The Un-Bee-lievable Chemistry of Bees

Keywords: bee, pheromones, venom, honey, wax, brood food, pollen

Abstract

As a superorganism, a bee colony needs to efficiently feed its members, construct a solid and weatherproof structure, allow the flow of information between individual bees, and defend itself. Bees use an arsenal of chemical processes to respond to their needs. Nourishment for its members is processed into shelf-stable, nutrient rich honey, bee bread, and brood jelly. Beeswax is impermeable, strong, and flexible, which is all critical for a solid hive structure. Propolis, or bee glue, is also used in construction for its impermeability, anti-intruder, and antiseptic properties. Members of the colony use pheromones to communicate and ensure proper division of labor. Pheromones also allow the recognition of intruders and the defence organization, where the intruders can be stung by a potent venom. All these complex substances vary in composition and are made by bees from available plant compounds, which makes bees un-bee-lievable chemists of nature.

Introduction

Beekeeping is an ancient and intrinsic human activity (Wachkoo et al., 2024). Over the years, bee products have continued to serve our needs and fascinate us. Honey, bee pollen, and royal jelly were luxurious foods and medicines the ancient Greeks thought were ingredients of ambrosia, the nectar of gods (Khalil et al., 2021). Beeswax and propolis are other harvested derivatives used for candle-making, therapeutics, and preservatives (Wachkoo et al., 2024). Even bee venom was extracted for medicinal purposes in ancient Egypt, Greece, and China (Wachkoo et al., 2024). These substances’ incredible properties are chemically tailor-made by bees for the benefit of the colony. Honey and bee pollen are processed to be viable forms of food storage to feed the entire colony day after day. Their compositions are also uniquely affected by the different floral origins of the produce. Bee jelly is bee’s own form of “milk” that helps epigenetically differentiate queens from ordinary workers. Beeswax forms the solid structure of the hive. Propolis is applied to the hive as a barrier against environmental elements and invaders. Bees also produce not-yet-harvested chemicals called pheromones to communicate with each other and enhance the functioning of the colony’s superorganism. Special pheromones allow guard bees to detect intruders and defend the nest. Lest we forget that their sting contains a potent venom capable of effectively neutralizing enemies both large and small. These chemicals, which vary widely in function, are masterfully used by bees to feed their members, build their homes, organize their society, and defend the colony.

1. Bee-licious foods

Most bees are herbivores, as both larvae and adults subsist solely on plant nutrients (Johnson & Seeley, 2023). The foragers mainly hop from flower to flower because flowers contain their primary sources of carbohydrates (from nectar), protein and lipids ( from pollen). Highly social bees, like honeybees, create long-term storage reserves of plant products in the forms of honey (concentrated nectar) and bee bread (stored pollen preserves) to prepare for colonial periods of scarcity. Furthermore, nurse honeybees secrete a nutrient-rich jelly to feed their brood (and sometimes other adults too), the composition of which aids in determining if the larvae will develop into a queen or an ordinary worker (Johnson & Seeley, 2023). Considering these products’ un-Bee-lievable shelf life and nutrient content, it is unsurprising that written sources have shown that ancient cultures have used bee substances for food and medicine (Wachkoo et al., 2024).

a. Honey, oh honey

Bees make honey in two steps. On the forager bee’s way back to the nest, digestive enzymes are added to nectar in its crop (Johnson & Seeley, 2023). These enzymes, notably invertase, diastase, and glucose oxidase, break down larger sugars into simpler sugars, which acidifies the nectar, making it resistant to bacterial growth. Back in the hive, the forager transfers the processed nectar to a middle-aged bee, who then stores it in a comb cell and dehydrates it down to less than 18% water by fanning its wings on the cell. Dehydration further prevents bacterial attack and shrinks the volume of the produced honey for efficient storage. The comb cell is then quickly capped like a can so that the stored honey can be viable for many years (Johnson & Seeley, 2023).

Since bees produce honey from plant nectar, honey composition varies depending on the available plants in a hive’s proximity (Johnson & Seeley, 2023). Nectars contain sugar ranging from 5-80% of its content, but most nectars are 20-40% sugar. The primary sugars in nectar are fructose, glucose, and sucrose, but their specific percentages vary across plant species. Large sugars, like maltose, trehalose, and melezitose, are also present and give honey its sweet (but not too sweet) flavour. Nectar also contains some amount of amino acids, vitamins, minerals, and ash, which can make honey darker in colour. Bees may also collect honeydew from plant-sucking insects, but it is typically a minor component of honey (Johnson & Seeley, 2023).

