Honey Bee Larvae Research Articles

Micronutrient Deficiency May Be Associated with the Onset of Chalkbrood Disease in Honey Bees.

Chalkbrood is a disease of honey bee brood caused by the fungal parasite Ascosphaera apis. Many factors such as genetics, temperature, humidity and nutrition influence the appearance of clinical symptoms. Poor nutrition impairs the immune system, which favors the manifestation of symptoms of many honey bee diseases. However, a direct link between dietary ingredients and the symptoms of chalkbrood disease has not yet been established. We show here that the elemental composition of chalkbrood mummies and healthy larvae from the same infected hives differ, as well as that mummies differ from larvae from healthy hives. Chalkbrood mummies had the highest concentration of macroelements such as Na, Mg, P, S, K and Ca and some microelements such as Rb and Sn, and at the same time the lowest concentration of B, As, Sr, Ag, Cd, Sb, Ba and Pb. Larvae from infected hives contained less Pb, Ba, Cs, Sb, Cd, Sr, As, Zn, Cu, Ni, Co, Mn, Cr, V and Al in contrast to healthy larvae from a disease-free apiary. This is the first study to demonstrate such differences, suggesting that an infection alters the larval nutrition or that nutrition is a predisposition for the outbreak of a chalkbrood infection. Though, based on results obtained from a case study, rather than from a controlled experiment, our findings stress the differences in elements of healthy versus diseased honey bee larvae.

Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.

Probiotic Potential of Bacillus Subtilis Strain I3: Antagonistic Activity Against Chalkbrood Pathogen and Pesticide Degradation for Enhancing Honeybee Health.

To explore the potential of probiotic candidates beneficial for honeybee health through the modulation of the gut microbiome, bee gut microbes were isolated from bumblebee (Bombus terrestris) and honeybee (Apis mellifera) using diverse media and cultural conditions. A total of 77 bee gut bacteria, classified under the phyla Proteobacteria, Firmicutes, and Actinobacteria, were identified. The antagonistic activity of the isolates against Ascosphaera apis, a fungal pathogen responsible for chalkbrood disease in honeybee larvae, was investigated. The highest growth inhibition percentage against A. apis was demonstrated by Bacillus subtilis strain I3 among the bacterial strains. The presence of antimicrobial peptide genes in the I3 strain was detected using PCR amplification of gene fragments encoding surfactin and fengycin utilizing specific primers. The export of antimicrobial peptides by the I3 strain into growth medium was verified using liquid chromatography coupled with mass spectroscopy. Furthermore, the strain's capabilities for degrading pesticides, used for controlling varroa mites, and its spent growth medium antioxidant activity were substantiated. The survival rate of honeybees infected with (A) apis was investigated after feeding larvae with only medium (fructose + glucose + yeast extract + royal jelly), (B) subtilis I3 strain, A. apis with medium and I3 strain + A. apis with medium. Honeybees receiving the I3 strain + A. apis exhibited a 50% reduction in mortality rate due to I3 strain supplementation under experimental conditions, compared to the control group. In silico molecular docking revealed that fengycin hydrolase from I3 strain effectively interacted with tau-fluvalinate, suggesting its potential in bee health and environmental protection. Further studies are needed to confirm the effects of the I3 strain in different populations of honey bees across several regions to account for genetic and environmental variations.

Fetal Alcohol Spectrum Disorder: The Honey Bee as a Social Animal Model.

Animal models have been essential for advancing research of fetal alcohol spectrum disorder (FASD) in humans, but few animal species effectively replicate the behavioural and clinical signs of FASD. The honey bee (Apis mellifera) is a previously unexplored research model for FASD that offers the distinct benefit of highly social behaviour. In this study, we chronically exposed honey bee larvae to incremental concentrations of 0, 3, 6, and 10% ethanol in the larval diet using an in vitro rearing protocol and measured developmental time and survival to adult eclosion, as well as body weight and motor activity of newly emerged adult bees. Larvae reared on 6 and 10% dietary ethanol demonstrated significant, dose-responsive delays to pupation and decreased survival and adult body weight. All ethanol-reared adults showed significantly decreased motor activity. These results suggest that honey bees may be a suitable social animal model for future FASD research.

