Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.

The dual coding of a single sex pheromone receptor in Asian honeybee Apis cerana.

In Asian honeybees, virgin queens typically only mate during a single nuptial flight before founding a colony. This behavior is controlled by the queen-released mandibular pheromone (QMP). 9-oxo-(E)-2-decenoic acid (9-ODA), a key QMP component, acts as sex pheromone and attracts drones. However, how the queens prevent additional mating remains elusive. Here, we show that the secondary QMP component methyl p-hydroxybenzoate (HOB) released by mated queens inhibits male attraction to 9-ODA. Results from electrophysiology and in situ hybridization assay indicated that HOB alone significantly reduces the spontaneous spike activity of 9-ODA-sensitive neurons, and AcerOr11 is specifically expressed in sensilla placodea from the drone's antennae, which are the sensilla that narrowly respond to both 9-ODA and HOB. Deorphanization of AcerOr11 in Xenopus oocyte system showed 9-ODA induces robust inward (regular) currents, while HOB induces inverse currents in a dose-dependent manner. This suggests that HOB potentially acts as an inverse agonist against AcerOr11.

The Effects of Artificial Diets on the Expression of Molecular Marker Genes Related to Honey Bee Health.

Honey bees are commonly used to study metabolic processes, yet the molecular mechanisms underlying nutrient transformation, particularly proteins and their effects on development, health, and diseases, still evoke varying opinions among researchers. To address this gap, we investigated the digestibility and transformation of water-soluble proteins from four artificial diets in long-lived honey bee populations (Apis mellifera ligustica), alongside their impact on metabolism and DWV relative expression ratio, using transcriptomic and protein quantification methods. Diet 2, characterized by its high protein content and digestibility, was selected for further analysis from the other studied diets. Subsequently, machine learning was employed to identify six diet-related molecular markers: SOD1, Trxr1, defensin2, JHAMT, TOR1, and vg. The expression levels of these markers were found to resemble those of honey bees who were fed with Diet 2 and bee bread, renowned as the best natural food. Notably, honey bees exhibiting chalkbrood symptoms (Control-N) responded differently to the diet, underscoring the unique nutritional effects on health-deficient bees. Additionally, we proposed a molecular model to elucidate the transition of long-lived honey bees from diapause to development, induced by nutrition. These findings carry implications for nutritional research and beekeeping, underscoring the vital role of honey bees in agriculture.

Exploring the Formation of Chemical Markers in Chaste Honey by Comparative Metabolomics: From Nectar to Mature Honey.

Identification of chemical markers is important to ensure the authenticity of monofloral honey; however, the formation of chemical markers in honey has received little attention. Herein, using comparative metabolomics, we first identified chemical markers in chaste honey and then explored their formation and accumulation from nectar to mature honey. We identified agnuside and p-hydroxybenzoic acid glucosides as chemical markers for chaste honey. Besides, we developed an UHPLC-MS/MS method for quantifying these markers and found that their levels varied significantly across sample sources. We compared the presence of these compounds in chaste nectar and mature honey. The outcomes underscore that these characteristic compounds are not simply delivered from nectar to mature honey, and activities of honeybees (collecting and processing) play a pivotal role in their formation and accumulation. These observations shed light on how mature honey can form its unique qualities with a rich assortment of natural bioactive compounds, potentially supporting health benefits.

Investigating the thermal sensitivity of key enzymes involved in the energetic metabolism of three insect species.

The metabolic responses of insects to high temperatures have been linked to their mitochondrial substrate oxidation capacity. However, the mechanism behind this mitochondrial flexibility is not well understood. Here, we used three insect species with different thermal tolerances (the honey bee, Apis mellifera; the fruit fly, Drosophila melanogaster; and the potato beetle, Leptinotarsa decemlineata) to characterize the thermal sensitivity of different metabolic enzymes. Specifically, we measured activity of enzymes involved in glycolysis (hexokinase, HK; pyruvate kinase, PK; and lactate dehydrogenase, LDH), pyruvate oxidation and the tricarboxylic acid cycle (pyruvate dehydrogenase, PDH; citrate synthase, CS; malate dehydrogenase, MDH; and aspartate aminotransferase, AAT), and the electron transport system (Complex I, CI; Complex II, CII; mitochondrial glycerol-3-phosphate dehydrogenase, mG3PDH; proline dehydrogenase, ProDH; and Complex IV, CIV), as well as that of ATP synthase (CV) at 18, 24, 30, 36, 42 and 45°C. Our results show that at high temperature, all three species have significantly increased activity of enzymes linked to FADH2 oxidation, specifically CII and mG3PDH. In fruit flies and honey bees, this coincides with a significant decrease of PDH and CS activity, respectively, that would limit NADH production. This is in line with the switch from NADH-linked substrates to FADH2-linked substrates previously observed with mitochondrial oxygen consumption. Thus, we demonstrate that even though the three insect species have a different metabolic regulation, a similar response to high temperature involving CII and mG3PDH is observed, denoting the importance of these proteins for thermal tolerance in insects.

