Introduction

Bees are the world’s most important pollinators of plants, including food crops. It is estimated that as much as a third of the world’s food depends mainly on bees’ pollination1,2. But bees are not only involved in food production. As a high-protein additive, they can also be a direct food source for humans and animals. These insects are renowned for their nutritional value. Bee brood (defined as the eggs, larvae and pupae of honey bees) is rich in proteins, carbohydrates and fats. It is also a good source of essential amino acids. In addition, bee brood contains beneficial B vitamins, vitamin C and choline, as well as important chemical elements known as macro and micronutrients such as P, Mg, K, Fe, Zn, Co and Se3,4.

The life cycle of a honey bee is complex and includes the stages of egg, larva, pre-pupa, pupa and imago (Fig. 1). We commonly refer to eggs and larvae (from egg laying to the ninth day) as open brood. At the age of 6 days (9 days after egg laying), the larva is sufficiently nourished and enclosed in a cell with a wax cap and a porous structure that allows gas exchange. Under the cover, the larva transforms into a pupa and then into an adult insect. A brood in these stages of development is called a capped brood. The transition from egg to adult, called complete metamorphosis, takes 21 days in a worker bee and 24 days in a drone bee. The single step of larval development, from hatching to pupation, takes eight days in worker bees and ten days in drone bees.

Fig. 1
figure 1

The life cycle of a worker honey bee.

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The larval stage is the longest stage of honey bee development. It is a highly complex period of biochemical regulation. As larvae are specialised for feeding, they develop rapidly. They grow up to 1500 times their original size. So far, age-related increases in protein transporters, receptors and protein nutrient stores have been observed in the bodies of bee larvae. Protein expression in energy metabolism and response to internal and external stimuli has also increased with age5. However, a study of honeybees should consider environmental, individual and social factors.

Bees have clearly defined roles in the hive. Worker (female) bees are responsible for gathering nectar (essential for humans to pollinate crops), caring for the queen and larvae, keeping the hive clean and producing honey6. The queen bee is responsible for laying eggs by mating with drones7. Drones - male bees - play a crucial role in reproducing, thermoregulating the hive and removing some parasites8. Due to adaptation to their functions, the bodies of drones are much larger than those of worker bees. Larvae also differ in size - drone larvae are up to 2.6 times larger than worker larvae. This disparity is supported by a higher absolute protein content observed in male larvae, approximately 34 mg in drones versus 13 mg in worker larvae9. It is also worth noting that the larvae, unlike the pupae, do not contain any chitinous elements and, immediately before pupation, contain the maximum amount of nutrients. All these characteristics make drone larvae a rich source of easily digestible dietary protein and other valuable nutrients.

Honeybees (Apis mellifera) are bred all over the world. Honeybee brood are eaten in many countries, particularly Asia, Central America and Australia. Eating insects continues to grow in popularity, as evidenced by European Union (EU) legislation (Regulation (EU) 2015/2283, effective from 1 January 2018), which states that edible insects (including honeybees) and ingredients derived from them can be legally placed on the EU market as novel foods - but only after pre-market authorisation. Therefore, insects need to be thoroughly tested for safe consumption, especially as insects can be expected to be consumed by adults, children and older people. This issue is critical, mainly because the effects of foreign proteins and other molecules (characteristic of insects but not found in humans) on the human body are not fully understood and may cause both beneficial and adverse effects. Allergy sufferers, children and people with various medical conditions may be particularly vulnerable to harmful insect components. The adverse effects of insect-derived foods can be avoided, but it is essential to understand their characteristics and potential offending biomolecules, including proteins and peptides.

The omics strategy is one of the greatest approaches to the in-depth characterisation of natural products. One branch of omics - proteomics - helps to increase knowledge of biochemical pathways, the specific physiological function of individual proteins, their structure and their impact on living organisms. Honeybee larvae are rich in proteins, so studying proteins’ and peptides’ nutritional, pharmacological or toxic properties is essential, especially as they would be treated as a novel food. Unfortunately, there is limited data on the proteomic composition of honeybee drone larvae. Thus, this study focused on searching for and characterising proteins and peptides in honeybee drone larvae. The goal of the proteomic analysis was to identify new proteins (uncharacterised or hypothetical) that may be dangerous (e.g. toxins and allergens) or, on the contrary, a beneficial component of the human diet. The analyses aimed to test the hypothesis that bee drone larvae contain various proteins with different functions, including those involved in metabolic processes during intensive development, as well as proteins potentially dangerous to humans who consume the larvae, such as allergens.