Developing a method to rear Varroa destructor in vitro.

Varroa destructor is a significant mite pest of western honey bees (Apis mellifera). Developing a method to rear and maintain populations of V. destructor in vitro would provide year-round access to the mites, allowing scientists to study their biology, behavior, and control more rapidly. In this study, we determined the impact of various rearing parameters on V. destructor survival and reproduction in vitro. This was done by collecting V. destructor from colonies, placing them in gelatin capsules containing honey bee larvae, and manipulating the following conditions experimentally: rearing temperature, colony source of honey bee larva, behavioral/developmental stages of V. destructor and honey bee larva, and mite:bee larva ratio. Varroa destructor survival was significantly impacted by temperature, colony source of larvae and mite behavioral stage. In addition, V. destructor reproduction was significantly impacted by mite: larva ratio, larval developmental stage, colony source of larva, and temperature. The following conditions optimized mite survival and reproduction in vitro: using a 4:1 mite:larva ratio, beginning the study with late stage uncapped larvae, using mites collected from adult bees, maintaining the rearing temperature at 34.5° C, and screening larval colony source. Ultimately, this research can be used to improve V. destructor in vitro rearing programs.

Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs’ expression between uninoculated and A. apis–inoculated larval guts were confirmed based on molecular methods. These findings not only enrich our understanding of bee host–fungal pathogen interactions but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. c. cerana larvae against A. apis invasion.Key points• The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection.• Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis–challenged host guts.

Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

A New Isolated Fungus and Its Pathogenicity for Apis mellifera Brood in China.

In this article, we report the pathogenicity of a new strain of fungus, Rhizopus oryzae to honeybee larvae, isolated from the chalkbrood-diseased mummies of honeybee larvae and pupae collected from apiaries in China. Based on morphological observation and internal transcribed spacer (ITS) region analyses, the isolated pathogenic fungus was identified as R. oryzae. Koch's postulates were performed to determine the cause-and-effect pathogenicity of this isolate fungus. The in vitro pathogenicity of this virulent fungus in honeybees was tested by artificially inoculating worker larvae in the lab. The pathogenicity of this new fungus for honeybee larvae was both conidial-concentration and exposure-time dependent; its highly infectious and virulent effect against the larvae was observed at 1 × 105 conidia/larva in vitro after 96 h of challenge. Using probit regression analysis, the LT50 value against the larvae was 26.8 h at a conidial concentration of 1 × 105 conidia/larva, and the LC50 was 6.2 × 103 conidia/larva. These results indicate that the new isolate of R. oryzae has considerable pathogenicity in honeybee larvae. Additionally, this report suggests that pathogenic phytofungi may harm their associated pollinators. We recommend further research to quantify the levels, mechanisms, and pathways of the pathogenicity of this novel isolated pathogen for honeybee larvae at the colony level.

Atmospheric non-thermal plasma inactivation of Ascosphaera apis, the causative agent of chalkbrood disease in honeybee

Ascosphaera apis is a worldwide pathogenic fungi of honeybees that can cause a decline in bee populations. In this study, we investigated the antifungal activity of non-thermal plasma on fungal growth. Spore inactivation after exposure to gas plasma by liquid phase and plasma activated water (PAW) and pathogenicity of A. apis in vivo were also examined. The results demonstrated that the mycelial growth of fungi was completely inhibited after argon plasma treatment. Both gas plasma and PAW exposures resulted in a significant decrease of A. apis spore numbers, maximum reduction of 1.71 and 3.18-fold, respectively. Germinated fungal spores on potato dextrose agar were also reduced after plasma treatment. SEM analysis revealed a disruption in the morphological structure of the fungal spores. The pathogenicity of A. apis on honeybee larvae was decreased after spores treated by gas plasma and PAW with a disease inhibition of 63.61 ± 7.28% and 58.27 ± 5.87%, respectively after 7 days of cultivation. Chalkbrood in honey bees have limited control options and our findings are encouraging. Here, we demonstrate a possible alternative control method using non-thermal plasma for chalkbrood disease in honeybees.

Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Short communication: Ascorbyl/ascorbate ratio as a marker of oxidative stress in larvae (Apis mellifera) exposed to Paenibacillus larvae.