Combined pesticides in field doses weaken honey bee (Apis cerana F.) flight ability and analyses of transcriptomics and metabolomics.

Imidacloprid, chlorpyrifos, and glyphosate rank among the most extensively employed pesticides worldwide. The effects of these pesticides and their combined on the flight capability of Apis cerana, and the potential underlying mechanisms remain uncertain. To investigate these effects, we carried out flight mill, transcriptome, and metabolome experiments. Our findings reveal that individual acute oral treatments with pesticides, specifically 20 µL of 10 ng/g imidacloprid (0.2 ng per bee), 30 ng/g chlorpyrifos (0.6 ng per bee), and 60 ng/g glyphosate (1.2 ng per bee), did not impact the flight capability of the bees. However, when bees were exposed to a combination of two or three pesticides, a notable reduction in flight duration and distance was observed. In the transcriptomic and metabolomic analyses, we identified 307 transcripts and 17 metabolites that exhibited differential expression following exposure to combined pesticides, primarily associated with metabolic pathways involved in energy regulation. Our results illuminate the intricate effects and potential hazards posed by combined pesticide exposures on bee behavior. These findings offer valuable insights into the synergistic potential of pesticide combinations and their capacity to impair bee behavior. Understanding these complex interactions is essential for comprehending the broader consequences of pesticide formulations on honey bee populations.

Changes in health-promoting metabolites associated with high-altitude adaptation in honey.

The levels of metabolites in honey are influenced by floral origin, production region, and bee species. However, how environmental factors affect honey quality remains unclear. Based on untargeted metabolomics and using UPLC Q-Orbitrap MS, we analyzed 3596 metabolites in 51 honey samples from Yunnan and Shennongjia. Comparative analysis revealed that geniposidic acid, kynurenic acid and caffieine accumulated at significantly different levels between Shennongjia and Yunnan honey. Based on cluster structure analysis, 36 Yunnan honey samples were divided into two distinct groups by altitude. Notably, quercetin, hyperoside, taxifolin, rutin, tryptophan, astragalin and phenylalanine were higher levels in high-altitude honey (>1700 m), whereas abscisic acid was higher levels in low-altitude honey (≤1700 m). Among these, significantly elevated levels of hyperoside, taxfolin, astragalin, and tryptophan were observed in honey collected from high-altitude areas in Shennongjia. Our findings highlight the effect of altitude on honey health-promoting components, providing valuable insights into honey quality.

Functional orthologs of honeybee CYP6AQ1 in stingless bees degrade the butenolide insecticide flupyradifurone.

Flupyradifurone (FPF), a novel butenolide insecticide binding to nicotinic acetylcholine receptors (nAChRs), has been shown to be less acutely toxic to western honey bees (Apis mellifera) than other insecticides such as neonicotinoids sharing the same target-site. A previous study revealed that this is due to enhanced oxidative metabolism of FPF, mediated by three cytochrome P450 monooxygenases (P450s), including CYP6AQ1. Therefore, we followed a toxicogenomics approach and investigated the potential role of functional CYP6AQ1 orthologs in FPF metabolism from eight different bee species, including stingless bees (Tribe: Meliponini). We conducted a phylogenetic analysis on four stingless bee species, including Frieseomelitta varia, Heterotrigona itama, Melipona quadrifasciata and Tetragonula carbonaria to identify CYP6AQ1-like functional orthologs. Three non-Meliponini, but tropical bee species, i.e., Ammobates syriacus, Euglossa dilemma and Megalopta genalis were analyzed as well. We identified candidate P450s in all (neo)tropical species with greater than 61% and 67% predicted protein sequence identities when compared to A. mellifera CYP6AQ1 and Bombus terrestris CYP6AQ26, respectively. Heterologous expression in High Five insect cells of these functional orthologs revealed a common coumarin substrate profile and a preference for the O-debenzylation of bulkier substrates. Competition assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) with these enzymes indicated inhibition of BOMFC metabolism by increasing concentrations of FPF. Furthermore, UPLC-MS/MS analysis revealed the capacity of all CYP6AQ1-like orthologs to metabolize FPF by hydroxylation in vitro at various levels, indicating a conserved FPF detoxification potential in different (neo)tropical bee species including Meliponini. This research, employing a toxicogenomics approach, provides important insights into the potential of stingless and other tropical bee species to detoxify FPF, and highlights the significance of investigating the detoxification mechanisms of insecticides in non-Apis bee species by molecular tools to inform risk assessment and conservation efforts.