Honey comprises about 200 different components, including water, vitamins, minerals, carbohydrates, amino acids, enzymes, pigments, organic acids, and volatile compounds (Wachkoo et al., 2024). Honey’s dry weight is 96% carbohydrates, 85% of which is fructose and glucose. Due to its high monosaccharide content, honey provides an immediate energy boost, with 100 grams of honey providing 304 kcal of energy, almost as much as three boiled eggs. The remaining 5% of honey varies widely in content and determines the specific honey’s physical and therapeutic properties. For example, honey with high polyphenol content exhibits high antioxidant, antimicrobial, anti-inflammatory, and anticarcinogenic activities and is of a dark colour. Depending on the botanical origin of honey, other metabolite agents can also provide medicinal properties. Rhododendron (derived from Rhododendron ponticum) honey contains diterpenoids, which lowers blood pressure. Manuka (derived from Leptospermum scoparium) honey has high antimicrobial activity due to its high methylglyoxal content (Fig. 1) (Wachkoo et al., 2024).

Fig. 1. Comparison between the physical color of manuka honey (left) with organic amber honey (right) made by a contributing writer. Manuka honey is darker than organic multi-floral honey.

Besides its polyphenol and various metabolite actions, honey has three basic antimicrobial properties which aid in its preservation and wound-healing effects. Honey’s viscous structure causes reverse osmosis in bacteria, its acidic pH range of 3.5-5.5 prevents bacterial growth, and its small but sufficient hydrogen peroxide content due to the glucose oxidase enzyme present in honey also combats bacteria (Wachkoo et al., 2024). Honey’s basic chemical composition is therefore optimized for its purpose: being nutrient-dense and shelf-stable. Its diverse botanical origins provide it with various medicinal compounds for the good health of bees.

b. Bee bread

Although honey contains small amounts of protein, vitamins, and minerals, pollen is the bees' primary source of lipids, protein, vitamins, and minerals (Johnson & Seeley, 2023). Forager bees collect pollen grains by rubbing their bodies against the flower (Wachkoo et al., 2024). Pollen is then transferred to the short, stiff hairs on their hind legs, called scopae, where it is compressed into balls using saliva and honey, and placed in specialized structures known as pollen baskets (Fig. 2) (Ghouizi et al., 2023). Back at the hive, the foragers cover the pollen balls with saliva, place and compact the balls along with some honey in comb alveoli, and cap the cell with a wax layer for storage. The mixture in the pollen cell undergoes lactic fermentation due to naturally occurring lactic bacteria in the hive. Pseudomonas consume oxygen and create an anaerobic environment, Lactobacillus convert carbohydrates into lactic acid, and Saccharomyces ensure the metabolism of the other sugars in the mixture. These reaction chains reduce the cell’s pH, prevent pollen germination, and improve bee pollen absorption capacity and nutritional value. Entirely fermented bee pollen is called bee bread and is used in royal jelly production, larvae nutrition, and worker sustenance as a significant source of protein (Fig. 3) (Ghouizi et al., 2023).

Fig. 2 Spanish bee pollen balls purchased by a contributing writer.

Like with nectar, pollen composition varies from plant to plant, which translates into bee bread variations too (Johnson & Seeley, 2023). Pollen’s protein content ranges from 10-60%, while bee bread varies between 10-30% protein. Bees need 10 essential amino acids in their diet, the most limiting being leucine, isoleucine, and valine. Bees are adept at extracting protein from pollen, as they extract 75% of available proteins during digestion. Pollen is 7-20% lipids, and bee bread averages at 3-8% in dry weight. Generally, lipids on the pollen grain surface have antimicrobial properties to protect the pollen from degradation, while the inner lipids are more nutritious. Like with proteins, bees need essential (cannot be self-synthesized) and non-essential (can be self-synthesized) fatty acids. The bulk of lipids in pollen consists of two essential fatty acids, alpha-linolenic acid (an omega-3) and linoleic acid (an omega-6), as well as non-essential fatty acids like palmitic acid. Because bees need a diverse range of essential amino acids and fatty acids, they collect pollen from many sources to create a balanced diet. For example, an imbalance of fatty acids (too high of a ratio of omega-6 to omega-3) causes learning and memory deficits and low survivorship in brood and adult bees (Johnson & Seeley, 2023).

The principal mineral elements in bee pollen are potassium, phosphorus, calcium, and magnesium, but pollen balls also contain copper, iron, zinc, manganese, and selenium (Wachkoo et al., 2024). Minerals have essential health benefits. For example, zinc and copper aid in the functioning of antioxidant enzymes like superoxide dismutase. Other vital enzymes, like catalases, aconitases, and peroxidases depend on iron to function.

Bee pollen also contains various phenolic compounds, vitamins, and organic acids, depending on the types of flowers they harvest their pollen from (Wachkoo et al., 2024).

