Honey Bee Immunity Research Articles

Spermidine supplementation influence on protective enzymes of Apis mellifera (Hymenoptera: Apidae).

Dietary supplementation has been proposed as a sustainable way to improve the health and resilience of honey bees (Apis mellifera, L.), as the decline in their numbers in recent decades has raised scientific, environmental, and economic concerns. Spermidine, a natural polyamine, has been shown to be a promising substance for honey bee supplementation, as its health-promoting effects have been demonstrated in numerous studies and in different organisms. As already shown, supplementation with spermidine at a certain concentration prolonged lifespan, reduced oxidative stress, and increased antioxidative capacity in honey bees. The aim of the present study was to investigate whether spermidine supplementation affects gene expression and/or enzyme activity of antioxidative and detoxification enzymes and immune response markers in honey bee workers. The different gene expression and enzyme activity patterns observed in abdominal and head tissues in response to spermidine supplementation suggest tissue-specific and concentration-dependent effects. In addition, the immune response markers suggest that spermidine has the ability to boost honey bee immunity. The observed changes make a valuable contribution to understanding the molecular mechanisms by which spermidine may exert its beneficial effects on the bee's health and lifespan. These results support the idea of the use of spermidine supplementation to promote bee health and resilience to environmental stressors, emphasizing that the dose must be carefully chosen to achieve a balance between the pro- and antioxidant effects of spermidine.

Modeling seasonal immune dynamics of honey bee (Apis mellifera L.) response to injection of heat-killed Serratia marcescens.

The honey bee, Apis mellifera L., is one of the main pollinators worldwide. In a temperate climate, seasonality affects the life span, behavior, physiology, and immunity of honey bees. In consequence, it impacts their interaction with pathogens and parasites. In this study, we used Bayesian statistics and modeling to examine the immune response dynamics of summer and winter honey bee workers after injection with the heat-killed bacteria Serratia marcescens, an opportunistic honey bee pathogen. We investigated the humoral and cellular immune response at the transcriptional and functional levels using qPCR of selected immune genes, antimicrobial activity assay, and flow cytometric analysis of hemocyte concentration. Our data demonstrate increased antimicrobial activity at transcriptional and functional levels in summer and winter workers after injection, with a stronger immune response in winter bees. On the other hand, an increase in hemocyte concentration was observed only in the summer bee population. Our results indicate that the summer population mounts a cellular response when challenged with heat-killed S. marcescens, while winter honey bees predominantly rely on humoral immune reactions. We created a model describing the honey bee immune response dynamics to bacteria-derived components by applying Bayesian statistics to our data. This model can be employed in further research and facilitate the investigating of the honey bee immune system and its response to pathogens.

Biochemical Indicators and Mortality in Honey Bee (Apis mellifera) Workers after Oral Exposure to Plant Protection Products and Their Mixtures

The honey bee obtains food from bee forage, which comprises crops grown in multi-hectare agricultural fields where various types of plant protection products such as pesticides are used. Some of these negatively affect the honey bee organism. In our research, we aimed to evaluate the effects of three pesticide groups: fungicides (tebuconazole), insecticides (acetamiprid), herbicides (glyphosate), and their mixtures on the functioning of honey bee workers (A. mellifera carnica). Pesticides in various proportions and dilutions were added to sugar syrups and then fed to the bees. Mortality and food intake were recorded daily, while hemolymph analysis was performed after seven days of exposure. Food intake, mortality, and the results of various biochemical analyses differed between the experimental group and the control group receiving untreated sugar syrup. PPP’s mixture of glyphosate tebuconazole and acetamiprid is more toxic to bees than single pesticides. Certain protection products such as pesticides can disrupt the antioxidant and detoxification systems associated with immunity in honey bees. Consequently, honey bees experience weaker conditions and their proper functioning deteriorates. The results obtained from biochemical changes provide a basis for field studies.