The study’s novelty was using sophisticated sample preparation techniques coupled with mass spectrometry and depicting potentially harmful proteins, such as allergens, among those identified. Using an advanced pre-treatment tool based on a combinatorial library of hexapeptide ligands and solid-phase extraction (SPE) for the first time, according to the available literature, has significantly improved the identification capabilities of the MALDI-TOF/TOF (matrix-assisted laser desorption/ionisation-time of flight/time of flight) mass spectrometer. Knowing the full composition of bee larvae and assessing the safety of their consumption is necessary before the potential industrial production of high-protein foods based on bee larvae can begin.

Materials and methods

Sample collection

This study analysed the proteomic composition of honeybee (Apis mellifera) drone larvae’s water extracts. The larvae (n = 3) were collected in May 2020 in the non-commercial apiary located in Góry Złotnickie village (N 51°87′504′′, E 18°12′431′′) in Greater Poland Voivodeship, Poland. Drone larvae were collected directly from drone frames within healthy colonies, with larvae identified at specific developmental stages to standardize protein profiles and ensure reproducibility in proteomic analysis. Following the removal of larvae using sterilized forceps, samples were placed in pre-labeled, sterile containers, maintaining aseptic handling to minimize contamination. The collected samples were then immediately transported on dry ice to a freezer set at -80 °C to prevent protein degradation before laboratory analysis. This methodology supports a consistent and contamination-free framework, preserving protein structure and facilitating accurate proteomic studies. Sample pre-treatment (preparation of aqueous extracts and their depletion, purification and digestion) was performed immediately after sample delivery to the laboratory. Eluates prepared directly for MS analyses were stored in the darkness at -80 ºC until analysis.

Sample pre-treatment

The extracts were prepared by suspending 300 mg of larvae, crushed in an agate mortar, in 1 mL of ultrapure fresh water. The suspensions were vortexed for 1 min and then sonicated for 30 min. After sonication, the suspensions were remixed for 1 min and spun at 13,000 rpm (9600 RCF) for 20 min. The volumes of 0.2 mL of the collected supernatants (referred to as ‘extracts’ in this study) were subjected to pre-treatment with ProteoMiner™ Sequential Elution Small Capacity Kit (Bio-Rad, Hercules, CA, USA). This kit utilises the combinatorial hexapeptide ligand library to enrich the proteomic fraction, reduce the concentration of high-abundant proteins, and purify and desalt the samples. All steps were performed strictly according to the manufacturer’s instructions. All four fractions obtained in this procedure from one sample (a total of 12 fractions from three biological replicates) were subjected to proteolytic digestion with trypsin and then purified with solid-phase extraction ZipTip C18 (Millipore, Bedford, MA, USA) reverse phase chromatography micropipette tips. Our previous articles describe the detailed procedure10,11.

Proteomic identification using nanoLC-MALDI-TOF/TOF MS/MS

The detailed procedure of the MS analysis with appropriate conditions and parameters is described elsewhere10,11. Briefly, the ZipTip C18 eluates were subjected to separation by nano-liquid chromatography (nanoLC) (Bruker Daltonics, Bremen, Germany). The resulting fractions were mixed with the α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution and automatically spotted onto the AnchorChip 384 MALDI target plate (Bruker Daltonics, Bremen, Germany). Proteomic identification was performed using an UltrafleXtreme (Bruker Daltonics, Bremen, Germany) mass spectrometer in the m/z range of 700–3500. FlexControl 3.4 software (Bruker Daltonics) managed the spectrometer’s operation, data acquisition, and initial spectral processing. Precursor ion selection for identification was facilitated using WARP-LC 1.3 software (Bruker Daltonics). MS analyses of all ProteoMiner™ eluates obtained from three larvae samples were performed in three technical replicates. An NCBInr database and Mascot 2.4.1 search engine with taxonomic restriction to Apis spp. were used for the identification of discriminative proteins and peptides. Proteomic data evaluation was conducted with ProteinScape 3.1 software. For deeper protein identification, the BLAST (Basic Local Alignment Search Tool) algorithm was used to compare the acquired protein sequence against an NCBI database. This approach allowed us to identify regions of similarity between the experimentally obtained sequences and those included in the database. Based on this homology, some hypothetical proteins were identified. The workflow of the study is presented in Fig. 2.

Fig. 2
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Workflow of the study (created with BioRender.com).