Fig. 3 Bee bread stored in honeycomb (Pollen, J.).

c. Jelly for brood (and adults too)

In opposition to almost all other bee species, honeybees feed their young a glandular secretion (comparable to milk) rather than directly providing them a pollen nectar mixture (Johnson & Seeley, 2023). Young nurse bees (aged 5 to 14 days) create brood jelly in the hypopharyngeal and mandibular salivary glands (Wachkoo et al., 2024). This jelly is a white-yellow, gelatinous, viscous, sour-tasting fluid with a faint phenol scent and is produced in the stomachs of worker bees due to their inability to digest honeydew fully (Wachkoo et al., 2024). Nurse bees create two types of jellies: royal jelly and worker jelly. Royal jelly is fed to all brood aged 3 days or less and continues to be fed to queen bees throughout their entire lives (Fig. 4) (Johnson & Seeley, 2023). Nurse bees feeding larvae destined to become worker bees will switch their feeding to worker jelly, which is mainly produced by the hypopharyngeal gland (Johnson & Seeley, 2023).

Fig. 4. Royal jelly product for human consumption (API CO. Ltd.)

Differential feeding of bee larvae is a major signal contributing to caste differentiation between worker and queen bees (Johnson & Seeley, 2023). A key difference between worker and royal jelly relates to sugar content. Royal jelly contains more sugar than worker jelly, and experiments showed that larvae fed with worker jelly supplemented with sugar developed into queen bees. Also, worker jelly contains more microRNA than royal jelly and may be a factor altering each caste’s brain development. Indeed, worker bees that are fed royal jelly develop superior learning abilities and memory, akin to a queen (Shi & al., 2018). Furthermore, a fatty substance called (E)-10-hydroxy-2-decenoic acid (10-HDA), a histone deacetylase inhibitor, makes up 5% of royal jelly, but only 1.5% of worker jelly. Acetylation of histones is a core epigenetic modification affecting gene expression. Experiments showed that increasing 10-HDA in brood food leads to lower bee body size. Additionally, queens are fed a larger quantity of food in general, while worker bees are fed just enough (Johnson & Seeley, 2023).

Overall, royal jelly is about 63% water, 18% carbohydrates, 14% protein, and 6% fats (Johnson & Seeley, 2023). It also contains sterols, and all the necessary micronutrients needed for growth and health (like vitamins, minerals, organic acids, and phenols) (Johnson & Seeley, 2023; Wachkoo et al., 2024). While the carbohydrates in royal jelly are like those of honey, its protein contents are widely different (mainly consisting of major royal jelly proteins) (Johnson & Seeley, 2023). Studies show that major royal jelly proteins increase the life expectancy of D. melanogaster that feed on it (Wachkoo et al., 2024). The nutritious composition of royal jelly serves to properly feed honeybee larvae and queens, being a key factor in the epigenetic caste differentiation between worker (infertile, smaller, shorter-lived) and queen bees (Wachkoo et al., 2024).

2. Beeswax

Beeswax is a naturally occurring, chemically complex substance produced by worker bees within the hive (Fig. 5). It plays an important role in the construction and maintenance of the hive’s structure, forming the honeycomb cells that hold honey, pollen, and larvae. Although beeswax contains over 300 different components, the most abundant are esters, hydrocarbons, and fatty acids (Dobrosielska et al., 2023).

Fig. 5 The honeycomb structure, which is made of beeswax (Svečnjak et al., 2019).

a. Chemical composition

Beeswax is a complex mixture of esters, hydrocarbons and free fatty acids (Dobrosielska et al., 2023). Its chemical composition is dependent on several factors, such as the age of wax, the climate in which it was produced, and the bee subspecies. However, the variation mainly impacts the proportion of components present, rather than the chemical structure of them.

Beeswax is made up of five main components, each occurring at different concentrations (Fig. 6). Linear wax monoesters and hydroxymonoesters make up 35-45% of the wax structure. The esters have carbon chain lengths ranging from 40-48 atoms and are derived from palmitic acid, 15-hydroxypalmitic acid, and oleic acid (Kuznesof & Whitehouse, 2005). Most of these wax esters are saturated, however unsaturated wax esters exist in the longer chain lengths (Kuznesof & Whitehouse, 2005).

The second most abundant components are complex wax esters with multiple ester bonds, making up 15-27% of the composition. These are formed through the esterification of hydroxyl groups from acylated hydroxy esters or diol diesters (JEFCA, 2005).

Straight-chain hydrocarbons with odd-numbered carbon chains of 27 to 33 atoms account for 12–16% (JEFCA, 2005). Free fatty acids, with carbon chain lengths of 24-32 atoms represent around 12-14%. Eight major and ten minor types of fatty acids were found, with lignoceric acid, hexacosanoic acid, and octacosanoic acid being the most common (JEFCA, 2005). Finally, free fatty alcohols occur in very small amounts, making up only about 1% of the structure (Kuznesof & Whitehouse, 2005).

