AChE) and honey enzyme invertase in imidacloprid‐treated survivors were mostly similar to those found in untreated bees. The immunity‐related phenoloxidase (PO) activity in imidacloprid‐treated survivors was also mostly similar to that of untreated control, but higher PO activity was detected in bees treated with higher concentrations for 3 weeks. By using both bioassays and enzymatic assays, this study revealed long‐term noneffective and effective concentrations of imidacloprid that may be useful for accurate assessment of toxicity risk of neonicotinoids to bees.     

The neonicotinoids thiacloprid,imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.)

A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24 h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50–200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.     

Conserving genetic diversity in the honeybee: Comments on Harpur et al. (2012)

The article by Harpur et al. (2012) ‘Management increases genetic diversity of honey bees via admixture’ concludes that ‘…honey bees do not suffer from reduced genetic diversity caused by management and, consequently, that reduced genetic diversity is probably not contributing to declines of managed Apis mellifera populations’. In the light of current honeybee and beekeeping declines and their consequences for honeybee conservation and the pollination services they provide, we would like to express our concern about the conclusions drawn from the results of Harpur et al. (2012). While many honeybee management practices do not imply admixture, we are convinced that the large-scale genetic homogenization of admixed populations could drive the loss of valuable local adaptations. We also point out that the authors did not account for the extensive gene flow that occurs between managed and wild/feral honeybee populations and raise concerns about the data set used. Finally, we caution against underestimating the importance of genetic diversity for honeybee colonies and highlight the importance of promoting the use of endemic honeybee subspecies in apiculture.     

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2 ng/bee) on the thorax temperatures of foragers exposed to low (22 °C) and high (33 °C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1–2 h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60–90 min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5 min at 4 °C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.     

The fungicide Pristine® inhibits mitochondrial function in vitro but not flight metabolic rates in honey bees

Honey bees and other pollinators are exposed to fungicides that act by inhibiting fungal mitochondria. Here we test whether a common fungicide (Pristine®) inhibits the function of mitochondria of honeybees, and whether consumption of ecologically-realistic concentrations can cause negative effects on the mitochondria of flight muscles, or the capability for flight, as judged by CO₂ emission rates and thorax temperatures during flight. Direct exposure of mitochondria to Pristine® levels above 5 ppm strongly inhibited mitochondrial oxidation rates in vitro. However, bees that consumed pollen containing Pristine® at ecologically-realistic concentrations (≈1 ppm) had normal flight CO₂ emission rates and thorax temperatures. Mitochondria isolated from the flight muscles of the Pristine®-consuming bees had higher state 3 oxygen consumption rates than control bees, suggesting that possibly Pristine®-consumption caused compensatory changes in mitochondria. It is likely that the lack of a strong functional effect of Pristine®-consumption on flight performance and the in vitro function of flight muscle mitochondria results from maintenance of Pristine® levels in the flight muscles at much lower levels than occur in the food, probably due to metabolism and detoxification. As Pristine® has been shown to negatively affect feeding rates and protein digestion of honey bees, it is plausible that Pristine® consumption negatively affects gut wall function (where mitochondria may be exposed to higher concentrations of Pristine®).     

Histological changes in the Dengue vector,Aedes aegypti (Diptera: Culicidae) larvae treated with neem oil loaded niosomes

Mosquitoes are one of the greatest threats to human health around the globe. They act as vectors for common diseases like Malaria, Dengue, Chikungunya, etc. Niosomes encapsulated with neem oil showed a significant mortality rate against Aedes aegypti larvae when treated for 24 h. In this study, the histological changes that led to the mortality of the Aedes aegypti mosquito larvae were studied. Organs of the late III-instar stage larvae such as head, optic lobes, cuticle, adipose tissue, midgut region, haemolymph were investigated. Several histological alterations such as disorientation of the brain and antenna in the head part, damage in the optic lobe and microvilli were observed. Total disruption was seen in the inner and outer retractor muscles of the larval body. The midgut and hindgut regions were disintegrated due to the damage to the fat bodies in the region. A Series of such histological changes in the body of mosquito larvae compared to the control larvae hindered metabolic functions leading to death. The results suggested that the neem oil loaded niosomes could be used as a biocontrol agent against the Dengue vector, Aedes aegypti larvae.     

Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid)

Currently, there is concern about declining bee populations and the sustainability of pollination services. One potential threat to bees is the unintended impact of systemic insecticides, which are ingested by bees in the nectar and pollen from flowers of treated crops. To establish whether imidacloprid, a systemic neonicotinoid and insect neurotoxin, harms individual bees when ingested at environmentally realistic levels, we exposed adult worker bumble bees, Bombus terrestris L. (Hymenoptera: Apidae), and honey bees, Apis mellifera L. (Hymenoptera: Apidae), to dietary imidacloprid in feeder syrup at dosages between 0.08 and 125 μg l^?1. Honey bees showed no response to dietary imidacloprid on any variable that we measured (feeding, locomotion and longevity). In contrast, bumble bees progressively developed over time a dose-dependent reduction in feeding rate with declines of 10–30% in the environmentally relevant range of up to 10 μg l^?1, but neither their locomotory activity nor longevity varied with diet. To explain their differential sensitivity, we speculate that honey bees are better pre-adapted than bumble bees to feed on nectars containing synthetic alkaloids, such as imidacloprid, by virtue of their ancestral adaptation to tropical nectars in which natural alkaloids are prevalent. We emphasise that our study does not suggest that honey bee colonies are invulnerable to dietary imidacloprid under field conditions, but our findings do raise new concern about the impact of agricultural neonicotinoids on wild bumble bee populations.     

Comparative laboratory toxicity of neem pesticides to honey bees (Hymenoptera: Apidae), their mite parasites Varroa jacobsoni (Acari: Varroidae) and Acarapis woodi (Acari: Tarsonemidae), and brood pathogens Paenibacillus larvae and Ascophaera apis

Laboratory bioassays were conducted to evaluate neem oil and neem extract for the management of key honey bee (Apis mellifera L.) pests. Neem pesticides inhibited the growth of Paenibacillus larvae (Ash, Priest & Collins) in vitro but had no effect on the growth of Ascophaera apis (Olive & Spiltoir). Azadirachtin-rich extract (neem-aza) was 10 times more potent than crude neem oil (neem oil) against P. larvae suggesting that azadirachtin is a main antibiotic component in neem. Neem-aza, however, was ineffective at controlling the honey bee mite parasites Varroa jacobsoni (Ouduemans) and Acarapis woodi (Rennie). Honey bees also were deterred from feeding on sucrose syrup containing > 0.01 mg/ml of neem-aza. However, neem oil applied topically to infested bees in the laboratory proved highly effective against both mite species. Approximately 50-90% V. jacobsoni mortality was observed 48 h after treatment with associated bee mortality lower than 10%. Although topically applied neem oil did not result in direct A. woodi mortality, it offered significant protection of bees from infestation by A. woodi. Other vegetable and petroleum-based oils also offered selective control of honey bee mites, suggesting neem oil has both a physical and a toxicological mode of action. Although oils are not as selective as the V. jacobsoni acaricide tau-fluvalinate, they nonetheless hold promise for the simultaneous management of several honey bee pests.     

Survival and health improvement of Nosema infected Apis florea (Hymenoptera: Apidae) bees after treatment with propolis extract

Nosema ceranae is now considered to be an emerging infectious disease of the European honey bee Apis mellifera. Only one antibiotic, Fumagillin, is commercially available to combat Nosema infections. This antibiotic treatment is banned from use in Europe and elsewhere there is a high probability for antibiotic resistance to develop. We are therefore interested in investigating the effects of a natural propolis extract on its ability to reduce N. ceranae infection loads in the dwarf honey bee, Apis florea, a native honey bee with a range that overlaps with Apis cerana and Apis mellifera that is at risk of infection. Experimentally infected caged bees were fed a treatment consisting of 0%, 50%, or 70% propolis extract. All 50% and 70% propolis treated bees had significantly lower infection loads, and the 50% treated bees had higher survival in comparison to untreated bees. In addition, propolis treated bees had significantly higher haemolymph trehalose levels and hypopharyngeal gland protein content similar to levels of uninfected bees. Propolis ethanolic extract treatment could therefore be considered as a possible viable alternative to Fumagillin to improve bee health. This natural treatment deserves further exploration to develop it as a possible alternative to combat N. ceranae infections distributed around the world.     

The effect of temperature on candidate gene expression in the brain of honey bee Apis mellifera (Hymenoptera: Apidae) workers exposed to neonicotinoid imidacloprid

Neonicotinoid insecticides are potent agonists of nicotinic acetylcholine receptors and are a major factor in the decline of pollinators worldwide. Several studies show that low doses of this neurotoxin influence honey bee physiology, however, little is known about how insecticides interact with other environmental variables. We studied the effects of two neonicotinoid Imidacloprid doses (IMD, 0, 2.5, and 10 ppb), and three temperatures (20, 28, and 36°C) on gene expression in the brains of worker honey bees (Apis mellifera). Using qRT-PCR we quantified the expression of eight key genes related to the nervous system, stress response, and motor and olfactory capacities. Gene expression tended to increase with the low IMD dose, which was further intensified in individuals maintained in the cold treatment (20°C). At 20°C the octopamine receptor gene (oa1) was underexpressed in bees that were not exposed to IMD, but overexpressed in individuals exposed to 2.5 ppb IMD. Also, heat shock proteins (hsp70 and hsp90) increased their expression at high temperatures (36°C), but not with IMD doses. These results suggest that despite the low insecticide concentrations used in this study (a field-realistic dose), changes in gene expression associated with honey bee physiological responses could be induced. This study contributes to the understanding of how neonicotinoid residual doses may alter honey bee physiology.     

Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.     

The accuracy of morphometric characteristic analysis depends on the type of the assessed traits of honey bees (Apis cerana F. and Apis mellifera L.)

Conservation of honey bees as pollinators and honey producers requires their assessment using convenient general methods. One of the most commonly used methods involves assessment of morphometric characteristics using random traits. The purpose of the study was to determine a set of morphometric characteristics for identifying the local (R, H: Apis cerana koreana) and adapted (A, C, F: Apis melifera ligustica; D: Apis mellifera carpathica; V: Apis mellifera) lines of honey bees bred in Korea. We found traits with significant differences (p < 0.001), high discriminant coefficient (>100), and range of variation (<50 %). The analysis demonstrated that the widths of the abdomen, forewing, and head, and the lengths of the antenna, body, proboscis, tergites 4 and 3, and forewing can be used as the nine main morphological characteristics for distinguishing the bred lines of honey bees. Furthermore, the last two traits and the length of the head can be used for distinguishing the adapted subspecies A. m. ligustica and A. m. carpathica. Our observations can be used for improving the morphometric method for the conservation of honey bees.     

Pollen diets and niche overlap of honey bees and native bees in protected areas

The decline of both managed and wild bee populations has been extensively reported for over a decade now, with growing concerns amongst the scientific community. Also, evidence is growing that both managed and feral honey bees may exacerbate threats to wild bees. In Australia, there are over 1600 native bee species and introduced European honey bees (Apis mellifera) have established throughout most landscapes. There is a major gap in knowledge of the interactions between honey bees and native bees in Australian landscapes, especially floral resource use.Here we report on the pollen diets of wild bees in protected areas of coastal heathland, an ecosystem characterised by mass flowering in late winter and spring. We sampled bees within three sites and DNA metabarcoding was used to compare the pollen diets of honey bees and native bees. We recorded 2, 772 bees in total, with 13 genera and 18 described species identified. Apis mellifera was the most common species across all locations, accounting for 42% of all bees collected. Native bee genera included eusocial Tetragonula (stingless bees) (37%), and semi-social Exoneura and Braunsapis (19.8% combined). Metabarcoding data revealed both Tetragonula and honey bees have wide foraging patterns, and the bipartite network overall was highly generalised (H₂’ = 0.24). Individual honey bees carried pollen of 7–29 plant species, and significantly more species than all other bees. We found niche overlap in the diets of honey bees and native bees generally (0.42), and strongest overlap with stingless bees (0.70) and species of Braunsapis (0.62). A surprising finding was that many species carried pollen from Restionaceae and Cyperaceae, families generally considered to be predominantly wind-pollinated in Australia. Our study showed introduced honey bee use of resources overlaps with that of native bees in protected heathlands, but there are clear differences in their diet preferences.     

Higher immunocompetence is associated with higher genetic diversity in feral honey bee colonies (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Honey bees are the most important managed pollinators as they provide key ecosystem services for crop production worldwide. Recent losses of honey bee colonies in North America and Europe have demonstrated a need to develop strategies to improve their health and conserve their populations. Previously, we showed that feral honey bees—colonies that live in the wild without human assistance—exhibit higher levels of immunocompetence than managed colonies in North Carolina (USA). In a first attempt to investigate the underlying mechanisms of this difference in immune response, here we characterize the genetic composition of feral and managed honey bees using microsatellite markers. Our results reveal significant but small genetic differentiation between feral and managed honey bee colonies (?_CT = 0.047, P?=?0.03) indicating admixture between these two groups. Higher genetic diversity was correlated with higher immune response in feral (P _MANOVA = 0.011) but not managed bees, despite the fact that the latter group showed significantly higher average genetic diversity (P _ANCOVA < 0.001). These findings suggest that genetic diversity is positively associated with immunocompetence in feral honey bee colonies, and that the benefits of genetic diversity are obscured in managed bees, perhaps as a result of artificial selection. We hypothesize that high genetic variability provides the raw material upon which natural selection acts and generates adaptive genotypes in unmanaged populations. Feral populations could be useful sources of genetic variation to use in breeding programs that aim to improve honey bee health.     

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.     

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 (0.02 ppm) decreased sperm viability by 50%, 7 days after treatment. Sperm viability was a downward trend (about 33%) in queens treated with high doses of coumaphos (100 ppm), 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 1 day 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 (5 ppm) and imidacloprid (0.02 ppm). 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.