Paenibacillus larvae, the causal agent of American foulbrood disease (AFB), affects Apis mellifera larvae and can induce oxidative stress by overproduction of radical oxygen species (ROS). This study aimed to assess the oxidative stress levels in larvae exposed to three different strains of P. larvae through their diet by examining the ascorbyl radical (A) to ascorbate anion (AH¯) ratio. The results revealed that larvae inoculated with P. larvae exhibited a lower value of this index compared to uninoculated ones. Interestingly, the level of A remained constant, while the concentration of AH¯ increased. Said increase correlated with the virulence of the specific P. larvae strain used in the inoculation. These findings suggest a potential link between AH¯ molecules and a defense response in A. mellifera larvae against infection, consistent with their resistance to P. larvae (LD50).

Review on Chalkbrood Disease of Honey Bee

Ascosphaera apis is the sole source of the invasive mycosis known as “chalkbrood disease”, which only affects honeybee larvae. Around 5-37% less honey is produced globally as a result of the illness, and 80% of broods perish. Because of the disease’s current severity, more pesticides are used, and colonies are frequently transported over great distances, which may have created new opportunities for Ascosphaera apis infection. There are signs that the incidence of this disease, which is now widespread worldwide, may be increasing. It is also highly unlikely that Ascosphaera apis will ever be completely eradicated due to its widespread distribution throughout the world, the fact that it can persist for more than 15-years in soil near infected apiaries, and the fact that viable spores can be found in stored honey, pollen, pollen capsules and tablets, used hive components, and used beekeeping tools and equipment. Safe control of the disease has been noted to be challenging, despite the fact that management techniques, medications, and the adoption of particular chalkbrood-resistant bees have all been demonstrated to have some benefits thus far. Thus, current research for a better understanding of chalkbrood will focus on genetic engineering for better fungus manipulation, molecular investigation of Ascosphaera apis pathogenesis pathways, and the potential identification of strategies to break the pathway through the identification of responsible genes for its pathogenesis. Some researchers have noted the prevalence of chalkbrood disease and linked chalkbrood diseases to bee colony losses and decreased productivity in affected bee colonies.

Early-Life Sublethal Thiacloprid Exposure to Honey Bee Larvae: Enduring Effects on Adult Bee Cognitive Abilities.

Honey bees have significant ecological and economic value as important pollinators, but they are continuously exposed to various environmental stressors, including insecticides, which can impair their health and cause colony decline. (1) Background: Cognitive abilities are vital for the functional maintenance of honey bees; however, it remains unknown if chronic, low-dose exposure to thiacloprid during the larval stage impairs the cognitive abilities of emerged adult honey bees. (2) Methods: To explore this question, honey bee larvae were fed 0, 0.5, and 1.0 mg/L thiacloprid during their developmental phase. Then, the cognitive (i.e., olfactory learning and memory) abilities of adult honey bees were quantified to assess the delayed impacts of early-stage thiacloprid exposure on adult honey bee cognition. Neural apoptosis and transcriptomic level were also evaluated to explore the neurological mechanisms underlying these effects. (3) Results: Our results revealed that chronic larval exposure to sublethal thiacloprid impaired the learning and memory abilities of adult honey bees by inducing neuronal apoptosis and transcriptomic alterations. (4) Conclusions: We highlighted a previously unknown impairment caused by thiacloprid in honey bees.

New insights into changing honey bee (Apis mellifera) immunity molecules pattern and fatty acid esters, in responses to Ascosphaera apis infection

Monitoring of metabolite changes could provide valuable insights into disturbances caused by an infection and furthermore, could be used to define the status of an organism as healthy or diseased and define what could be defensive elements against the infection. The present investigation conducted a gas chromatography–mass spectrometry (GC/MS) for haemolymph of larval honey bees (Apis mellifera L.) infected with the fungal pathogen Ascosphaera apis in comparison with control haemolymph non-infected insects. Results revealed that the pathogen caused a general disturbance of metabolites detected in the haemolymph of the honey bee. The majority of metabolites identified before and after infection were fatty acid esters. The disease caused an elevation in levels of methyl oleate, methyl palmitate, and methyl stearate, respectively. Further, the disease drove to the disappearance of methyl palmitoleate, and methyl laurate. Conversely, methyl linolelaidate, and ethyl oleate were identified only in infected larvae. A high reduction in diisooctyl phthalate was recorded after the infection. Interestingly, antimicrobial activities were confirmed for haemolymph of infected honey bee larvae. In spite of the presence of some previously known bioactive compounds in healthy larvae there were no antimicrobial activities.