Fig. 6. Chemical composition of some components of beeswax. This includes: (A) Hydrocarbon chains, (B) monoesters, (C) Diesters, (D) hydroxy monoesters, (F) free fatty acids, (G) free fatty alcohols, (H & I) triesters, (J) acid monoesters and (K) acid polyesters (Dobrosielska et al., 2023).

b. Production process

Bees have been found to start producing beeswax when they are around 1 week old, with peak production at two weeks old (Hepburn et al., 1991). To form the wax, bees consume large amounts of honey. It takes around 20 g of honey to create 1 g of wax (Xu et al., 2024). The sugars in honey are metabolized into fatty acids and lipids in the bee’s body, then secreted out of the bee's body as wax.

Beeswax is produced by eight abdominal glands that are located in the anterior sections of sterna IV to VII (Tinto et al., 2017). These glands secrete wax platelets in liquid form, which solidify into translucent scales when in contact with air (Fig. 7). Every segment of the sternum contains wax mirrors, which are two smooth surfaces that cover the wax glands. The wax mirrors help guide the secretion of wax and ensure proper formation of the scales (Svečnjak et al., 2019). The bees use their legs to scrape off the scales. They are then chewed and mixed with saliva and enzymes (Tinto et al., 2017). This process makes it easier for them to build the honeycomb, which provides storage for honey and protection for larvae.

Fig. 7. Wax scales emerging from the abdominal wax glands of a worker bee. Using the legs, the scales are brought to the mouth where they are nibbled and mixed with saliva (Klein, 2015).

There are several specialized cells that help with the production of beeswax, each having a distinct role in the process (Fig. 8). The conversion of carbohydrates from honey to the fatty acids and hydrocarbons components of beeswax is a complex and energy intensive process. Overall, there are three main types of cells that are involved in producing beeswax: Epithelial cells, oenocytes and adipocytes (Hepburn et al., 1991).

Epithelial cells are found around the wax glands and help transport the wax from the inside of the bee to the wax mirrors before secretion (Hepburn et al., 1991). Oenocytes are specialized cells that primarily help synthesize the hydrocarbon and fatty acid components of beeswax (Hepburn et al., 1991). Adipocytes, or fat cells, provide the energy that is needed for the production process and store lipids (Hepburn et al., 1991).

Fig. 8. Diagram of the wax gland in a bee. The main components include: WS = wax scale, WM = wax mirror, E = epidermal cells, O = oenocytes, F = adipocytes. (Hepburn et al., 1991).

The lifespan of a bee is around two to six weeks during the summer and around 20 weeks in the winter (Prado et al., 2020). In younger bees, oenocytes are inactive, but by the fourth day, they grow and start producing materials for wax. The cells stay large while the bees are producing wax, however by day 18 the cells start to shrink, and lysosomes begin to appear (Hepburn et al., 1991).

Adipocytes have many fat-producing structures but lack some wax-producing elements, such as a smooth endoplasmic reticulum. Younger bees have around 60% of these cells filled with fat droplets (Hepburn et al., 1991). Before wax is produced, the fat cells grow larger and store energy in the form of glycogen. When the bees use energy to create the wax, the cells start to shrink (Hepburn et al., 1991). The different cells are located close to each other. They are separated by small gaps that are filled with a thin layer of material called the basal lamina (Hepburn et al., 1991).

c. Properties of beeswax

Beeswax can be classified into two types: yellow and white, each with slightly different properties. White beeswax is a white solid that consists of thin, translucent layers and has a faint honey smell (Kuznesof & Whitehouse, 2005). Yellow beeswax is a yellow or light brown solid that is brittle when cold and has a prominent honey smell (Kuznesof & Whitehouse, 2005).

As the chemical components of beeswax differ based on variables such as bee species, its physical properties also vary; however, beeswax is generally hydrophobic due to its composition of hydrocarbons and esters. This is an important property, as being water-resistant helps protect materials inside of the hive from the moisture (Gupta & Anjali, 2023). Beeswax is also slightly insoluble in alcohols, soluble in ethers and fixed and volatile oils, and resistant to acids (Kuznesof & Whitehouse, 2005). Beeswax also has a low melting point that can range from 62-67°C, making it solid in cooler conditions and mouldable when warm, helping with the building of the honeycomb structure (Kuznesof & Whitehouse, 2005). The combination of esters, hydrocarbons, fatty acids and fatty alcohols provides structural stability. The chemical structure offers strength and flexibility, which is essential for creating the honeycomb structure (Svečnjak et al., 2019).

3. Communication by pheromones

To achieve such a high level of social organization, bees communicate by exchanging chemical messenger molecules called pheromones (Bortolotti & Costa, 2014). Bee colony pheromones are like the hormones of a single organism – guiding the colony's development, coordinating its response to specific triggers, and driving forward its life cycle. Individual bees synthesize pheromones in various exocrine glands, including their mandibular, Dufour, Koshevnikov, Tarsal, and Tergal exocrine glands (Fig. 9) (Bortolotti & Costa, 2014). Pheromones are transmitted throughout the colony by mechanisms which vary based on the chemical properties of pheromone compounds (Altstein, 2013). Volatile compounds, characterized by their smaller molecular weight and weaker intermolecular forces, vaporize and disperse through the air. Some pheromones are nonvolatile; these are called “passenger pheromones” and can only be dispersed via physical contact between bees (Bortolotti & Costa, 2014).

