Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees.

Taking into consideration that bees can be contaminated by pesticides through the ingestion of contaminated floral resources, we can utilize genetic techniques to assess effects that are scarcely observed in behavioral studies. This study aimed to investigate the genetic effects of ingesting lethal and sublethal doses of the insecticide fipronil in foraging honey bees during two periods of acute exposure. Bees were exposed to fipronil through contaminated honey syrup at two dosages (LD50 = 0.19 µg/bee; LD50/100 = 0.0019 µg/bee) and for two durations (1 and 4 h). Following exposure, we measured syrup consumption per bee, analyzed the transcriptome of bee brain tissue, and identified differentially expressed genes (DEGs), categorizing them functionally based on gene ontology (GO). The results revealed a significant genetic response in honey bees after exposure to fipronil, regardless of the dosage used. Fipronil affected various metabolic, transport, and cellular regulation pathways, as well as detoxification processes and xenobiotic substance detection. Additionally, the downregulation of several DEGs belonging to the olfactory-binding protein (OBP) family was observed, suggesting potential physiological alterations in bees that may lead to disoriented behaviors and reduced foraging efficiency.

Validation of a Questionnaire for Equine Personality Assessement.

We propose a practical method of assessing the personality of horses using five personality axes. Four evaluators empirically judged 19 horse individuals on specific adjectives for each axis. To validate the questionnaire, four behavioral tests were conducted with these same animals (social interactions, novel object test, bridge test,and arena test). In this tests, the frequency of specific behaviors were evaluated to create a scale related to the same personality adjectives and judge the animals based on their reactions.The questionnaire was reliable in determining the personality of horses, since the results were consistent with those obtained through behavioral tests. Additionally, in this group of horses attention reactions were more frequent than fear reactions, but significant differences occurred among tests. This study proposes a practical questionnaire for owners and trainers to assess the personality of their horses. The application of this tool can improve the relationship between humans and horses, directing a more empathic approach in the everyday routine with these animals.

Intake of imidacloprid in lethal and sublethal doses alters gene expression in Apis mellifera bees.

Bees are important pollinators for ecosystems and agriculture; however, populations have suffered a decline that may be associated with several factors, including habitat loss, climate change, increased vulnerability to diseases and parasites and use of pesticides. The extensive use of neonicotinoids, including imidacloprid, as agricultural pesticides, leads to their persistence in the environment and accumulation in bees, pollen, nectar, and honey, thereby inducing deleterious effects. Forager honey bees face significant exposure to pesticide residues while searching for resources outside the hive, particularly systemic pesticides like imidacloprid. In this study, 360 Apis mellifera bees, twenty-one days old (supposed to be in the forager phase) previously marked were fed syrup (honey and water, 1:1 m/v) containing a lethal dose (0.081 µg/bee) or sublethal dose (0.00081 µg/bee) of imidacloprid. The syrup was provided in plastic troughs, with 250 µL added per trough onto each plastic Petri dish containing 5 bees (50 µL per bee). The bees were kept in the plastic Petri dishes inside an incubator, and after 1 and 4 h of ingestion, the bees were euthanised and stored in an ultra-freezer (-80 °C) for transcriptome analysis. Following the 1-h ingestion of imidacloprid, 1516 genes (73 from lethal dose; 1509 from sublethal dose) showed differential expression compared to the control, while after 4 h, 758 genes (733 from lethal dose; 25 from sublethal) exhibited differential expression compared to the control. All differentially expressed genes found in the brain tissue transcripts of forager bees were categorised based on gene ontology into functional groups encompassing biological processes, molecular functions, and cellular components. These analyses revealed that sublethal doses might be capable of altering more genes than lethal doses, potentially associated with a phenomenon known as insecticide-induced hormesis. Alterations in genes related to areas such as the immune system, nutritional metabolism, detoxification system, circadian rhythm, odour detection, foraging activity, and memory in bees were present after exposure to the pesticide. These findings underscore the detrimental effects of both lethal and sublethal doses of imidacloprid, thereby providing valuable insights for establishing public policies regarding the use of neonicotinoids, which are directly implicated in the compromised health of Apis mellifera bees.

Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom.

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Biogenic amines and stable isotopes in the quality and authenticity of honeys from Brazil.

The identification of biogenic amines and some precursor amino acids and the adulteration through stable isotopes was carried out in 114 honey from different geographic regions in Brazil (states of São Paulo (SP) and Santa Catarina (SC)) as support for evaluating quality control and food safety. Serotonin was detected in all samples, while melatonin was quantified in 92.2% of honey from SP and in 94% of SC. l-Dopa, dopamine and histamine appeared at higher levels in honey from SP. Cadaverine, putrescine, spermidine and spermine, varied little according to botanical source. Three honey from the metropolitan region of SP were considered adulterated (C4SUGARS > 7%), 92 were authentic samples (C4SUGARS - 7 to 7%) and 19 unadulterated (C4SUGARS less than - 7%), with isotopic values of δ13CH and δ13CP > 7%. The data were important for differentiating quality as a function of biogenic amines and stable isotope technique was important in detecting honey adulteration.

Tryptophan and Biogenic Amines in the Differentiation and Quality of Honey.

Honey is a natural product with beneficial properties to health and has different characteristics depending on the region of production and collection, flowering, and climate. The presence of precursor amino acids of- and biogenic amines can be important in metabolomic studies of differentiation and quality of honey. We analyzed 65 honeys from 11 distinct regions of the State of Santa Catarina (Brazil) as to the profile of amino acids and biogenic amines by HPLC. The highest L-tryptophan (Trp), 5-hydroxytryptophan (5-OH-Trp), and tryptamine (Tryp) levels were detected in Cfb climate and harvested in 2019. Although we have found high content of serotonin, dopamine, and L-dopa in Cfb climate, the highest values occurred in honey produced during the summer 2018 and at altitudes above 900 m. Results indicate that the amino acids and biogenic amine levels in honeys are good indicators of origin. These data warrant further investigation on the honey as source of amino acids precursor of serotonin, melatonin, and dopamine, what can guide the choice of food as source of neurotransmitters.

Zinc supplementation modifies brain tissue transcriptome of Apis mellifera honeybees.

BACKGROUND: Bees are the most important group of pollinators worldwide and their populations are declining. In natural conditions, Apis mellifera depends exclusively on food from the field to meet its physiological demands. In the period of scarcity, available resources are insufficient and artificial supplementation becomes essential for maintaining the levels of vitamins, proteins, carbohydrates, and minerals of colonies. Among these minerals, zinc is essential in all living systems, particularly for the regulation of cell division and protein synthesis, and is a component of more than 200 metalloenzymes. RESULTS: The total RNA extracted from the brain tissue of nurse bees exposed to different sources and concentrations of zinc was sequenced. A total of 1,172 genes in the treatment that received an inorganic source of zinc and 502 genes that received an organic source of zinc were found to be differentially expressed among the control group. Gene ontology enrichment showed that zinc can modulate important biological processes such as nutrient metabolism and the molting process. CONCLUSIONS: Our results indicate that zinc supplementation modulates the expression of many differentially expressed genes and plays an important role in the development of Apis mellifera bees. All the information obtained in this study can contribute to future research in the field of bee nutrigenomics.

Supplementation with an Inorganic Zinc Source in the Metalloproteomic Profile of Royal Jelly in Apis mellifera L.

This study aimed to evaluate the quality of royal jelly produced by honeybees Apis mellifera supplemented with different concentrations of inorganic zinc (zinc sulfate monohydrate-0, 25, 50, and 75 ppm). Two-dimensional electrophoresis for the fractionation of royal jelly proteins was performed, and the zinc level was quantified by the flame atomic absorption spectrometry (FAAS) technique. Proteins were identified by electrospray ionization mass spectrometry (ESI MS MS). Analysis of variance followed by the Tukey test (P < 0.05) was used. Supplementation with the mineral zinc positively affected the quantification of proteins for treatments 50 and 75 ppm. However, all treatments independent of zinc concentrations showed fewer protein spots when compared to the control. All zinc-containing proteins were classified as major royal jelly proteins (MRJPs). The exposure of nursing bees to the mineral zinc in its inorganic form reduced the expression of six different MRJPs involved in larval and glands development of nursing bees (MRJP1, MRJP2, MRJP3, MRJP5, and MRJP7), however promoted an increase in the expression of royal jelly proteins involved in defense systems (MRJP8 and MRJP9). The results demonstrate that vital proteins and metabolic processes are impaired in nursing bees exposed to the mineral zinc in its inorganic form in all doses used affecting nutrition and maintenance of colonies.

