Honey Bee Larvae Research Articles

Glyphosate affects larval gut microbiota and metamorphosis of honey bees with differences between rearing procedures

The honey bee Apis mellifera is a sentinel species of the pollinator community which is exposed to a wide variety of pesticides. In the last half-century, the pesticide most applied worldwide has been the herbicide glyphosate (GLY) used for weed control and with microbiocide effects. After its application in crops, the GLY residues have been detected in flowers visited by honey bees as well as in the stored food of their hives. Therefore, the honey bee brood can ingest the herbicide during larval development. Recent studies proved that GLY has detrimental effects on adult honey bees and other insects associated with the disturbance of their gut microbiota. GLY induces changes in the growth, metabolism and survival of honey bees and stingless bees reared in vitro. However, the effect of GLY on larval microbiota is unknown so far and there are few studies with an in-hive exposure to GLY. For these reasons, this study aims to determine whether GLY induces dysbiosis in honey bee larvae and affects their metamorphosis during the exposure period (pre-defecation) and the post-exposure period. Furthermore, we assessed this herbicide in vitro and in the hive to compare its effects on different rearing procedures. Finally, we tested the pigment BLUE1 as an indirect exposure marker to detect and estimate the in-hive intake concentration of GLY. Our results indicate that the intake of field-relevant concentrations of GLY induced a slowdown in growth with dysbiosis in the larval gut microbiota followed by late effects on their metamorphosis such as teratogenesis and mortality of newly emerged bees. Nevertheless, brood from the same colonies expressed different signs of toxicity depending on the rearing procedure and in a dose-dependent manner.

Effects of fungicides propiconazole and Trichoderma spp. on the mortality and the physiological status of larvae and adult worker honey bees (Apis mellifera L.)

Exposure to pesticides may affect the survival and normal physiological functions of the honey bee (Apis mellifera L.). The fungicide propiconazole (PRO) and the biofungicide Trichoderma are widely used in agroecosystems, and their effects on the physiology of the honey bee have been largely neglected in risk assessment procedures. We investigated the mortality and variations in physiological biomarkers of honey bee larvae and newly emerged workers exposed to PRO and Trichoderma. Exceeding the field-recommended concentrations of Trichoderma decreased the survival of newly emerged bee workers significantly through oral exposure. PRO, however, showed no significant lethal effects. Contact or oral exposure to field-realistic concentrations of PRO and Trichoderma profoundly altered the activities of detoxification, antioxidative, and digestive enzymes along with energy reserve concentrations of honey bee larvae and adult workers. Integrated biological index (IBRv2) values suggested potential risks of PRO and Trichoderma to honey bees by impairing the physiological status of larvae and newly emerged workers.

Exploring RNA methylation as a promising biomarker for assessing sublethal effects of fipronil on honeybees (Apis mellifera L.)

Honeybees play a crucial role as pollinators for crops and are regarded as sensitive bioindicators of environmental health. The widespread use of pesticides poses a severe threat to honeybee survival. However, there is limited information available on the specific risks associated with fipronil exposure in honeybees, particularly concerning the impact on RNA methylation throughout their lifespan. This study aimed to evaluate the effects of sublethal concentrations of fipronil on RNA m6A and m5C methylations, along with the associated genes in honeybee larvae and newly emerged adults. LC-MS/MS analysis revealed a notable hypomethylation of m5C in larvae, while hypermethylation of m6A was observed in the adult brain. Significant changes in the expression of genes such as AmWTAP, AmYTHDF, AmALKBH4, AmALKBH6, AmALKBH8, AmNSUN5, AmNOP2, AmTET1, and AmYBX1 were observed in the adult brain, whereas alterations in the expression of AmNSUN2, AmMETTL14, AmALKBH1, AmALKBH4, AmALKBH6 AmALYREF, AmTET1, and AmYBX1 were observed in the larvae. Notably, the expression of AmALKBH1 was not detected in any fipronil-treated larvae, suggesting its potential as an early risk indicator for honeybee larvae in future assessments. This pioneering study provides insights into the effects of fipronil on RNA methylations in honeybees and explores the possibility of employing RNA methylation as a tool for assessing pesticide risks in this important pollinator species. These findings offer new perspectives on honeybee protection and the development of toxicity evaluation systems for pesticides.

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.