Chronic larval and adult honey bee laboratory testing: Which dietary additive should be considered when a test substance is not solubilized in acetone?

Chronic toxicity tests on adult and larval honey bees (Apis mellifera) can require the use of dietary additives (solvents, emulsifiers, adjuvants and viscosifier agents) when the active ingredient of plant protection products cannot be dissolved or does not remain stable and homogeneous within the test diets. Acetone is the widely used and accepted solvent allowed within the international regulatory guidelines, but it can be ineffective in keeping certain compounds in solution and can cause toxicity to adults and larvae. In this publication, we present an evaluation of alternative additives in adult and larval diets. Six dietary additives including five solvents (ethanol, isopropanol, n-propanol, propylene glycol and triethylene glycol) and a viscosifier agent (xanthan gum) at five concentrations along with a negative control and a solvent control (acetone) were investigated at seven laboratories. The safe levels for bees were determined for each of the additives used in the 10-day chronic adult and 22-day chronic larval tests. In the 10-day chronic adult study, ethanol and isopropanol were found to be safe at concentrations ≤ 5.0 %, while xanthan gum can be reliably used at concentrations ≤ 0.1 %. Greater variability across laboratories was observed for N-propanol, propylene glycol, and triethylene glycol and these agents may cause mortality when added to diets at concentrations above 0.25–0.5 %. The safe levels of additives to larval diet in the 22-day chronic larval test had a greater variability and were generally lower than what were observed for adult diet. Our results do not recommend the inclusion of ethanol or n-propanol into the larval diet, and isopropanol, propylene glycol, and triethylene glycol may cause mortality at concentrations above 0.25–0.5 %. Safe levels for xanthan gum were more variable than what was observed for adults, but it can be used reliably at concentrations ≤ 0.05 %. Our analyses conclude that several additives can be integrated successfully in honey bee laboratory bioassays at levels that cause low mortality to adults and larvae.

The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees.

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

Total Lipid Extracts of Honeybee Drone Larvae Are Modulated by Extraction Temperature and Display Consistent Anti-Inflammatory Potential.

Honeybee drone larvae are male bees that develop from unfertilized eggs and play a role in colony reproduction. The nutritional value of honeybee drone larvae is due to their high protein, lipid, and other nutrient contents, making them a profitable food source for humans in some cultures. Drone larvae lipids (DLLs) contribute to drone development; however, few studies have explored their substantial compositions and bioactive functions. In this study, we carried out DLL lipidomics analysis using UPLC-Q-Exactive-Orbitrap-MS prior to in vitro anti-inflammatory activity analysis. The results highlighted the importance of the extraction temperature on the DLL composition. A total of 21 lipids were found in the DLL extract, mostly categorized into five groups: nine phospholipids, three sphingolipids, two neutral lipids, one plant glycoglycerolipid, four lipid acyl, and others. Drying extraction at -20 °C produced more sphingolipids, phospholipids, and unsaturated fatty acids. Of 37 fatty acids, 18 were displayed at -20 °C degrees, as shown by GC-MS quantitative analysis. Myristic (246.99 ± 13.19 μg/g), palmitic (1707.87 ± 60.53 μg/g), stearic (852.32 ± 24.17 μg/g), and oleic (2463.03 ± 149.61 μg/g) acids were the predominant fatty acids. Furthermore, we examined the significant in vitro anti-inflammatory effects of DLL (-20 °C) using lipopolysaccharide (LPS)-challenged RAW264.7 cells. Nitric oxide (NO) and reactive oxygen (ROS) production and mRNA expression of IL-6, IL-10, COX-2, and iNOS were significantly decreased, demonstrating the anti-inflammatory function of DLL. Overall, this study provided insight into the lipid composition of DLL, revealed the influence of temperature, and explored the functionality of DLL (-20 °C), allowing for further application of DLLs as functional foods.