Defense Response in Brazilian Honey Bees (Apis mellifera scutellata × spp.) Is Underpinned by Complex Patterns of Admixture.

In 1957, an invasive and highly defensive honey bee began to spread across Brazil. In the previous year, Brazilian researchers hoped to produce a subtropical-adapted honey bee by crossing local commercial honey bees (of European origin) with a South African honey bee subspecies (Apis mellifera scutellata; an A-lineage honey bee subspecies). The resulting cross-African hybrid honey bees (AHBs)-escaped from their enclosure and spread through the Americas. Today, AHB is the most common honey bee from Northern Argentina to the Southern United States. AHBs are much more likely to sting nest intruders than managed European-derived honey bee colonies. Previous studies have explored how genetic variation contributes to differences in defense response between European-derived honey bee and AHB. Although this work demonstrated very strong genetic effects on defense response, they have yet to pinpoint which genes influence variation in defense response within AHBs, specifically. We quantified defense response for 116 colonies in Brazil and performed pooled sequencing on the most phenotypically divergent samples. We identified 65 loci containing 322 genes that were significantly associated with defense response. Loci were strongly associated with metabolic function, consistent with previous functional genomic analyses of this phenotype. Additionally, defense-associated loci had nonrandom and unexpected patterns of admixture. Defense response was not simply the product of more A-lineage honey bee ancestry as previously assumed, but rather an interaction between A-lineage and European alleles. Our results suggest that a combination of A-lineage and European alleles play roles in defensive behavior in AHBs.

Energetic supplementation for maintenance or development of Apis mellifera L. colonies.

BACKGROUND: The nutritional requirements of honeybees (Apis mellifera) for their complete development need to be supplied through food sources available in the environment, since honeybees are insects that depend directly on blossoming food sources. However, at certain times a food-supply reduction can promote nutritional stress, thus necessitating food supplementation for maintenance or production stimulus of the colonies. Thus, the determination of optimal energy supplementation can assist in the maintenance and production of colonies. METHODS: Twenty Apis mellifera beehives were used (with five beehives per treatment): CTL, control (without feeding); SJ, sugarcane juice; SS, sugar syrup; and IS, inverted sucrose. We evaluated the food consumption, population development, and physiological state (expression of vitellogenin and hexamerin 70a genes) of each colony. RESULTS: The results showed that the supplementation of colonies with sugar syrup resulted in an intermediate consumption level (894.6 ± 291 mL) and better development (384.9 ± 237.3 and 158.3 ± 171.6 cm2, sealed and open brood, respectively). Furthermore, this diet ensured that the colonies were in a good physiological state, as bees fed this diet presented the highest relative expression levels of vitellogenin and hexamerin 70a among all the diets tested. CONCLUSIONS: Therefore, sugar syrup is concluded to be the best artificial energetic food for use in the supplementation of honeybee colonies.

Author Correction: Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

Understanding the effect of pesticides on the survival of honeybee colonies is important because these pollinators are reportedly declining globally. In the present study, we examined the changes in the head proteome of nurse honeybees exposed to individual and combined pesticides (the fungicide pyraclostrobin and the insecticide fipronil) at field-relevant doses (850 and 2.5 ppb, respectively). The head proteomes of bees exposed to pesticides were compared with those of bees that were not exposed, and proteins with differences in expression were identified by mass spectrometry. The exposure of nurse bees to pesticides reduced the expression of four of the major royal jelly proteins (MRJP1, MRJP2, MRJP4, and MRJP5) and also several proteins associated with carbohydrate metabolism and energy synthesis, the antioxidant system, detoxification, biosynthesis, amino acid metabolism, transcription and translation, protein folding and binding, olfaction, and learning and memory. Overall, when pyraclostrobin and fipronil were combined, the changes in protein expression were exacerbated. Our results demonstrate that vital proteins and metabolic processes are impaired in nurse honeybees exposed to pesticides in doses close to those experienced by these insects in the field, increasing their susceptibility to stressors and affecting the nutrition and maintenance of both managed and natural colonies.