Antibacterial effects of propolis and brood comb extracts on the causative agent of European Foulbrood (Melissococcus plutonius) in honey bees (Apis mellifera)

Among a long list of parasites and pathogens that threaten the European honey bee (Apis mellifera), European Foulbrood (EFB) has become an urgent apiary disease, as epidemic outbreaks are becoming increasingly common. EFB is a bacterial disease of larval honey bees, caused by a gram-positive, anaerobic bacterium, Melissococcus plutonius. The most effective current treatment for EFB, oxytetracycline hydrochloride, can disrupt the bee microbiome, cause bee mortality and residues may persist in honey harvested for human consumption. In this study, we explore the efficacy of more sustainable bee-derived solutions, including propolis, honey comb, and brood comb ethanol extracts. Propolis has been shown to successfully inhibit the growth of a similar larval bee disease, American Foulbrood (AFB). Using a series of dilutions of these extracts, we determined the minimum inhibitory concertation (MIC) of each bee-derived product on M. plutonius, as well as two model bacterial species, Staphylococcus saprophyticus (gram-positive) and Escherichia coli (gram-negative). Overall, we found that propolis extract was most effective at inhibiting the growth of gram-positive bacteria, and that M. plutonius was also susceptible to honey comb (MIC = 16.00 mg/mL) and brood comb (MIC = 45.33 mg/mL) extracts, but at much higher concentrations than that of propolis (MIC = 1.14 mg/mL). As previously demonstrated for AFB, propolis is a promising natural defense against EFB, which could have important implications for hive management.

Effect of fungicidal contamination on survival, morphology, and cellular immunity of Apis mellifera (Hymenoptera: Apidae).

Pesticide residues have been reported in hive-stored products for long periods. Larvae of honey bees experience oral or contact exposure to these products during their normal growth and development inside the cells. We analyzed various toxicological, morphogenic, and immunological effects of residue-based concentrations of two fungicides, captan and difenoconazole, on the larvae of worker honey bees, Apis mellifera. Selected concentrations (0.08, 0.4, 2, 10, and 50ppm) of both fungicides were applied topically at a volume of 1µL/larva/cell as single and multiple exposures. Our results revealed a continuous, concentration-dependent decrease in brood survival after 24h of treatment to the capping and emergence stages. Compared to larvae with a single exposure, the multiply exposed youngest larvae were most sensitive to fungicidal toxicity. The larvae that survived higher concentrations, especially multiple exposures, showed several morphological defects at the adult stage. Moreover, difenoconazole-treated larvae showed a significantly decreased number of granulocytes after 1h of treatment followed by an increase after 24h of treatment. Thus, fungicidal contamination poses a great risk as the tested concentrations showed adverse effects on the survival, morphology, and immunity of larval honey bees.

Use of Dicranum polysetum extract against Paenibacillus larvae causing American Foulbrood under in vivo and in vitro conditions.

Recent research shows that Dicranum species can be used to ameliorate the negative effects of honeybee bacterial diseases and that novel compounds isolated from these species may have the potential to treat bacterial diseases. This study aimed to investigate the efficacy of Dicranum polysetum Sw. against American Foulbrood using toxicity and larval model. The effectiveness of D. polysetum Sw. ethanol extract in combating AFB was investigated in vitro and in vivo. This study is important in finding an alternative treatment or prophylactic method to prevent American Foulbrood disease in honey bee colonies. Spore and vegetative forms of Paenibacillus larvae PB31B with ethanol extract of D. polysetum were tested on 2040 honey bee larvae under controlled conditions. Total phenolic and flavonoid contents of D. polysetum ethanol extracts were determined as 80.72mg/GAE(Gallic acid equivalent) and 303.20µg/mL, respectively. DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging percent inhibition value was calculated as 4.32%. In Spodoptera frugiperda (Sf9) and Lymantria dispar (LD652) cell lines, the cytotoxic activities of D. polysetum extract were below 20% at 50µg/mL. The extract was shown to considerably decrease infection in the larvae, and the infection was clinically halted when the extract was administered during the first 24h after spore contamination. The fact that the extract contains potent antimicrobial/antioxidant activity does not reduce larval viability and live weight, and does not interact with royal jelly is a promising development, particularly regarding its use to treat early-stage AFB infection.

Chronic larval exposure to thiacloprid impairs honeybee antennal selectivity, learning and memory performances.