No impact of hygienic behavior and viral coinfection on the development of European foulbrood in honey bee (Apis mellifera) colonies during blueberry pollination in Michigan.

European foulbrood (EFB) is a severe disease of honey bee (Apis mellifera) larvae caused by the bacterium Linnaeus [Hymenoptera: Apidae]) Melissococcus plutonius (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae). Many beekeepers in North America report severe EFB following blueberry pollination, but it is not clear what factors during pollination are related to clinical disease. Additionally, the impact that other factors such as viral load and hygienic behavior have on EFB has not been studied. In Spring of 2020 we enrolled 60 commercial honey bee colonies in a prospective cohort study. Colonies were inspected 3 times over the season with hive metrics and samples taken for viral testing. Each colony was tested for hygienic behavior twice and the score was averaged. Viral loads were determined by qPCR for deformed wing virus (DWV) A and B. We found no statistical difference in the EFB prevalence or severity between the 2 yards at any timepoint; 50% (n = 16) of the colonies in the holding yard and 63% (n = 17) in blueberry developed moderate to severe EFB over the study period. When colonies from both yards were pooled, we found no relationship between viral load or hygienic behavior and development of EFB. These results suggest that other factors may be responsible for driving EFB virulence and hygienic behavior is not likely helpful in managing this disease.

Risk assessment of honeybee larvae exposure to pyrethroid insecticides in beebread and honey

Honeybee is an essential pollinator to crops, evaluation to the risk assessment of honeybee larvae exposure to pesticides residue in the bee bread and honey is an important strategy to protect the bee colony due to the mixture of these two matrices is main food for 3-day-old honeybee larvae. In this study, a continuous survey to the residue of five pyrethroid insecticides in bee bread and honey between 2018 and 2020 from 17 major cultivation provinces which can be determined as Northeast, Northwest, Eastern, Central, Southwest, and Southern of China, there was at least one type II pyrethroid insecticide was detected in 54.7 % of the bee bread samples and 43.4 % of the honey. Then, we assayed the acute toxicity of type II pyrethroid insecticides based on the detection results, the LD50 value was 0.2201 μg/larva (beta-cyhalothrin), 0.4507 μg/larva (bifenthrin), 2.0840 μg/larva (fenvalerate), 0.0530 μg/larva (deltamethrin), and 0.1640 μg/larva (beta-cypermethrin), respectively. Finally, the hazard quotient was calculated as larval oral ranged from 0.046 × 10-3 to 2.128 × 10-3. Together, these empirical findings provide further insight into the accurate contamination of honey bee colonies caused by chemical pesticides, which can be used as a valuable guidance for the beekeeping industry and pesticide regulation.

Honey Bee Larval Hemolymph as a Source of Key Nutrients and Proteins Offers a Promising Medium for Varroa destructor Artificial Rearing.

Varroa destructor, a major ectoparasite of the Western honey bee Apis mellifera, is a widespread pest that damages colonies in the Northern Hemisphere. Throughout their lifecycle, V. destructor females feed on almost every developmental stage of their host, from the last larval instar to the adult. The parasite is thought to feed on hemolymph and fat body, although its exact diet and nutritional requirements are poorly known. Using artificial Parafilm™ dummies, we explored the nutrition of V. destructor females and assessed their survival when fed on hemolymph from bee larvae, pupae, or adults. We compared the results with mites fed on synthetic solutions or filtered larval hemolymph. The results showed that the parasites could survive for several days or weeks on different diets. Bee larval hemolymph yielded the highest survival rates, and filtered larval plasma was sufficient to maintain the mites for 14 days or more. This cell-free solution therefore theoretically contains all the necessary nutrients for mite survival. Because some bee proteins are known to be hijacked without being digested by the parasite, we decided to run a proteomic analysis of larval honey bee plasma to highlight the most common proteins in our samples. A list of 54 proteins was compiled, including several energy metabolism proteins such as Vitellogenin, Hexamerin, or Transferrins. These molecules represent key nutrient candidates that could be crucial for V. destructor survival.