Fig. 9. The exocrine glands of a honeybee queen. Glands associated with pheromone secretion are indicated by the grey lines. (Bortolotti & Costa, 2014)

Bee chemosensory organs consist of groups of innervated pores along their antennae, called sensillae (Sandoz et al., 2007). When a pheromone arrives at the sensillae, it binds to a transmembrane olfactory receptor protein corresponding to its chemical structure. This binding event triggers a cascade of reactions, leading to depolarization and ultimately, the transduction of the chemical signal into an electrical action potential. These signals can illicit both transient neural responses and changes in gene expression (Bortolotti & Costa, 2014). Bee pheromones can be classified either as a releaser pheromone, which elicit weaker, temporary behavioral responses, or a primer pheromone, which evoke long-term physiological and behavioral changes (Bortolotti & Costa, 2014).

a. The Queen’s signal

To ensure the social cohesion of very large colonies, a queen bee relies on the spread of her pheromonal signals. The Queen Mandibular Pheromone (QMP), secreted by exocrine glands inside her head and at the base of her mandibles, is the most powerful component of her signal (Bortolotti & Costa, 2014). The chemical composition of QMP varies by bee species; in honeybees, it is composed of five main compounds (Fig. 10). The blended nature of QMP allows for the pheromone’s chemical ratios to fluctuate as the queen mates and ages, eliciting different colony responses at different times.

Fig. 10. The five primary components of the Queen Mandibular Pheromone. (Wade, 2022)

QMP has attractive releaser effects towards drones during mating, as well as to her hive during swarming (Bortolotti & Costa, 2014). Since the queen must assert these effects from a distance, these processes involve more volatile components of QMP, such as 9-HDA. QMP also exerts a releaser effect on worker bees in her near proximity and attracts the queen’s “court” - an ever-changing retinue of 8-10 worker bees who feed and groom her (Fig. 11). This behavior is not self-indulgent, but necessary; since not all components of QMP are volatile, physical contact between the queen and her retinue initializes the spread of her pheromones throughout the hive. The retinue only forms when all five QMP compounds, as well as four auxiliary compounds from other glands, act in synergy with one another (Sandoz et al, 2007).

Fig. 11. A queen bee and her retinue of worker bees, attracted by the release of QMP. (Jarriault & Mercer, 2012).

The queen’s pheromones also maintain the hive’s stable social structure by regulating reproduction and preventing the rise of additional queens (Bortolotti & Costa, 2014). QMP causes an overexpression of nursing genes and represses forager genes in worker bees (Sandoz et al., 2007). This decreases their levels of Juvenile Hormone and inhibits workers from developing or laying their unfertilized eggs (Bortolotti & Costa, 2014). Furthermore, QMP prompts nurse bees to refrain from building queen cups, thus preventing the rearing of queen larvae. The presence of a QMP signal also increases the hive’s productivity by boosting wax production, stimulating foragers to carry larger pollen loads, and reducing defensive and stinging behaviors of the colony.

While (E)-9-oxo-2-decenoic acid (9-ODA) is uniquely produced in the mandibular gland, the queen’s other glands also produce components of QMP, both as volatiles and oily secretions (Bortolotti & Costa, 2014). This redundancy serves to further amplify the queen’s signal throughout the colony. As the queen ages and her exocrine glands – in particular, the Koschevnikov gland - deteriorate, her signal decays. Once her signal drops below a certain threshold – or is removed completely due to her death or disappearance - workers begin building specialized cells to raise new queens or developing their own ovarian systems and mandibular gland pheromones. This leads to the creation of a new, or false, queen.

b. Worker pheromones

Foraging is a dangerous task for worker bees because they face predators, extreme temperatures, pesticides, and dehydration. As a result, the daily mortality rate for foraging honeybees can reach up to 36% (Prado et al., 2020).To compensate for forager death, the colony coordinates the replenishment of its forager workforce using a primer pheromone called ethyl oleate (Bortolotti & Costa, 2014). Outside the hive, foraging bees are naturally exposed to ethanol when they consume fermented nectar (Castillo et al., 2012). Ethanol undergoes a condensation reaction with oleic acid to synthesize ethyl oleate, which accumulates on forager exoskeletons, and then diffuses throughout the hive by close or physical contact. Ethyl oleate changes the development of young nurse bees by increasing the age at which they leave the nest to forage (Bortolotti & Costa, 2014). Thus, if part of the forager bee population dies, causing a drop in ethyl oleate levels, bee nurses will develop more quickly into replacement foragers.