Exploring the impact of fungicide exposure and nutritional stress on the microbiota and immune response of the Cape honey bee (Apis mellifera capensis)

Honey bees (Apis mellifera) harbour a stable core microbial community within their gut, that is suggested to play a role in metabolic functioning, immune regulation, and host homeostasis. This microbiota presents a unique opportunity to observe the effects of stressors on honey bee health. We examined the effects of two common honey bee stressors: indirect fungicide contamination and nutrient limitation. These effects were observed through changes in their hind- and midgut microbiota using Automated Ribosomal Intergenic Spacer Analysis (ARISA), alongside high-throughput amplicon sequencing. Expression of the honey bees’ immune response was examined through the expression of three immune-related genes, namely, immune deficiency (imd), proPhenolOxidase (proPO), and spaetzle (spz). Additionally, longevity of the honey bees was monitored through observation of the expression levels of Vitellogenin (Vg). Both treatment groups were compared to a negative control, and a diseased positive control. There was no effect on the hindgut microbiota due to the stressors, while significant changes in the midgut was observed. This was also observed in the expression of the immune-related genes within the treatment groups. The Imd pathway was substantially downregulated, with upregulation in the prophenoloxidase pathway. However, no significant effect was observed in the expression of spz, and only the pollen treatment group showed reduced longevity through a downregulation of Vg. Overall, the effect of these two common stressors indicate a compromise in honey bee immunity, and potential vulnerabilities within the immune defence mechanisms.

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.

Imidacloprid increases the prevalence of the intestinal parasite Lotmaria passim in honey bee workers

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 μg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

A clash on the Toll pathway: competitive action between pesticides and zymosan A on components of innate immunity in Apis mellifera.

The immune system of honeybees includes multiple pathways that may be affected by pesticide exposure decreasing the immune competencies of bees and increasing their susceptibility to diseases like the fungal Nosema spp. infection, which is detected in collapsed colonies. To better understand the effect of the co-presence of multiple pesticides that interact with bees like imidacloprid and amitraz, we evaluated the expression of immune-related genes in honeybee hemocytes. Imidacloprid, amitraz, and the immune activator, zymosan A, mainly affect the gene expression in the Toll pathway. Imidacloprid, amitraz, and zymosan A have a synergistic or an antagonistic relationship on gene expression depending on the level of immune signaling. The presence of multiple risk factors like pesticides and pathogens requires the assessment of their complex interaction, which has differential effects on the innate immunity of honeybees as seen in this study.

Honey bee immunity and physiology are enhanced by consuming high-fat diets

Honey bee immunity and physiology are enhanced by consuming high-fat diets

Effects of Vairimorpha (Nosema) spp. on Humoral Immunity of Honey Bees via Antimicrobial Peptides

Arılar, dünya çapındaki tüm tarımsal türlerin büyük çoğunluğu ve yabani flora için önemli tozlayıcılardır. Son yıllarda dünyadaki arı kolonilerinin sayısında hızlı bir düşüş yaşanmıştır. Bal arıları sosyal böceklerdir, bu da onları mikrobiyal patojenler ve parazitlerin hızla yayılmasına karşı hassas hale getirir. Koloni çöküşüne yol açan tek bir nedensel ajan tanımlanamaz ve işçi arılardaki azalmayla ilgili en yaygın biyolojik etkenlerden biri parazitik microsporidia Vairimorpha (Nosema) spp., esas olarak Vairimorpha (Nosema) apis ve Vairimorpha (Nosema) ceranae, her ikisi de Apis türlerinde görülen Nosemosis hastalığından sorumludur. Vairimorpha ceranae daha yaygındır ve koloni çöküşü ile ilişkili olan arı bağışıklık tepkisi üzerinde etkilidir. Bu mikrosporidiaların immünosupresif etkisi ve kovan organizasyonun bozulması koloniyi zayıflatır ve koloni kayıplarına yol açar. Bunun da ekolojik, tarımsal ve ekonomik sonuçları oldukça fazladır. Bal arıları mikrobiyal patojenlerin zararlı etkilerini en aza indiren, doğuştan ve sonradan kazanılmış bağışıklığı içeren son derece etkili savunma mekanizmalarına sahiptirler. Bal arılarının en temel savunma sistemi olan hümoral tepki, doğuştan gelen bağışıklığın ikinci kategorisidir ve antimikrobiyal peptidler (AMP' ler) aracılık eder. Stres faktörleri ile başa çıkabilme yeteneğine sahip bal arılarının bağışıklık mekanizmalarına odaklanan araştırmalar, kolonilerin gücünü ve verimliliğini arttırmalarına yardımcı olabilir. Vairimorpha (Nosema) spp’nin arıların bağışıklık sistemi üzerindeki etkisi, karşılıklı ilişkilerini daha iyi anlamak ve etkili arı koruma yöntemleri geliştirmek için daha ayrıntılı bir şekilde anlaşılmalıdır. Bal arısı bağışıklık sistemleri çözümlendikçe, sosyal böcekler ve bağışıklık fonksiyonları arasındaki potansiyel evrimsel ilişki belirlenebilir. Böylece arı kayıplarını azaltmak için yerel alttür ve ekotipleri koruma stratejileri geliştirilebilir.