Acute and chronic effects of sublethal neonicotinoid thiacloprid to Asian honey bee (Apis cerana cerana).

Pesticide pollution is one of the most important factors for global bee declines. Despite many studies have revealed that the most important Chinese indigenous species，Apis cerana, is presenting a high risk on exposure to neonicotinoids, the toxicology information on Apis cerana remain limited. This study was aimed to determine the acute and chronic toxic effects of thiacloprid (IUPAC name: {(2Z)-3-[(6-Chloro-3-pyridinyl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide) on behavioral and physiological performance as well as genome-wide transcriptome in A. cerana. We found the 1/5 LC50 of thiacloprid significantly impaired learning and memory abilities after both acute and chronic exposure, nevertheless, has no effects on the sucrose responsiveness and phototaxis climbing ability of A. cerana. Moreover, activities of detoxification enzyme P450 monooxygenases and CarE were increased by short-term exposure to thiacloprid, while prolonged exposure caused suppression of CarE activity. Neither acute nor chronic exposure to thiacloprid altered honey bee AChE activities. To further study the potential defense molecular mechanisms in Asian honey bee under pesticide stress, we analyzed the transcriptomes of honeybees in response to thiacloprid stress. The transcriptomic profiles revealed consistent upregulation of immune- and stress-related genes by both acute or chronic treatments. Our results suggest that the chronic exposure to thiacloprid produced greater toxic effects than a single administration to A. cerana. Altogether, our study deepens the understanding of the toxicological characteristic of A. cerana against thiacloprid, and could be used to further investigate the complex molecular mechanisms in Asian honey bee under pesticide stress.

Floral Pollen Bioactive Properties and Their Synergy in Honeybee Pollen.

Honeybee pollen (HBP) is a mixture of floral pollen collected by honeybees near the hive. It is characterized by a composition rich in phenolic compounds, carotenoids and vitamins that act as free radicals scavengers, conferring antioxidant and antibacterial capacity to the matrix. These bioactive properties are related to the botanical origin of the honeybee pollen. Honeybee pollen samples were collected from different geographical locations in central Chile, and their total carotenoid content, polyphenols profile by HPLC/MS/MS, DPPH radical scavenging capacity, and antimicrobial capacity against S. pyogenes, E. coli, S. aureus, and P. auriginosa strains were evaluated. Our results showed a good carotenoids content and polyphenols composition, while antioxidant capacity presented values between 0-95 % for the scavenging effect related to the botanical origin of the samples. Inhibition diameter for the different strains presented less variability among the samples, Furthermore, binary mixtures representing the two most abundant species in each HBP were prepared to assess the synergy effect of the floral pollen (FP) present in the samples. Data shows an antagonist effect was observed when assessing the carotenoid content, and a synergy effect often presents for antimicrobial and antioxidant capacity for bee pollen samples. The bioactive capacities of the honeybee pollen and their synergy effect could apply to develop new functional ingredients for the food industry.

Investigation of Botanical Origin, Phenolic Compounds, Carotenoids, and Antioxidant Properties of Monofloral and Multifloral Bee Bread.

Bee bread is a unique natural product made by bees and good for human health. It has many bioactive molecules that can treat or prevent diseases. In this study, melissopalynological methods were used to examine five bee bread samples. Major plant sources found in bee bread were Lotus spp., Trifolium spp., and Xeranthemum spp., which are from the Fabaceae and Asteraceae families. Then, the amount of phenolic compounds and major carotenoids in bee bread (BB) samples were quantified. Gallic acid, caffeic acid, quercetin, and kaempferol were found in all BB samples, with ß-carotene being the most abundant carotenoid in all but BB1. In addition, the total phenolic/flavonoid content and antioxidant activities of all BB samples were determined. Total flavonoid, total phenolic, DPPH⋅, and ABTS⋅+ values were varied between 5.6-10.00 mg GAE/g DW, 1.2-4.3 mg QE/g DW, 1.2-5.5 mg TEAC/g DW, and 2.6-15.4 mg TEAC/g DW, respectively.