The use of agricultural neonicotinoid insecticides has sub-lethal chronic effects on bees that are more prevalent than acute toxicity. Among these insecticides, thiacloprid, a commonly used compound with low toxicity, has attracted significant attention due to its potential impact on the olfactory and learning abilities of honeybees. The effect of sub-lethal larval exposure to thiacloprid on the antennal activity of adult honeybees (Apis mellifera L.) is not yet fully understood. To address this knowledge gap, laboratory-based experiments were conducted in which honeybee larvae were administered thiacloprid (0.5mg/L and 1.0mg/L). Using electroantennography (EAG), the impacts of thiacloprid exposure on the antennal selectivity to common floral volatiles were evaluated. Additionally, the effects of sub-lethal exposure on odor-related learning and memory were also assessed. The results of this study reveal, for the first time, that sub-lethal larval exposure to thiacloprid decreased honeybee antenna EAG responses to floral scents, leading to increased olfactory selectivity in the high-dose (1.0mg/L) group compared to the control group (0mg/L vs. 1.0mg/L: p = 0.042). The results also suggest that thiacloprid negatively affected odor-associated paired learning acquisition, as well as medium-term (1h) (0mg/L vs. 1.0mg/L: p = 0.019) and long-term memory (24h) (0mg/L vs. 1.0mg/L: p = 0.037) in adult honeybees. EAG amplitudes were dramatically reduced following R-linalool paired olfactory training (0mg/L vs. 1.0mg/L: p = 0.001; 0mg/L vs. 0.5mg/L: p = 0.027), while antennal activities only differed significantly in the control between paired and unpaired groups. Our results indicated that exposure to sub-lethal concentrations of thiacloprid may affect olfactory perception and learning and memory behaviors in honeybees. These findings have important implications for the safe use of agrochemicals in the environment.

H3K4me1 Modification Functions in Caste Differentiation in Honey Bees.

Honey bees are important species for the study of epigenetics. Female honey bee larvae with the same genotype can develop into phenotypically distinct organisms (sterile workers and fertile queens) depending on conditions such as diet. Previous studies have shown that DNA methylation and histone modification can establish distinct gene expression patterns, leading to caste differentiation. It is unclear whether the histone methylation modification H3K4me1 can also impact caste differentiation. In this study, we analyzed genome-wide H3K4me1 modifications in both queen and worker larvae and found that H3K4me1 marks are more abundant in worker larvae than in queen larvae at both the second and fourth instars, and many genes associated with caste differentiation are differentially methylated. Notably, caste-specific H3K4me1 in promoter regions can direct worker development. Thus, our results suggest that H3K4me1 modification may act as an important regulatory factor in the establishment and maintenance of caste-specific transcriptional programs in honey bees; however, the potential influence of other epigenetic modifications cannot be excluded.

Bioactive metabolites from autochthonous lactic acid bacteria inhibit the growth of Melissococcus plutonius, causal agent of European foulbrood disease in honey bees

AbstractEuropean foulbrood (EFB), a serious disease affecting honey bee larvae, Apis mellifera L. (Hymenoptera: Apidae), is caused by the Gram‐positive bacterium Melissococcus plutonius corrig. (ex White) Bailey & Collins. In this in‐vitro study, we evaluated the inhibitory potential of microbial metabolites produced by lactic acid bacteria (LAB), isolated from the honey bee gut, against M. plutonius. Several LAB species were identified by biochemical tests and 16S rRNA sequencing. Their filtered cell‐free supernatant (CFS) fluids were evaluated individually and in combination against growth and survival of three concentrations of M. plutonius (104–106 colony‐forming units mL−1), for various time intervals (24–96 h), in agar‐well diffusion and broth micro‐dilution assays. We also assessed whether the bioactive metabolites from selected LAB isolates consisted of acids, bacteriocins, and/or hydrogen peroxide, and quantified the presence of short‐chain fatty acids (SCFA; i.e., acetic, butyric, lactic, and propionic acid) by means of high‐performance liquid chromatography. Six LAB isolates inhibited M. plutonius growth: Lactobacillus acidophilus (Moro) Hansen & Mocquot, Lacticaseibacillus rhamnosus (Hansen) Zheng et al., Lactiplantibacillus plantarum (Orla‐Jensen) Zheng et al. (two isolates), Lactobacillus apis Killer et al., and Pediococcus acidilactici Lindner. The inhibitory effects on M. plutonius were larger at the lowest than at the highest M. plutonius concentration, and P. acidilactici showed the strongest antibacterial activity. Exposure to a mixture of CFS fluids of the four most potent isolates could reduce M. plutonius survival to 0% within 96 h. The antagonistic activity in the CFS of L. rhamnosus and L. apis appeared to be related to the presence of acids, whereas the antagonistic activity in L. plantarum and P. acidilactici appeared to be related to the presence of bacteriocins or bacteriocin‐like inhibitory substances (BLIS). Hydrogen peroxide did not seem to play a role. Total SCFA levels were highest in L. rhamnosus CFS fluid. These results can serve as a basis for in vivo studies on the use of LAB isolates as potential biocontrol agents against EFB in honey bees.