The Nasonov gland, positioned at the tip of a worker bee’s abdomen, secretes attractive pheromones which assist with another key worker bee function: navigation (Bortolotti & Costa, 2014). At the entrances of hives, temporary cluster locations, and new hive sites, worker bees will assume a characteristic stance - wings splayed, abdomen lifted – and release a bouquet of seven volatile compounds, often fanning their wings to promote dispersion (Fig. 12). These Nasonov pheromones help guide the foragers home and prompt migrating swarms to coalesce into clusters (see Bee Physics). Honeybees also release these pheromones at profitable water and food resources, to communicate their location to other foragers.

Fig. 12 (A) Forager honeybee secretes Nasonov pheromones at an artificial food source. (B) Compounds of the Nasonov pheromone. (Reinhard & Srinivasan, 2009)

c. Brood pheromones

The nutritional, nursing, and reproductive needs of a colony vary based on a complicated combination of factors – for example, the time of year, the number of eggs laid, and the developmental stage of brood (Bortolotti & Costa, 2014). To appropriately address these changing needs, workers bees respond to pheromones produced by the brood. Brood bees secrete a blend of ten non-volatile fatty acid esters from their salivary glands, and rely on physical contact between larvae and workers to spread them. Alone, the different compounds act as releaser pheromones and regulate how the nursing bees care for the brood. For example, when larvae are ready to have their cells capped with a wax seal, they begin to produce higher proportions of methyl linolenate, methyl linoleate, methyl oleate, and methyl palminate, which stimulate capping behaviors in the nurse bees. Together, all ten compounds make up the Brood Pheromone (BP), which acts as a primer to slow the development of nurse bees into foragers. Thus, young bee larvae, with higher nutritional needs, emit a low BP signal and promote foraging, while older larvae produce more BP to increase the population of nursing bees. Alongside QMP, BP also alters the reproductive development of worker bees by inhibiting ovarian maturation and diverting nutritional protein supplies away from the nurse bee ovaries and towards their hypopharyngeal gland, where brood food is produced (Bortolotti & Costa, 2014).

4. Defense techniques

Bees are eusocial insects, and the colony lives in a nest constructed with beeswax, where all the resources are contained, including food stores, the queen, the brood, and the bees themselves. Thus, defending the nest against potential predators and parasites becomes one of the colony’s main tasks. The defense techniques employed by the bees include not only propolis to strengthen and maintain the hive but also the recruitment of specialized workers, called guard bees, to perform nest defense.

a. Propolis

Propolis, or bee glue, refers to the resinous mixture produced by the bees from plant resins (Wagh, 2013). Bees collect resins from tree buds, masticate them to add salivary enzymes, and then combine the mixture with beeswax to produce this natural glue, whose color varies from yellow-green to dark brown depending on its source and age. In general, raw propolis is composed of 50% resins, 30% waxes, 10% essential oils, 5% pollen, and 5% various organic compounds, yet the precise composition varies with the resin source (Burdock, 1998). In turn, propolis is lipophilic in nature and is a soft, pliable, and sticky substance at 25°C to 45°C, while hard and brittle in cold weather (Wagh, 2013).

The word propolis is derived from Greek, in which pro stands for “in front of” or “in defense” and polis for “city”, which means hive defense for bees (Burdock, 1998). Due to its waxy and sticky properties, propolis is used by bees to strengthen and repair their hives. A smooth layer of propolis envelope between 0.3 and 0.5 mm thick lines on the interior walls of the hive to serve as a waterproof barrier against wind and rain (Simone-Finstrom et al., 2017) (Fig. 13). Bees manipulate the pliable glue to seal openings and cracks, especially at the joints between hive chambers, making them difficult to separate (Wagh, 2013). Bees also benefit from the repellent properties of resins by depositing propolis in vicinity of hive entrance, narrowing the entrance as well as entangling termites and ants, thus, preventing invasions (Popova et al., 2021).

Fig. 13. Comb cell edges coated with propolis (Bell & Sadenwater, 2017).

Moreover, propolis serves as an antiseptic meant to prevent the hive from microbial infections and is vital in the collective immune defense at the colony-level against pathogens and parasites (Simone-Finstrom et al., 2017). Although the chemistry of propolis varies, a class of natural polyphenols called flavonoids (Fig. 14) are by far the largest group of compounds identified in propolis and are ubiquitous in the plant kingdom. The antimicrobial activity of propolis has been reported to be primarily due to flavonoids, aromatic acids, and their esters (Burdock, 1998). For example, American Foulbrood, caused by the bacterial pathogen Paenibacillus larvae, is a severe larval disease that kills the honeybee (Apis mellifera) colony. Experiments have shown significant inhibition of P. larvae growth in vitro correlated with samples of propolis that contain a high content of total flavonoids. It was also demonstrated that the interaction effects among flavonoids are particularly important to the strength of propolis’s antibacterial activity (Mihai et al., 2012). Upon encountering large and immovable foreign objects in the hive, such as dead lizards, bees also cover and seal them up with propolis to remove the smell and embalm the intruders, preventing decomposition (Burdock, 1998).