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Results and discussion

The results of our study confirmed that bee drone larvae are high in protein, as we identified 194 unique proteins in this product, 109 of which were taxonomically assigned to the genus Apis (bees). Each protein was identified based on at least two peptides. A total of 1080 unique peptides were used for protein identification (see Table S1 and Table S2 in the Supplementary Materials). To the best of our knowledge, in this study, some proteins and peptides have been identified for the first time in the drone larvae of the honey bee. The achievement of such valuable results was made possible by using advanced methodology, particularly sample pre-treatment with ProteoMiner™ and SPE enrichment techniques. It has been confirmed that the use of the ProteoMiner™ strategy significantly increases the number of identified proteomic features in complex samples of natural origin10,12. Therefore, the high number of proteins identified in this study is due to the novel use of this approach. To the best of our knowledge, this study is the first attempt to exploit the advantages of the ProteoMiner™ in the analysis of honeybees.

The most significant number of proteins (n = 17) in drone larvae was identified based on three unique peptides. Moreover, based on four unique peptides, n = 16 proteins were identified and based on two unique peptides, n = 12 proteins were identified. Other proteins were identified by ten or more peptides, including one (n = 1) protein identified based on the most significant number of 116 individual fragments (Fig. 3).

Fig. 3
figure 3

Number of Apis proteins identified by a given number of unique peptide fragments.

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At least two unique peptide fragments of the protein are required for reliable proteomic identification using a MALDI-TOF mass spectrometer13. Detection of peptides depends on several factors, including the experimental protocol, effective cleavage by trypsin, the method of proteins and peptides depletion, the capabilities of the equipment (liquid chromatography and mass spectrometry, LC-MS) and the power of the software used for analysis. Not all of the peptides obtained by enzymatic digestion will be detected in a sample analysed by MALDI-TOF MS/MS14. However, the more significant number of unique peptides identified within a single protein reduces the risk of false positives. Therefore, in this study, we chose to discard all proteins identified on the basis of a single peptide, even if they reached certain scoring thresholds based on the native results of the MASCOT database search algorithms.

It should be noted that the MALDI-TOF/TOF-MS/MS method is categorised as a qualitative method, and no correlation could be made between the number of peptides and protein concentration.

The proteins identified based on the adopted criteria were classified into functional classes (Fig. 4, Supplementary Materials Table S1). Among the proteins identified, enzymes were the largest group. Forty-one (n = 41; 38% of all identified proteins in this study) proteins classified as enzymes were identified, with this number also representing isoforms. The next functional classes of the identified proteins included ribosomal proteins (n = 22; 20%), regulatory proteins (n = 17; 16%), binding proteins (n = 8;7%), storage proteins (n = 4, 4%), transport proteins (n = 4, 4%), as well as uncharacterised proteins (n = 3; 3%) and others (n = 5; 5%). Of particular note are hypothetical proteins (n = 5; 5%) whose presence in bee products and larvae has not yet been experimentally confirmed, although the amino acid sequence of these proteins matches the nucleotide sequence of the genes. All of the hypothetical proteins identified in this study are first-time discoveries in bee larvae, according to the available scientific literature. The above results support the hypothesis that the bee drone’s larvae contain several proteins with different functions.

Fig. 4
figure 4

Functional classes of proteins identified in drone bee larvae.

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The most numerous functional groups of proteins were enzymes and ribosomal proteins. These functional protein groups indicate an intense period of larval development, during which many new biomolecules are synthesised. This stage requires large amounts of energy, resulting in the presence of many enzymes involved in energy-producing metabolic pathways (Fig. 5). Among the proteins identified in this study were those involved in metabolic pathways that lead to the production of energy, namely glycolysis (fructose-bisphosphate aldolase-like, glyceraldehyde-3-phosphate dehydrogenase 2 isoform 1), glycogenolysis (glycogen phosphorylase-like), and citric acid cycle (ATP-citrate synthase)15,16,17. In addition, many of the identified enzymes are involved in the synthesis or metabolism of endogenous compounds – lipids, carbohydrates, amino acids, peptides, proteins, and others. These pathways may also lead directly or indirectly to energy production in cells.

Fig. 5
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Glycolytic enzymes involved in metabolic pathways leading to energy production.