Monitoring of Paenibacillus larvae in Lower Austria through DNA-Based Detection without De-Sporulation: 2018 to 2022

American foulbrood is caused by the spore-forming Paenibacillus larvae. Although the disease effects honey bee larvae, it threatens the entire colony. Clinical signs of the disease are seen at a very late stage of the disease and bee colonies are often beyond saving. Therefore, through active monitoring based on screening, an infection can be detected early and bee colonies can be protected with hygiene measures. As a result, the pressure to spread in an area remains low. The cultural and molecular biological detection of P. larvae is usually preceded by spore germination before detection. In this study, we compared the results of two methods, the culture detection and RT-PCR detection of DNA directly isolated from spores. Samples of honey and cells with honey surrounding the brood were used in a five-year voluntary monitoring program in a western part of Lower Austria. DNA-extraction from spores to speed up detection involved one chemical and two enzymatic steps before mechanical bashing-beat separation and additional lysis. The results are comparable to culture-based methods, but with a large time advantage. Within the voluntary monitoring program, the proportion of bee colonies without the detection of P. larvae was high (2018: 91.9%, 2019: 72.09%, 2020: 74.6%, 2021: 81.35%, 2022: 84.5%), and in most P. larvae-positive bee colonies, only a very low spore content was detected. Nevertheless, two bee colonies in one apiary with clinical signs of disease had to be eradicated.

A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious bacterial disease affecting developing honeybee larvae and pupas. In this study, a library of 24 (thio)glycosides, glycosyl sulfones, 6-O-esters, and ethers derived from d-mannose, d-glucose, and d-galactose having C10 or C12 alkyl chain were evaluated for their antibacterial efficacy against two P. larvae strains. The efficacy of the tested compounds determined as minimal inhibitory concentrations (MICs) varied greatly. Generally, dodecyl derivatives were found to be more potent than their decylated analogs. Thioglycosides were more efficient than glycosides and sulfones. The activity of the 6-O-ether derivatives was higher than that of their ester counterparts. Seven derivatives with dodecyl chain linked (thio)glycosidically or etherically at C-6 showed high efficacy against both P. larvae strains (MICs ranged from 12.5 μM to 50 μM). Their efficacies were similar or much higher than those of selected reference compounds known to be active against P. larvae-lauric acid, monolaurin, and honeybee larval food components, 10-hydroxy-2-decenoic acid, and sebacic acid (MICs ranged from 25 μM to 6400 μM). The high efficacies of these seven derivatives suggest that they could increase the anti-P. larvae activity of larval food and improve the resistance of larvae to AFB disease through their application to honeybee colonies.

Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016–2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

Metabolic profiling of Apis mellifera larvae treated with sublethal acetamiprid doses