Simultaneous Administration of Bevacizumab with Bee-Pollen Extract-Loaded Hybrid Protein Hydrogel NPs Is a Promising Targeted Strategy against Cancer Cells.

Bevacizumab (Bev) a humanized monoclonal antibody that fights vascular endothelial growth factor A (VEGF-A). It was the first specifically considered angiogenesis inhibitor and it has now become the normative first-line therapy for advanced non-small-cell lung cancer (NSCLC). In the current study, polyphenolic compounds were isolated from bee pollen (PCIBP) and encapsulated (EPCIBP) inside moieties of hybrid peptide-protein hydrogel nanoparticles in which bovine serum albumin (BSA) was combined with protamine-free sulfate and targeted with folic acid (FA). The apoptotic effects of PCIBP and its encapsulation (EPCIBP) were further investigated using A549 and MCF-7 cell lines, providing significant upregulation of Bax and caspase 3 genes and downregulation of Bcl2, HRAS, and MAPK as well. This effect was synergistically improved in combination with Bev. Our findings may contribute to the use of EPCIBP simultaneously with chemotherapy to strengthen the effectiveness and minimize the required dose.

Thin-layer chromatography in the authenticity testing of bee-products.

Quality control, nutritional value and the monitoring of hazardous residues in honey bee- products have become major topics for both producers and consumers. Due to its potential role in human health, bee-products rich in bioactive compounds are becoming increasingly popular. This review aims to provide an overview of thin-layer chromatography methods used in quality control,authenticity testing and chemical profiling of bee-products in order to help scientists engaged in the field of bee-products chemistry to utilize the advantages of this technique in the detection and elimination of fraudulent practices in bee-product manufacturing. Recently, hyphenation of thin-layer chromatography, image analysis and chemometrics support bee-products analysisbysimultaneousdeterminationofanalytes with different detection principles, identification of individual bioactive compounds as well as structure elucidation of compounds. Highlighted opportunities of thin-layer chromatography could encourage further investigations that would lead to improvements in the detection and elimination of marketing fraudulent practices.

Chronic Effects of Imidacloprid on Honey Bee Worker Development-Molecular Pathway Perspectives.

Sublethal dosages of imidacloprid cause long-term destructive effects on honey bees at the individual and colony levels. In this review, the molecular effects of sublethal imidacloprid were integrated and reported. Several general effects have been observed among different reports using different approaches. Quantitative PCR approaches revealed that imidacloprid treatments during the adult stage are expressed as changes in immuneresponse, detoxification, and oxidation-reduction response in both workers and queens. In addition, transcriptomic approaches suggested that phototransduction, behavior, and somatic muscle development also were affected. Although worker larvae show a higher tolerance to imidacloprid than adults, molecular evidence reveals its potential impacts. Sublethal imidacloprid treatment during the larval stage causes gene expression changes in larvae, pupae, and adults. Transcriptome profiles suggest that the population and functions of affected differentially expressed genes, DEGs, vary among different worker ages. Furthermore, an early transcriptomic switch from nurse bees to foragers was observed, suggesting that precocious foraging activity may occur. This report comprehensively describes the molecular effects of sublethal dosages of imidacloprid on the honey bee Apis mellifera. The corresponding molecular pathways for physiological and neurological responses in imidacloprid-exposed honey bees were validated. Transcriptomic evidence suggests a global and sustained sublethal impact of imidacloprid on honey bee development.

Adulteration of beeswax: A first nationwide survey from Belgium.

Beeswax is intended for use in the beekeeping sector but also in the agro-food, pharmaceutical or cosmetics sectors. The adulteration of beeswax is an emerging issue that was reported lately at several occasions in the scientific literature. This issue tends to become more frequent and global, but its exact extent is not accurately defined. The present study aims to assess the current situation in Belgium through a nationwide survey. Randomized beeswax samples originating from Belgian beekeepers (N = 98) and commercial suppliers (N = 9) were analysed with a Fourier transform infrared spectroscopy (FTIR) coupled with Attenuated Total Reflectance (ATR) accessory (FTIR-ATR spectroscopy) for adulteration. The survey revealed a frequency of 9.2% and 33.3% of adulteration in beekeepers beeswax samples (9 samples out of 98: 2 with paraffin and 7 with stearin/stearic acid) and commercial beeswax samples (3 samples out of 9: all adulterated with stearin/stearic acid), respectively. The analysed samples were adulterated with various percentages of paraffin (12 to 78.8%) and stearin/stearic acid (1.2 to 20.8%). This survey indicates that in the beekeepers samples, beeswax adulteration was more frequent in comb foundation and crude beeswax than in comb wax. With the example of this nationwide survey conducted in Belgium, this study shows the emergence of the issue and the urgent need for action to safeguard the health of both honey bees health and humans, in particular with the setting of a proper regulation legal framework and a specific routine analytical testing of commercial beeswax to ensure beeswax quality.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee.