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In the lipid pathway, glucosylceramidase isoform 1 is involved in sphingolipid metabolism and glycan structure degradation18,19. Fatty acid synthase, ATP-citrate synthase isoform 1, and acetyl-CoA carboxylase-like isoform 2 participate in fatty acid synthesis20,21,22. In addition, by converting citrate to acetyl-CoA, the latter enzyme links carbohydrate metabolism, which produces citrate as an intermediate, to fatty acid synthesis, which requires acetyl-CoA23.

Among enzymes involved in carbohydrate metabolism, in this study, we identified transketolase isoform 1 and 6-phosphogluconate dehydrogenase, decarboxylating - the enzymes involved in the pentose phosphate pathway, which leads to the generation of NADPH, pentoses and ribose 5-phosphate, a precursor for nucleotide synthesis24,25,26. We also identified UTP-glucose-1-phosphate uridylyltransferase isoform 1 involved in glycogenesis (via synthesis of UDP-glucose from glucose-1-phosphate and UTP) and 4-hydroxybutyrate coenzyme A transferase-like - enzyme involved in ketogenesis (via transferring of coenzyme A group from an acyl-CoA to a carboxylic acid)27,28. Another identified enzyme, 3-ketoacyl-CoA thiolase, mitochondrial-like, is known to be involved in the mevalonate pathway – a crucial metabolic pathway leading to the production of cholesterol, steroid hormones, vitamin K and coenzyme Q10 29,30. Other enzymes identified in this study involved in carbohydrate metabolism include sorbitol dehydrogenase-like isoform 2; aldose reductase-like isoform 1; and alpha-glucosidase precursor31,32,33.

Enzymes identified in this study also include those involved in protein and amino acid metabolism. We identified cytosolic non-specific dipeptidase-like isoform 1, engaged in dipeptide hydrolysis; arginine kinase, involved in arginine and proline metabolism, which plays a critical role in the ATP buffering systems of animal cells that have a high and variable rate of ATP turnover; and casein kinase II subunit alpha, involved in acidic protein phosphorylation34,35,36. Moreover, we detected glutaryl-CoA dehydrogenase, a mitochondrial enzyme involved in the degradation pathway of L-hydroxylysine, L-lysine and L-tryptophan metabolism, and arginase-1-like - a vital enzyme of the urea cycle that catalyses the conversion of L-arginine to L-ornithine and urea37,38.

The presence of enzymes involved in the synthesis and metabolism of carbohydrates and amino acids may be evidence not only of the intense development of the larvae of bee drones but also of the development of mechanisms that protect cell stability and prevent stress caused by heat and humidity. According to Xinyu et al.39, the presence of some sugars, polyols and free amino acids can effectively protect bees from shock caused by high temperatures and elevated humidity. The presence of substances that stabilise and protect the larvae from harmful factors can be crucial to the survival rate of bees, as the colony is exposed to threats that can lead to colony loss. Such factors include changes in climatic conditions, bee management, pests and diseases, and exposure to environmental pollution and pesticides40. In particular, anthropogenic activities negatively affect the survival of bees, as bees come into contact with many environmental factors in their daily lives and are exposed to many human activities and their consequences41. It has been reported that honeybees have a limited number of detoxifying enzymes compared to other insects, making them very sensitive to toxins, including pesticides, which directly target their central nervous system42. However, thanks to the development of appropriate mechanisms, including those based on the accumulation of carbohydrates, polyols and amino acids, bees are able to survive in increasingly difficult conditions.

Moreover, in this study, enzymes that are responsible for the regulation of the oxidative stress response, namely peroxiredoxin 1 and superoxide dismutase 1 were identified in honeybee drone larvae. Antioxidant enzymes reduce life-shortening oxidative damage and combat pathological tissue damage caused by various factors43. A study by Sidor et al.44 on different stages of drone brood larval development showed that the homogenate’s antioxidant potential depends on the larval stage. It was found that the shorter the hatching time of the larvae studied, the higher the activity of antioxidant enzymes.

In addition, in honeybee drone larvae, we identified enzymes involved in regulation and signalling (glutamate dehydrogenase, mitochondrial isoform 1; retinal dehydrogenase 1-like isoform 1) and pyrimidine biosynthesis and metabolism crucial for nucleic acid synthesis (glucosylceramidase-like isoform 1, CAD protein-like).

Ribosomal proteins were the second most abundant functional group of proteins identified in drone bee larvae. By translating the genetic information encoded in mRNA into an amino acid sequence, ribosomes are responsible for protein synthesis45. The presence of ribosomal proteins in the drone larvae body may also indicate the larvae’s intensive development and biomass growth.