Acetamiprid is a neonicotinoid insecticide used in crop protection worldwide. Such widespread application can pose risks to pollinator insects, particularly to honeybees (Apis mellifera); therefore, the evaluation of the harmful effects of acetamiprid is necessary. Recent studies report behavior and gene expression dysfunction in honeybees, related to acetamiprid contamination. However, most studies do not consider potential metabolism disorders. To examine the effects of sublethal acetamiprid doses on the hemolymph metabolism of honeybees, worker bee larvae(2 days old) were fed with sucrose water containing different concentrations of acetamiprid (0, 5, and 25 mg/L) until capped (6 days old). The hemolymph (200 μL) of freshly capped larvae was collected for liquid chromatography-mass spectrometry (LC-MS). Overall, increasing acetamiprid exposure induced greater metabolic variations in worker bee larvae(treated groups compared to untreated). In the positive ion mode, 36 common differential metabolites in the acetamiprid-treated groups were screened from the identified differential metabolites. Of these, 19 metabolites were upregulated, and 17 were downregulated. 10 common differential metabolites were screened in the negative ion mode. 3 metabolites were upregulated, and 7 metabolites were downregulated. These common metabolites included traumatic acid, indole etc. These commonly differentiated metabolites were classified as compounds with biological roles, lipids, and phytochemical compounds, and others. The metabolic pathways of common differentiated metabolites with significant differences (P < 0.05) included the metabolism of tryptophan, purines, phenylalanine, etc. As the concentration of acetamiprid increased, the content of traumatic acid increased, the content of tryptophan metabolite l-kynurenine and indole decreased, and the content of lipids also decreased. Our results revealed that the damage to honeybee larvae increased when the acetamiprid solution formulations residue in their food had a concentration higher than 5 mg/L, causing metabolic disorders in various substances in larvae. Analysis of these metabolic processes can provide a theoretical basis for further research on the metabolism of acetamiprid-treated honeybees and elucidate the detoxification mechanisms.

Black queen cell virus detected in Canadian mosquitoes.

Black queen cell virus (BQCV) is a ubiquitous honeybee virus and a significant pathogen to queen bee (Apis mellifera) larvae. However, many aspects of the virus remain poorly understood, including the transmission dynamics. In this study, we used next-generation sequencing to identify BQCV in Aedes vexans (n = 4,000) collected in 2019 and 2020 from Manitoba, Canada. We assembled de novo the nearly complete (>96%) genome sequence of the virus, which is the first available from North America and the first report of BQCV being harbored by mosquitoes. Phylogenetic tree reconstructions indicated that the genome had 95.5% sequence similarity to a BQCV isolate from Sweden. Sequences of a potential vector (Varroa destructor) and a microsporidian associated with BQCV (Nosema apis) were not identified in the mosquito samples, however, we did detect sequences of plant origin. We, therefore, hypothesize that the virus was indirectly acquired by mosquitoes foraging at the same nectar sources as honeybees.

Honey bee larval culture in vitro: gut emptying determines the transition from larva to prepupa and recombinant AccApidaecin improves antibacterial activity.

In vitro rearing of honey bee larvae is ideal for bioassay studies; no honey bee stable cell lines are available. Inconsistency of internal development staging of reared larvae and a susceptibility to contamination are common problems encountered. Standardized protocols on rearing larvae in vitro to make the larvae growth and development more similar to that of natural colonies are necessary to ensure the accuracy of experimental results and promote honey bee research as a model organism. Here, we concluded that when larval fasting weight was >160 mg, the time point of gut emptying can be defined as the critical point separating the larval and prepupal stages. In this way, we can conduct precise studies on the prepupal stage, such as organ remodeling during metamorphosis. Simultaneously, we further verified that recombinant AccApidaecin in genetic engineered bacteria added to the larval diet upregulated antibacterial peptide gene expression, and did not stimulate the stress response in larvae, nor did it affect the pupation rate or eclosion rate. This demonstrated that feeding recombinant AccApidaecin can enhance the individual antibacterial ability at the molecular level.

American Foulbrood-Old and Always New Challenge.

American foulbrood (AFB) is exclusively an infectious disease of honey bee larvae (Apis mellifera) and their subspecies that is spread easily and rapidly and is often present in apiaries. Due to the resistance and pathogenicity of the bacterial causative agent of the disease, which has considerable epizootiological and economic significance for beekeeping, AFB was classified as a highly dangerous, infectious animal disease by the World Organization for Animal Health (WOAH). Considering the severity of the infection, a frequent occurrence, rapid and easy spread, epizooty and enzooty are common. We tried to present an overview of the latest information related to AFB through several chapters. In addition to the latest data on the etiology of the causative agent, the most important elements of the clinical signs of the disease are also listed. Along with an overview of classic microbiological and the latest molecular methods of diagnosis, we also discuss AFB treatment from its differential diagnostic aspect. We hope that through demonstrating the mentioned preventive measures and measures of good beekeeping practice, the review will contribute to the preservation of the health of bees and therefore the overall biodiversity of the planet.