Influence of apitoxin and melittin from Apis mellifera bee on Staphylococcus aureus strains.

The antibacterial activities of apitoxin, a venom produced by Apis mellifera bee, and melittin, an antimicrobial peptide from apitoxin, were tested against planktonic and biofilm states of Staphylococcus aureus methicillin-resistant (MRSA), including clinical, and enterotoxin-producing isolates. Also, the synergism of apitoxin and melittin in combination with oxacillin were evaluated as well. The induced morphological changes on S. aureus cells of both products were detected by transmission electronic microscopy (TEM). The minimum inhibitory concentration (MIC) values were 7.2 µg/mL, and 6.7 µg/mL, for apitoxin and melittin, respectively. The minimum bactericidal concentration (MBC) values were 28.7 µg/mL, and 26 µg/mL for apitoxin and melittin, respectively. The time-kill curve assays of apitoxin or melittin with oxacillin exhibited bactericidal synergism against MRSA isolates. TEM images showed cell distortion, cell disintegration with leakage of cytoplasmic content and loss of cytoplasm content. However, apitoxin and melittin did not interfere with staphylococcal enterotoxin production or release. Thus, apitoxin and melittin are potential agents against MRSA that can serve as possible models for new antibacterial drugs.

Supplementation with an Inorganic Iron Source Modulates the Metalloproteomic Profile of the Royal Jelly Produced by Apis mellifera L.

This study aimed to evaluate the quality of the royal jelly produced by Apis mellifera bees in the presence of different iron concentrations (ferrous sulfate heptahydrate-0, 25, 50, and 100 mg L-1). Two-dimensional electrophoresis was used for the fractionation of royal jelly proteins, and iron level was quantified using flame atomic absorption spectrometry technique. The proteins were identified using electrospray ionisation mass spectrometry. Analysis of variance followed by the Tukey test (P < 0.05) was utilised. Dietary supplementation with mineral Fe affected the protein content and number of proteins in the experimental period. Further, the diet containing the highest iron concentration showed a greater number of spots containing iron, as well as in the abdomen of the bees. The most protein containing Fe were classified as major royal jelly proteins. These results showed that Fe influenced the quality of royal jelly and can improve its nutritional value.

Energetic supplementation for maintenance or development of Apis mellifera L. colonies

The nutritional requirements of honeybees (Apis mellifera) for their complete development need to be supplied through food sources available in the environment, since honeybees are insects that depend directly on blossoming food sources. However, at certain times a food-supply reduction can promote nutritional stress, thus necessitating food supplementation for maintenance or production stimulus of the colonies. Thus, the determination of optimal energy supplementation can assist in the maintenance and production of colonies. Methods: Twenty Apis mellifera beehives were used (with five beehives per treatment): CTL, control (without feeding); SJ, sugarcane juice; SS, sugar syrup; and IS, inverted sucrose. We evaluated the food consumption, population development, and physiological state (expression of vitellogenin and hexamerin 70a genes) of each colony. Results: The results showed that the supplementation of colonies with sugar syrup resulted in an intermediate consumption level (894.6 ± 291 mL) and better development (384.9 ± 237.3 and 158.3 ± 171.6 cm2, sealed and open brood, respectively). Furthermore, this diet ensured that the colonies were in a good physiological state, as bees fed this diet presented the highest relative expression levels of vitellogenin and hexamerin 70a among all the diets tested. Conclusions: Therefore, sugar syrup is concluded to be the best artificial energetic food for use in the supplementation of honeybee colonies.(AU)

Toxicity and motor changes in Africanized honey bees (Apis mellifera L.) exposed to fipronil and imidacloprid.