Hypothetical proteins are a particularly interesting group of proteins identified for the first time in bee drone larvae in this study. These are proteins whose amino acid sequence matches the known nucleotide sequence of the honeybee genome but whose presence has not yet been experimentally confirmed. In this study, we identified five hypothetical proteins. A BLAST analysis was performed on the hypothetical proteins to estimate the homology of the proteins identified in bee larvae with proteins taxonomically classified to Apis mellifera or other Apis species according to the NCBI database (Table 1).

Table 1 Results of BLAST analysis [accession date: 17.01.2024; https://blast.ncbi.nlm.nih.gov/Blast.cgi].
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Based on the homology assignment, we identified the AP-3 complex subunit beta-1 as the homologous protein corresponding to the hypothetical protein LOC409479 with query coverage and per cent identity values of 100%. High homology was also observed between the hypothetical protein gi|77415558 detected in this study and the chemosensory protein 3 precursor [Apis mellifera] (query coverage = 100%, per cent identity = 99.23%) or the odorant binding protein ASP3 [Apis cerana cerana] (query coverage = 100%, percent identity = 98.46%). In addition, the hypothetical protein LOC551211 isoform 2 was shown to be homologous to the leucine-rich repeat-containing protein found in several bee species (query coverage 83%, per cent identity 99.27–98.63%).

Adaptins (subunits of clathrin adaptor protein (AP) complexes), including the AP-3 complex, are involved in forming intracellular transport vesicles and selecting which cargo to incorporate into them. Thus, these proteins are responsible for intracellular trafficking and secretion46. Chemosensory proteins and odorant binding proteins are thought to increase the solubility of hydrophobic odours and are involved in the olfactory recognition and release of chemical stimuli in insects47,48. These proteins are expressed according to the caste and age of the bees49. Leucine-rich repeat-containing protein is involved in forming protein-protein interactions50. The identified hypothetical proteins, classified for the first time into specific functional groups, may further contribute to understanding honeybee physiology. In addition, this study tested whether the previously unknown proteins are harmful after ingestion. However, there is no evidence that the current hypothetical proteins could be harmful to humans.

The available reports focusing on the composition of bee larvae are very scarce. Many of the papers published to date have focused on the analysis of the nutritional value of whole bee brood (i.e. eggs, larvae and pupae) or apilarnil, which is prepared from the contents of the cells of drone larvae3,51,52,53,54. Therefore, our study should be regarded as a novel one. However, the results of this study are consistent with those reported in the available literature. In the study by Li et al.55, as in our study, the most abundant group of proteins identified in bee larvae were those involved in carbohydrate, amino acid and fatty acid metabolism and energy production. However, it should be noted that our study analysed drone larvae, whereas Li’s study analysed worker and queen bee larvae. Nevertheless, the metabolic processes leading to larval development are similar in male and female larvae, so the results are comparable.

Because of their rich composition and high protein content, drone larvae can be considered a valuable food source for both humans and livestock. As they are not involved in honey production, drone larvae can, to some extent, be removed from the hive to collect them for food purposes without causing much damage56. From the honeybee colony’s point of view, harvesting drone larvae for food is less harmful than reducing the number of worker bees. This may actually be beneficial to the colony. Removing drone brood helps reduce infection by the Varroa mite. Varroa is one of the most dangerous diseases in bees, the parasite attacks adult bees and brood57. Although beekeepers are increasingly turning to organic products (formic acid, oxalic acid, lactic acid) to control the parasite, drone brood removal is still practised. By removing the brood, part of the brood becomes a by-product, a large waste that can be used in the food, medical or cosmetic industries7. However, it is still vital to identify and select the most appropriate stages of colony development for drone harvesting so as to cause the least damage to the bee family.

Considering drone larvae as a food source requires prior determination of their protein components’ immunogenic and allergenic properties. In the available literature, the composition of bee larvae has not been considered concerning their allergenicity. Therefore, in this study, we analysed the identified proteins in terms of their negative effects on human and animal bodies. Since allergens cause inflammatory allergic reactions, which in the worst cases can lead to serious health problems or even death, the analysis of the composition of the products treated as a new food is of utmost importance. This new and up-to-date knowledge will help avoid side effects in people susceptible to specific allergens.