Fig. 14. Flavonoids in propolis (Loveridge, 2001).

b. Chemical detection of intruders

With its rich honey reservoirs, the hive is under constant threat of invasion by "robber” bees from other colonies (Nouvian et al, 2016). To protect against unwelcome intruders, the colony stations “guard” worker bees at hive entrances to identify and attack non-colony members. The precise method of identification involves the cuticular layer, a thin coat of lipids secreted onto the exoskeleton of most insects (Bortolotti & Costa, 2014). This matrix is made up of a blend of compounds – usually long, saturated fatty acids – which act as an impermeable barrier to protect insects against drying out. When it comes to bees, members of the same colony share a similar cuticular composition, due to their similar genetics and environment, (i.e. diet and exposure to the same wax components). Furthermore, their cuticular layer contains a higher concentration of unsaturated lipids, which reduces its impermeability, but increases the distinguishability of its molecules. As a result, the bee cuticular layer can be used as a pheromone for nestmate recognition.

When a bee arrives at the hive entrance, guard bees touch their antennae to its cuticular layer to detect and compare the individual’s chemical profile with that of the hive (Nouvian et al., 2016). If the incoming bee’s profile do not match, the guard will trigger a defensive response using alarm pheromones. Interestingly, younger bees contain fewer lipids in their cuticles and thus have a more ambiguous chemical identity; as a result, they are more easily adopted into foreign bee colonies (Bortolotti & Costa, 2014).

Pheromonal recognition is also used to evict drones from the hive at the end of the season (Free & Williams, 1975). High ratios of certain lipid compounds, such as branched and short-chained alkanes, are characteristic of males (Bortolotti & Costa, 2014). Furthermore, the cuticular lipid patterns of males change as they age. Guard bees use these trends to identify sexually mature drones and turn them away from the hive, conserving the hive’s food resources.

c. Sting alarm pheromone

Alarm pheromones serve as communication in a defensive context, specifically to alert nestmates of potential threat and to recruit a larger number of bees for colony defense. To spread easily, alarm pheromones generally consist of low-molecular-weight and highly volatile compounds, which, at the same time, allow quick evaporation to terminate the aggression when danger no longer exists (Klowden, 2008).

Sting alarm pheromone, with its characteristic banana-like scent, is produced by both the Koschewnikow glands and the glandular areas of the sting sheaths of bees (Fig. 15, highlighted in orange). Produced as a pheromonal blend, the secretions flow into the sting chamber and accumulate on the setaceous membrane (Fig. 15, red), of which the hairs provide a large surface area that enables quick volatilization (Nouvian et al., 2016). Guard bees, as mentioned, are specialized workers recruited as the first defensive line. During defensive behavior, they arrive at the hive entrance, raise their abdomen, expose the sting chamber to release the alarm pheromone, and in the meantime, fan their wings to facilitate the dispersion of volatile compounds (Bortolotti & Costa, 2014).

While over 40 compounds have been identified in this pheromonal blend, isoamyl acetate (IAA) is one of the main active components (Fig. 14) (Nouvian et al., 2016). IAA marks on stationary objects at the hive entrance attract and alert the guards most effectively, and thus elicit defensive responses (Bortolotti & Costa, 2014). Although bees react strongly to IAA, tests have shown that IAA alone does not account for the full response observed with sting extracts. Its mixture in a 1: 1 ratio with (Z)-11-eicosen-1-ol is sufficient to trigger a full response. (Z)-11-eicosen-1-ol attracts bees to a moving item but not to a stationary one, unlike IAA, and it is identified as the effective component in inducing stinging (Nouvian et al., 2016). Also, being less volatile, it prolongs the activity of IAA on stationary targets, presumably by slowing down its evaporation (Bortolotti & Costa, 2014).

Besides eliciting immediate and short-term responses, sting alarm pheromone is also involved in prompting long-term physiological and behavioral changes in bees. IAA has been reported to induce analgesia through activation of an opioid system and impair appetitive learning for up to 24 hours. These mechanisms, as a design solution, facilitate recruitment during intense and/or long-lasting colony defense, by ensuring the soldiers are unlikely to withdraw from the fight and priming them for defensive events (Nouvian et al., 2016).

Fig. 15. The sting apparatus in the bee abdomen, and the chemical structure of isoamyl acetate. Koschewnikow gland (Kg) and sheath lobe (Sh) that produce the sting alarm pheromone are shown in orange, and setaceous membrane (Sm) that disperses the pheromone is shown in red. In this image, Sh is abnormally folded upwards instead of laying along the stylet (St) (Nouvian et al., 2016).

d. The chemistry of ouch!