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S-23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose's osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

Elementary calcium release events in the skeletal muscle cells of the honey bee Apis mellifera.

Calcium sparks are involved in major physiological and pathological processes in vertebrate muscles but have never been characterized in invertebrates. Here, dynamic confocal imaging on intact skeletal muscle cells isolated enzymatically from the adult honey bee legs allowed the first spatio-temporal characterization of subcellular calcium release events (CREs) in an insect species. The frequency of CREs, measured in x-y time lapse series, was higher than frequencies usually described in vertebrates. Honey bee CREs had a larger spatial spread at half maximum than their vertebrate counterparts and a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs' duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation-contraction coupling specificities in honey bee skeletal muscle fibres. The first characterization of CREs from an arthropod which shows strong genomic, ultrastructural and physiological differences with vertebrates may help in improving the research field of sparkology and more generally the knowledge in invertebrates cell Ca2+ homeostasis, eventually leading to a better understanding of their roles and regulations in muscles but also the myotoxicity of new insecticides targeting ryanodine receptors.

Non-targeted lipidomics and transcriptomics analysis reveal the molecular underpinnings of mandibular gland development in Apis mellifera ligustica.

The mandibular gland is an important exocrine gland of worker bees, which mainly secretes fatty acids and pheromones. Lipids have important roles in energy storage, membrane structure stabilization, and signaling. However, molecular underpinnings of mandibular gland development and lipid remodeling at the different physiological stages of worker bees is still lacking. In this study, we used scanning and transmission electron microscopy to reveal the morphological changes in secretory cells, and liquid chromatography-mass spectrometry and RNA-seq to investigate the lipidome and gene transcripts during development. The morphology of secretory cells was flat in newly emerged workers, becoming vacuolated and turgid when they were activated in nurse bees and foragers. Transport vesicles became denser from newly emerged bees to 21-day worker bees. Concentrations of 10-HDA reached a maximum within 15d workers and changes in genes expression were consistent with 10-HDA content. Non-targeted lipidomics analysis of newly emerged, 6d, and 15d worker bees revealed that PC and TAG were the main lipids in mandibular gland, and lipids dramatically altered across developmental stages. TAG 54:4 was increased most strongly at 6d and 15d worker bees, meanwhile, the abundances of TAG 54:1 and TAG 54:2 were decreased sharply. Further, transcriptomics analysis showed that differentially expressed genes were significantly enriched in key nutrient metabolic pathways, particularly lipid metabolism, in 6d and 15d bees. This multi-omic perspective provides a unique resource and deeper insight into bee mandibular gland development and baseline data for further study of the mandibular gland in worker bees.

Size constrains oxygen delivery capacity within but not between bumble bee castes.

Bumble bees are eusocial, with distinct worker and queen castes that vary strikingly in size and life-history. The smaller workers rely on energetically-demanding foraging flights to collect resources for rearing brood. Queens can be 3 to 4 times larger than workers, flying only for short periods in fall and again in spring after overwintering underground. These differences between castes in size and life history may be reflected in hypoxia tolerance. When oxygen demand exceeds supply, oxygen delivery to the tissues can be compromised. Previous work revealed hypermetric scaling of tracheal system volume of worker bumble bees (Bombus impatiens); larger workers had much larger tracheal volumes, likely to facilitate oxygen delivery over longer distances. Despite their much larger size, queens had relatively small tracheal volumes, potentially limiting their ability to deliver oxygen and reducing their ability to respond to hypoxia. However, these morphological measurements only indirectly point to differences in respiratory capacity. To directly assess size- and caste-related differences in tolerance to low oxygen, we measured critical PO2 (Pcrit; the ambient oxygen level below which metabolism cannot be maintained) during both rest and flight of worker and queen bumble bees. Queens and workers had similar Pcrit values during both rest and flight. However, during flight in oxygen levels near the Pcrit, mass-specific metabolic rates declined precipitously with mass both across and within castes, suggesting strong size limitations on oxygen delivery, but only during extreme conditions, when demand is high and supply is low. Together, these data suggest that the comparatively small tracheal systems of queen bumble bees do not limit their ability to deliver oxygen except in extreme conditions; they pay little cost for filling body space with eggs rather than tracheal structures.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.