As it is well known, honeybee venom contains allergens58. The World Health Organization’s Allergen Nomenclature Sub-Committee (WHO/IUIS) has registered 12 honey bee protein fractions as allergenic59. Most of them are highly immunogenic glycoproteins, causing a variety of symptoms, from local reactions to systematic, life-threatening anaphylaxis and even death58. In this study, we identified a few proteins in the drone larvae extract that, according to WHO/IUIS, may be considered in the context of allergens. The identified arginine kinase is an ATP phosphotransferase found in invertebrates. It catalyses the phosphorylation of arginine residues60. Arginine kinase is a monomeric protein with a molecular weight of approximately 40 kDa and is quite thermally stable. The enzyme purified from cockroaches retained 50% of its activity when heated to 50 °C for 10 min. Arginine kinase is considered a food allergen. It has been found in various crustaceans, such as crabs, different species of shrimp and lobsters61. This protein is highly conserved both functionally and structurally within the Arthropoda. The high degree of homology in the protein domain causes cross-reactivity within crustacean species and between crustacea and insects. Arginine kinase has also recently been discovered in the extract of Dermatophagoides farinae (Der f 20). Although arginine kinase-induced allergic diseases have been described, there are few studies on the characterisation of the Dermatophagoides farinae-derived arginine kinase62.

Another cross-reactive molecular component identified in the drone larvae extract was glyceraldehyde-3-phosphate dehydrogenase. Its homologue was first discovered in the American cockroach and named Per a 13. The allergenicity of glyceraldehyde-3-phosphate dehydrogenase was confirmed by immunoblot and basophil activation test in cockroach-allergic patients63. Thioredoxin found in drone larvae represents a family of cross-reactive allergens, which, according to literature data, may contribute to the symptoms of baker’s asthma64. Isoforms of major royal jelly protein - Api m 11.0101 (MRJP8) and Api m 11.0201 (MRJP9) have been identified as bee venom allergens, while Api m 11.0301 (MRJP1) has been described as a food allergen in honey65. In turn, alpha-glucosidase (Aed a 4) detected in the drone larvae extract has been previously identified in Aedes aegypti mosquito saliva66,67.

Data on the allergenic potential of drone larvae are limited. However, Stoevesandt et al.68 reported anaphylaxis caused by drone larvae juice in a 29-year-old beekeeper. The patient had an anaphylactic reaction after consuming a freshly prepared beverage from raw drone larvae. Larvae-specific sensitisation was confirmed using the prick-to-prick skin test and basophil activation test. The patient tolerated bee stings and classic bee products, including honey and royal jelly. To our knowledge, this is the only report on IgE-mediated allergy to drone larvae.

Although, as mentioned above, there is some risk of anaphylactic reaction after consuming broodstock, there are also benefits to growing broodstock for food. The advantage is undoubtedly a relatively small area of agricultural land and a small financial investment for larvae rearing, in contrast to livestock farming. Furthermore, compared to insects, raising animals produces more greenhouse gases and requires more feed to produce 1 kg of body weight69. There is a growing interest and demand for insect-based foods. Bee brood is an attractive breeding option, similar to other insects reared for food. Insects such as honeybees, flies, beetles and crickets are a good source of protein for humans and livestock. Insect foods are readily available at low cost and are high in protein, fat and minerals70. The protein content of honeybee brood (15.4–18.2%) is comparable to that of beef (17.7%), highlighting its nutritional value70,71. This suggests that drone larvae can be used as a substitute for meat and other protein products.

Conclusions

The results of this study support the hypothesis that the bee drone’s larvae contain several proteins with different functions. The substantial results presented in this article were obtained by using advanced analytical techniques: nanoLC-MALDI-TOF/TOF MS/MS and sophisticated sample preparation techniques for proteomic analyses. Since no previous studies in the available literature have utilized this methodology, this study’s results are unique and advance substantially existing knowledge. The larvae are a potential source of antioxidant components. Their qualitative composition may indicate their early developmental stage, which requires a high-energy input and the synthesis of many new proteins. The present study may be a step towards a better understanding of the physiological processes occurring in the bee body during the early stages of development. Studies of bee larvae also provide relevant information on their nutritional value and potential use in the food industry. The results of this study, which identified over 100 unique proteins, suggest that bee larvae are a valuable protein-rich product. On the other hand, the larvae also contains allergens, which, in the worst case, can cause life-threatening anaphylaxis, as reported in the scientific literature. Therefore, further research into the safety of consuming bee brood, including the larvae themselves, is essential if bee larvae are to be widely used by the food industry in Europe, America and other regions of the world.