Viral Co-Infections and Antiviral Immunity in Honey Bees.

Over the past few decades, honey bees have been facing an increasing number of stressors. Beyond individual stress factors, the synergies between them have been identified as a key factor in the observed increase in colony mortality. However, these interactions are numerous and complex and call for further research. Here, in line with our need for a systemic understanding of the threats that they pose to bee health, we review the interactions between honey bee viruses. As viruses are obligate parasites, the interactions between them not only depend on the viruses themselves but also on the immune responses of honey bees. Thus, we first summarise our current knowledge of the antiviral immunity of honey bees. We then review the interactions between specific pathogenic viruses and their interactions with their host. Finally, we draw hypotheses from the current literature and suggest directions for future research.

Effects of Acetylsalicylic Acid, Echinacea Purpurea Extract, and Vitamin C On Survival, Immunity and Performance of Honey Bees

Effects of Acetylsalicylic Acid, Echinacea Purpurea Extract, and Vitamin C On Survival, Immunity and Performance of Honey Bees

Gut microbiota composition and gene expression changes induced in the Apis cerana exposed to acetamiprid and difenoconazole at environmentally realistic concentrations alone or combined.

Apis cerana is an important pollinator of agricultural crops in China. In the agricultural environment, A. cerana may be exposed to acetamiprid (neonicotinoid insecticide) and difenoconazole (triazole fungicide), alone or in combination because they are commonly applied to various crops. At present, our understanding of the toxicological effects of acetamiprid and difenoconazole on honey bee gut microbiomes is limited. The primary objective of this study was to explore whether these two pesticides affect honey bees' gut microbiota and to analyze the transcriptional effects of these two pesticides on honey bees' head and gut. In this study, adults of A. cerana were exposed to acetamiprid and/or difenoconazole by contaminated syrup at field-realistic concentrations for 10days. Results indicated that acetamiprid and/or difenoconazole chronic exposure did not affect honey bees' survival and food consumption, whereas difenoconazole decreased the weight of honey bees. 16S rRNA sequencing suggested that difenoconazole and the mixture of difenoconazole and acetamiprid decreased the diversity index and shaped the composition of gut bacteria microbiota, whereas acetamiprid did not impact the gut bacterial community. The ITS sequence data showed that neither of the two pesticides affected the fungal community structure. Meanwhile, we also observed that acetamiprid or difenoconazole significantly altered the expression of genes related to detoxification and immunity in honey bees' tissues. Furthermore, we observed that the adverse effect of the acetamiprid and difenoconazole mixture on honey bees' health was greater than that of a single mixture. Taken together, our study demonstrates that acetamiprid and/or difenoconazole exposure at field-realistic concentrations induced changes to the honey bee gut microbiome and gene expression.

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.

Decoupling the effects of nutrition, age, and behavioral caste on honey bee physiology, immunity, and colony health.