Hexanic extract of Achyrocline satureioides: antimicrobial activity and in vitro inhibitory effect on mechanisms related to the pathogenicity of Paenibacillus larvae.

Paenibacillus larvae is a spore-forming bacillus, the most important bacterial pathogen of honeybee larvae and the causative agent of American foulbrood (AFB). Control measures are limited and represent a challenge for both beekeepers and researchers. For this reason, many studies focus on the search for alternative treatments based on natural products. The objective of this study was to determine the antimicrobial activity of the hexanic extract (HE) of Achyrocline satureioides on P. larvae and the inhibitory activity on some mechanisms related to pathogenicity. The Minimum Inhibitory Concentration (MIC) of the HE was determined by the broth microdilution technique and the Minimum Bactericidal Concentration (MBC) by the microdrop technique. Swimming and swarming motility was evaluated in plates with 0.3 and 0.5% agar, respectively. Biofilm formation was evaluated and quantified by the Congo red and crystal violet method. The protease activity was evaluated by the qualitative technique on skim milk agar plates. It was determined that the MIC of the HE on four strains of P. larvae ranged between 0.3 and 9.37µg/ml and the MBC between 1.17 and 150µg/ml. On the other hand, sub-inhibitory concentrations of the HE were able to decrease swimming motility, biofilm formation and the proteases production of P. larvae.

Exposure of chlorothalonil and acetamiprid reduce the survival and cause multiple internal disturbances in Apis mellifera larvae reared in vitro.

Background: Chlorothalonil and acetamiprid are chemical pesticides commonly used in agricultural production and have been shown to have negative effects on bee's fitness. Despite many studies have revealed that honey bee (Apis mellifera L.) larvae are posting a high risk on exposure to pesticides, but the toxicology information of chlorothalonil and acetamiprid on bee larvae remain limited. Results: The no observed adverse effect concentration (NOAEC) of chlorothalonil and acetamiprid for honey bee larvae were 4μg/mL and 2μg/mL, respectively. Except for CarE, the enzymic activities of GST and P450 were not influenced by chlorothalonil at NOAEC, while chronic exposure to acetamiprid slightly increased the activities of the three tested enzymes at NOAEC. Further, the exposed larvae showed significantly higher expression of genes involved in a series of different toxicologically relevant process following, including caste development (Tor (GB44905), InR-2 (GB55425), Hr4 (GB47037), Ac3 (GB11637) and ILP-2 (GB10174)), immune system response (abaecin (GB18323), defensin-1 (GB19392), toll-X4 (GB50418)), and oxidative stress response (P450, GSH, GST, CarE). Conclusion: Our results suggest that the exposure to chlorothalonil and acetamiprid, even at concentrations below the NOAEC, showed potentially effects on bee larvae's fitness, and more important synergistic and behavioral effects that can affect larvae fitness should be explored in the further.

Field recommended concentrations of pyraclostrobin exposure disturb the development and immune response of worker bees (Apis mellifera L.) larvae and pupae.

The strobilurin fungicide pyraclostrobin is widely used to prevent and control the fungal diseases of various nectar and pollen plants. Honeybees also directly or indirectly contact this fungicide with a long-term exposure period. However, the effects of pyraclostrobin on the development and physiology of Apis mellifera larvae and pupae during continuous exposure have been rarely known. To investigate the effects of field-realistic concentrations of pyraclostrobin on honeybee survival and development, the 2-day-old larvae were continuously fed with different pyraclostrobin solutions (100mg/L and 83.3mg/L), and the expression of development-, nutrient-, and immune-related genes in larvae and pupae were examined. The results showed that two field-realistic concentrations of pyraclostrobin (100 and 83.3mg/L) significantly decreased the survival and capped rate of larvae, the weight of pupae and newly emerged adults, and such decrease was a positive correlation to the treatment concentrations. qPCR results showed that pyraclostrobin could induce the expression of Usp, ILP2, Vg, Defensin1, and Hymenoptaecin, decrease the expression of Hex100, Apidaecin, and Abaecin in larvae, could increase the expression of Ecr, Usp, Hex70b, Vg, Apidaecin, and Hymenoptaecin, and decreased the expression of ILP1, Hex100 and Defensin1in pupae. These results reflect pyraclostrobin could decrease nutrient metabolism, immune competence and seriously affect the development of honeybees. It should be used cautiously in agricultural practices, especially in the process of bee pollination.