Bees employ various defensive mechanisms against predators and intruders, including fanning wings to dislodge small insects or forming “hot bee balls” to fry hornets alive (see Bee Physics). However, when confronting larger predators, such as birds, apes, and bears, some guards will extrude their venomous sting as the last resort.

In the act of stinging, barbed lancets (La) of the sting apparatus (Fig. 15), along with its weak connection to the rest of the abdomen, cause this apparatus and the associated venom sac as well as muscles to be torn from the bee and stay in the wound (Nouvian et al., 2016). The muscles continue to penetrate the sting deeper into the wound and inject further venom, thereby one sting becomes equivalent to many injections (Loveridge, 2001). Interestingly, this phenomenon of sting autotomy, in which bee death is followed by stinging behavior, is observed only in eusocial insects where the loss of a sterile worker does not have a direct effect on its reproductive fitness (Nouvian et al., 2016).

Bee venom (BV) induces allergic reactions due to the presence of multiple protein allergens, most of which possess an enzymatic activity. In addition, unlike many other insect venoms, BV is a hydrolytic mixture with an acidic pH of 4.5 to 5.5 and 88% water, thus injection into moist tissue, such as elastic mammalian tissue, allows effective dispersion (Wehbe et al., 2019). BV contains an extremely complex blend of peptides, enzymes, histamine, pheromones, amino acids, and other acids, with 63 components in total. 50% of the venom’s dry weight comes from melittin, a 26-residue peptide (Loveridge, 2001). The amphipathic property of melittin enables its insertion into cell membranes, mediating pore formation and disrupting the phospholipid bilayers (Wehbe et al., 2019). In this way, melittin acts to destroy red blood cells at the sting site, lowering blood pressure and causing histamine release, which in turn causes itching and pain (Loveridge, 2001). Another protein in the BV that destroys cells is phospholipase A, accounting for 12% of dry weight. Being the most lethal enzyme in BV, it was demonstrated that melittin helps in exposing membrane phospholipids to the catalytic site of phospholipase A via opening melittin-induced channels. These effects are cytotoxic and eventually lead to pain and inflammation (Wehbe et al., 2019). Moreover, toxicity of BV is strengthened by apamin and hyaluronidase (Loveridge, 2001). Apamin makes up 3% of dry weight and is the smallest neurotoxin in bee venom, allowing this polypeptide to cross the blood-brain barrier and destroy nerve tissue by affecting the permeability of the cell membrane to potassium ions (Wehbe et al., 2019), whereas hyaluronidase, representing 2% of dry weight, acts as a spreading factor by breaking down hyaluronic acid—a polysaccharide found in interstitial fluid in connective tissue—thereby weakening tissue structure and increasing blood flow in the area (Loveridge, 2001). These two actions are combined to intensify the widespread of BV (Wehbe et al., 2019).

As previously mentioned, the sting alarm pheromone influences the stinging behavior of bees. It can be released while a bee extrudes its sting without actually stinging, during stinging, and from stingers left behind in the victim (Bortolotti & Costa, 2014). Interestingly, a study has shown that, upon stinging, the alarm pheromones left in the target by previous defenders serve as a powerful attractant to quickly direct other guard bees toward the single most stung target, ensuring effective nest defense (Nouvian et al., 2016).

Conclusion

The chemical processes behind superorganisms like bees provide a foundation for understanding their behaviors and survival mechanisms. From communication and defense systems to honey and beeswax production, bees use chemically complex interactions to help their colonies.

Bees depend heavily on plant-derived nutrients, with flowers providing their main sources of carbohydrates from nectar and proteins/lipids from pollen. Socials bees can create reserves of these plant products in the forms of honey and bee bread for long term storage. In addition, nurse honeybees can secrete a nutrient rich jelly that is fed to their brood, with the composition determining if the larvae will develop into a worker or queen (Johnson & Seeley, 2023). Worker bees can produce beeswax, which plays a vital role in building the hive's honeycomb structure. The honeycomb cells are made from beeswax, which has primary components of esters, hydrocarbon chains, fatty acids, and alcohols (Joint & Additives, 2005). Colonies use pheromones to coordinate their division of labor, reproduction, and navigation. By detecting the chemical composition of their cuticles, bees recognize their nestmates and selectively attack intruder bees. Bees can use various chemical defense mechanisms to protect their hive. For example, they collect resins from plants, which are rich in antimicrobial compounds, to produce propolis, a waxy and lipophilic substance that strengthens and protects the hive structure. Moreover, bees communicate in a defensive context using highly volatile sting alarm pheromone, containing compounds that trigger defensive responses and/or stinging behaviours. Stinging with the poisonous and powerful venom acts as the last resort of bees against larger predators.

Understanding these mechanisms allows for further insight into the fascinating world of bees and shows how important chemistry is in the organization and behaviour of colonies.