Nutritional stress, especially a dearth of pollen, has been linked to honey bee colony losses. Colony-level experiments are critical for understanding the mechanisms by which nutritional stress affects individual honey bee physiology and pushes honey bee colonies to collapse. In this study, we investigated the impact of pollen restriction on key markers of honey bee physiology, main elements of the immune system, and predominant honey bee viruses. To achieve this objective, we uncoupled the effects of behavior, age, and nutritional conditions using a new colony establishment technique designed to control size, demography, and genetic background. Our results showed that the expression of storage proteins, including vitellogenin (vg) and royal jelly major protein 1 (mrjp1), were significantly associated with nursing, pollen ingestion, and older age. On the other hand, genes involved in hormonal regulation including insulin-like peptides (ilp1 and ilp2) and methyl farnesoate epoxidase (mfe), exhibited higher expression levels in young foragers from colonies not experiencing pollen restriction. In contrast, pollen restriction induced higher levels of insulin-like peptides in old nurses. On the other hand, we found a strong effect of behavior on the expression of all immune genes, with higher expression levels in foragers. In contrast, the effects of nutrition and age were significant only the expression of the regulatory gene dorsal. We also found multiple interactions of the experimental variables on viral titers, including higher Deformed wing virus (DWV) titers associated with foraging and age-related decline. In addition, nutrition significantly affected DWV titers in young nurses, with higher titers induced by pollen ingestion. In contrast, higher levels of Black queen cell virus (BQCV) were associated with pollen restriction. Finally, correlation, PCA, and NMDS analyses proved that behavior had had the strongest effect on gene expression and viral titers, followed by age and nutrition. These analyses also support multiple interactions among genes and virus analyzed, including negative correlations between the expression of genes encoding storage proteins associated with pollen ingestion and nursing (vg and mrjp1) with the expression of immune genes and DWV titers. Our results provide new insights into the proximal mechanisms by which nutritional stress is associated with changes in honey bee physiology, immunity, and viral titers.

Insights into the effects of sublethal doses of pesticides glufosinate-ammonium and sulfoxaflor on honey bee health

Insect pollinators are threatened worldwide, being the exposure to multiple pesticides one of the most important stressor. The herbicide Glyphosate and the insecticide Imidacloprid are among the most used pesticides worldwide, although different studies evidenced their detrimental effects on non-target organisms. The emergence of glyphosate-resistant weeds and the recent ban of imidacloprid in Europe due to safety concerns, has prompted their replacement by new molecules, such as glufosinate-ammonium (GA) and sulfoxaflor (S). GA is a broad-spectrum and non-selective herbicide that inhibits a key enzyme in the metabolism of nitrogen, causing accumulation of lethal levels of ammonia; while sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and generates excitatory responses including tremors, paralysis and mortality. Although those molecules are being increasingly used for crop protection, little is known about their effects on non-target organisms. In this study we assessed the impact of chronic and acute exposure to sublethal doses of GA and S on honey bee gut microbiota, immunity and survival. We found GA significantly reduced the number of gut bacteria, and decreased the expression of glucose oxidase, a marker of social immunity. On the other hand, S significantly increased the number of gut bacteria altering the microbiota composition, decreased the expression of lysozyme and increased the expression of hymenoptaecin. These alterations in gut microbiota and immunocompetence may lead to an increased susceptibility to pathogens. Finally, both pesticides shortened honey bee survival and increased the risk of death. Those results evidence the negative impact of GA and S on honey bees, even at single exposition to a low dose, and provide useful information to the understanding of pollinators decline.

Effect of carbendazim on honey bee health: Assessment of survival, pollen consumption, and gut microbiome composition

Gut microbiota and nutrition play major roles in honey bee health. Recent reports have shown that pesticides can disrupt the gut microbiota and cause malnutrition in honey bees. Carbendazim is the most commonly used fungicide in China, but it is not clear whether carbendazim negatively affects the gut microbes and nutrient intake levels in honey bees. To address this research gap, we assessed the effects of carbendazim on the survival, pollen consumption, and sequenced 16 S rRNA gene to determine the bacterial composition in the midgut and hindgut. Our results suggest that carbendazim exposure does not cause acute death in honey bees even at high concentrations (5000 mg/L), which are extremely unlikely to exist under field conditions. Carbendazim does not disturb the microbiome composition in the gut of young worker bees during gut microbial colonization and adult worker bees with established gut communities in the mid and hindgut. However, carbendazim exposure significantly decreases pollen consumption in honey bees. Thus, exposure of bees to carbendazim can perturb their beneficial nutrition homeostasis, potentially reducing honey bee immunity and increasing their susceptibility to infection by pathogens, which influence effectiveness as pollinators, even colony health.

Bee Stressors from an Immunological Perspective and Strategies to Improve Bee Health.