This study evaluated the in vitro toxicity and motor activity changes in African-derived adult honey bees (Apis mellifera L.) exposed to lethal or sublethal doses of the insecticides fipronil and imidacloprid. Mortality of bees was assessed to determine the ingestion and contact lethal dose for 24 h using probit analysis. Motor activities in bees exposed to lethal (LD50) and sublethal doses (1/500th of the lethal dose) of both insecticides were evaluated in a behavioral observation box at 1 and 4 h. Ingestion and contact lethal doses of fipronil were 0.2316 ? 0.0626 and 0.0080 ? 0.0021 µg/bee, respectively. Ingestion and contact lethal doses of imidacloprid were 0.1079 ? 0.0375 and 0.0308 ? 0.0218 µg/bee, respectively. Motor function of bees exposed to lethal doses of fipronil and imidacloprid was impaired; exposure to sublethal doses of fipronil but not imidacloprid impaired motor function. The insecticides evaluated in this study were highly toxic to African-derived A. mellifera and caused impaired motor function in these pollinators.

Toxicity and motor changes in Africanized honey bees (Apis mellifera L) exposed to fipronil and imidacloprid

ABSTRACT This study evaluated the in vitro toxicity and motor activity changes in African-derived adult honey bees (Apis mellifera L.) exposed to lethal or sublethal doses of the insecticides fipronil and imidacloprid. Mortality of bees was assessed to determine the ingestion and contact lethal dose for 24 h using probit analysis. Motor activities in bees exposed to lethal (LD50) and sublethal doses (1/500th of the lethal dose) of both insecticides were evaluated in a behavioral observation box at 1 and 4 h. Ingestion and contact lethal doses of fipronil were 0.2316 ? 0.0626 and 0.0080 ? 0.0021 μg/bee, respectively. Ingestion and contact lethal doses of imidacloprid were 0.1079 ? 0.0375 and 0.0308 ? 0.0218 μg/bee, respectively. Motor function of bees exposed to lethal doses of fipronil and imidacloprid was impaired; exposure to sublethal doses of fipronil but not imidacloprid impaired motor function. The insecticides evaluated in this study were highly toxic to African-derived A. mellifera and caused impaired motor function in these pollinators.

Harvesting Season and Botanical Origin Interferes in Production and Nutritional Composition of Bee Pollen

ABSTRACT We aimed to evaluate the frequency of bee pollen production, its botanical origin and chemical composition when collected in different seasons. Our results indicate that higher proteins (22.80 ± 3.09%) and flavonoids (2789.87 ± 1396.00 μg 100g-1) levels were obtained in the winter season, which also showed greater pollen production (134.50 ± 35.70 grams) and predominance of the Myrtaceae family. As for spring we found high concentrations of lipids (4.62 ± 2.26%) and low ash content (2.22 ± 0.39%). Regarding the amino acid composition and vitamin C content, we found no differences between the averages throughout the seasons. Our results highlight the importance of understanding not only the botanical origin and the chemical composition of bee pollen, but also the harvesting frequency of this product by bees, so that it becomes possible to supplement the colonies in times of natural food resources shortage.

Harvesting Season and Botanical Origin Interferes in Production and Nutritional Composition of Bee Pollen.

We aimed to evaluate the frequency of bee pollen production, its botanical origin and chemical composition when collected in different seasons. Our results indicate that higher proteins (22.80 ± 3.09%) and flavonoids (2789.87 ± 1396.00 µg 100g-1) levels were obtained in the winter season, which also showed greater pollen production (134.50 ± 35.70 grams) and predominance of the Myrtaceae family. As for spring we found high concentrations of lipids (4.62 ± 2.26%) and low ash content (2.22 ± 0.39%). Regarding the amino acid composition and vitamin C content, we found no differences between the averages throughout the seasons. Our results highlight the importance of understanding not only the botanical origin and the chemical composition of bee pollen, but also the harvesting frequency of this product by bees, so that it becomes possible to supplement the colonies in times of natural food resources shortage.