Honeybees are the most prevalent insect pollinator species; they pollinate a wide range of crops. Colony collapse disorder (CCD), which is caused by a variety of biotic and abiotic factors, incurs high economic/ecological loss. Despite extensive research to identify and study the various ecological stressors such as microbial infections, exposure to pesticides, loss of habitat, and improper beekeeping practices that are claimed to cause these declines, the deep understanding of the observed losses of these important insects is still missing. Honeybees have an innate immune system, which includes physical barriers and cellular and humeral responses to defend against pathogens and parasites. Exposure to various stressors may affect this system and the health of individual bees and colonies. This review summarizes and discusses the composition of the honeybee immune system and the consequences of exposure to stressors, individually or in combinations, on honeybee immune competence. In addition, we discuss the relationship between bee nutrition and immunity. Nutrition and phytochemicals were highlighted as the factors with a high impact on honeybee immunity.

Impacts of Diverse Natural Products on Honey Bee Viral Loads and Health

Western honey bees (Apis mellifera), a cornerstone to crop pollination in the U.S., are faced with an onslaught of challenges from diseases caused by parasites, pathogens, and pests that affect this economically valuable pollinator. Natural products (NPs), produced by living organisms, including plants and microorganisms, can support health and combat disease in animals. NPs include both native extracts and individual compounds that can reduce disease impacts by supporting immunity or directly inhibiting pathogens, pests, and parasites. Herein, we describe the screening of NPs in laboratory cage studies for their effects on honey bee disease prevention and control. Depending on the expected activity of compounds, we measured varied responses, including viral levels, honey bee immune responses, and symbiotic bacteria loads. Of the NPs screened, several compounds demonstrated beneficial activities in honey bees by reducing levels of the critical honey bee virus deformed wing virus (DWV-A and-B), positively impacting the gut microbiome or stimulating honey bee immune responses. Investigations of the medicinal properties of NPs in honey bees will contribute to a better understanding of their potential to support honey bee immunity to fight off pests and pathogens and promote increased overall honey bee health. These investigations will also shed light on the ecological interactions between pollinators and specific floral food sources.

Transcriptome analysis of Apis mellifera under benomyl stress to discriminate the gene expression in response to development and immune systems

The health and safety of the honeybees are seriously threatened due to the abuse of chemical pesticides in modern agriculture and apiculture. In this study, the RNA Seq approach was used to assess the effects of the honeybees treated with benomyl. The results showed that there were a total of 11,902 differentially expressed genes (DEGs). Among them, 5,759 DEGs were up-regulated and involved in the functions of immunity, detoxification, biological metabolism, and regulation. The DEGs were clustered in the GO terms of epidermal structure and response to external stimuli, and most of the DEGs were enriched in 15 pathways, such as light conduction, MAPK, calcium ion pathway, and so on. Moreover, the pathway of the toll signal transduction was activated. The data investigated that the expression of functional genes involved in the growth, development, foraging, and immunity of honeybees were significantly affected by benomyl stress, which would seriously threaten the health of the honeybees. This study provided a theoretical basis for revealing the response mechanism of honeybees to pesticides stress.

Physiological effects of the interaction between Nosema ceranae and sequential and overlapping exposure to glyphosate and difenoconazole in the honey bee Apis mellifera

Pathogens and pollutants, such as pesticides, are potential stressors to all living organisms, including honey bees. Herbicides and fungicides are among the most prevalent pesticides in beehive matrices, and their interaction with Nosema ceranae is not well understood. In this study, the interactions between N. ceranae, the herbicide glyphosate and the fungicide difenoconazole were studied under combined sequential and overlapping exposure to the pesticides at a concentration of 0.1 µg/L in food. In the sequential exposure experiment, newly emerged bees were exposed to the herbicide from day 3 to day 13 after emerging and to the fungicide from day 13 to day 23. In the overlapping exposure experiment, bees were exposed to the herbicide from day 3 to day 13 and to the fungicide from day 7 to day 17. Infection by Nosema in early adult life stages (a few hours post emergence) greatly affected the survival of honey bees and elicited much higher mortality than was induced by pesticides either alone or in combination. Overlapping exposure to both pesticides induced higher mortality than was caused by sequential or individual exposure. Overlapping, but not sequential, exposure to pesticides synergistically increased the adverse effect of N. ceranae on honey bee longevity. The combination of Nosema and pesticides had a strong impact on physiological markers of the nervous system, detoxification, antioxidant defenses and social immunity of honey bees.