A Closer Look: Venom/Venom Glands

by Clarence Collison

Worker honey bees begin their first use of venom when they attain an age of of about 14 days for defense against predators and intruders.

Honey bee workers use venom for the defense of the colony and themselves when they are exposed to dangers and predators. The venom injected into the victim when a worker stings is a mixture of toxic proteins and peptides, the major component being a protein called melittin (Winston 1987). Venom contains other compounds such as hyaluronidase, phospholipase A, acid phosphatase and histamine. The venom gland and reservoir secretes a mixture of at least 50 identified components. A number of these components have significant toxic effects on many insect and vertebrate species (Bridges and Owen 1984). The complex nature of the venom may be due to the wide variety of insect and vertebrate pests and predators which might attack a bee colony; different components of the venom seem to be important in repelling different species of attackers (Winston 1987).

There are two glands associated with the base of the sting apparatus, the venom or acid gland and the Dufour’s (alkaline) gland. The venom gland consists of a pair of long, slender, convoluted (intricately folded, twisted, coiled) tubules which float freely within the hemolymph of the posterior part of the abdomen (Stell 2012). Secretory cells occur along the length of the tubules, their small ducts opening into a common, chitin-lined duct. Each tubule ends with a small glandular enlargement, and the two tubules unite in a short common duct. The duct opens into the anterior end of the venom sac or reservoir and this in turn opens into the cavity of the bulb at the base of the sting (Figure 1). Muscle bands are attached to the venom gland and are reported to move the secretion down into the poison sac, where the venom accumulates. The epithelial walls of the poison sac have a thick, laminated cuticular intima (innermost lining of an organ) thrown into numerous high folds. In the neck of the sac the folds form regular transverse rings, holding the neck rigidly open. The poison sac walls have no muscles, and the venom therefore is not expelled by contraction of the sac; it is driven through the canal of the sting by the action of the sting lancets and their valves (Snodgrass 1956; Goodman 2003).

Along most of the length of the venom glands are similar secretory units that have four major components (secretory cells, duct cells, ducts, and end apparatuses), except in the part of the gland proximal to the venom reservoir, where the secretory units resemble those around the venom reservoir. In the latter secretory units a funnel structure occurs between the duct (which is shorter than that of the secretory units of the gland) and the end apparatus. This funnel may be important in protecting the secretory cells around the reservoir from the cytolytic activity (destruction of a cell) by the complex chemical mixture constituting the venom (Bridges and Owen 1984; Peiren et al. 2008).

“The Dufour’s gland, previously called the basic or alkaline gland is associated with the venom, sting sheath and Koschevnikov glands in the
sting apparatus.”

The Dufour’s gland, previously called the basic or alkaline gland is associated with the venom, sting sheath and Koschevnikov glands in the sting apparatus. Despite several studies, the precise role of the Dufour gland is still unclear (Martin et al. 2005). The Dufour’s gland is a short thick, slightly convoluted, opaquely whitish tube. The glandular wall consists of a thick epithelium of distinct cells lined by a thin cuticular intima (Snodgrass 1956). The Dufour gland exits between the sting lancets. The exit is very narrow and indistinct and is in the same position in both queens and workers. The gland’s exit is close to the setosa membrane, a region of cuticle, which acts as a platform for pheromone release. This is consistent with the idea that the Dufour gland secretes compounds that are utilized in defense by workers or reproduction in queens (Martin et al. 2005).

The venom glands of workers have a single secretory cycle, which begins at the end of pupation and reaches its maximum around the 16th day of the worker’s adult life (Roat et al. 2006a). The venom that is produced during this intense synthetic stage is stored in the reservoir (poison sac) and the gland enters the degeneration process (Owen and Bridges 1976), which is completed around 30 days after emergence. Thus, in foraging workers, the glandular cells have evident signs of degeneration, lacking most distinguishable cellular structures with the nuclei and the microvilli surrounding the canaliculi (small canal or duct) as the only discernable structures. Vesicles with irregular sizes and shapes are found in the remaining cytoplasm. In all, about 0.3 mg of venom is produced.

The venom gland is present in both the worker and the queen castes, but queens have significantly larger glands than the workers and produce more venom. Queens use venom during fights with other rival queens, an event that occurs as soon as the imago (mature adult stage) emerges, while fertilized queens rarely use venom. The queen’s venom is only half as lethal to mice as worker venom, and by the time queens are one to two years of age their venom has become essentially inactive (Schmidt 1995).

Worker honey bees begin their first use of venom when they attain an age of about 14 days for defense against predators and intruders (Seeley 1985). Queens never use their stings for defense of the colony. Instead queen stinging is reserved for fighting rival queens just prior to emergence or that emerge during the same time period. Queen venom is more lethal toward other honey bees than is worker venom (Kato 1994). The queen fights occur during the first weeks of adult life, after which time the successful queen has stung or destroyed the rival queens. Once a queen is mature and laying eggs not only has she no need to fight other queens, but also her swollen abdomen renders her incapable of fighting efficiently. Thus, venom toxinology accords with the biology that a queen needs a plentiful supply of active venom at emergence and shortly thereafter, but not in later life (Schmidt 1995).

The protein content of venom glands from worker and queen honey bees falls after the first week of adult life. Queen bee venom glands lose 90% and workers 50% of their protein. This protein loss precedes the ultrastructural changes in the morphology of the venom gland secretory cells (Owen and Bridges 1976).
Old laying queens that have been heading a colony for a year or more possess venom dramatically different than young queens. Venom from old queens was at least 15 times less lethal than the venoms of young queens (Schmidt 1995). In addition, dramatic changes in the venom of old queens were observed during dissection and venom collection. Young queens invariably contained reservoirs full of transparent, colorless, fluid venom. Old queens usually possessed venom of a tan to dark brown color that was viscous. In many old queens the venom had become a dark brown to black almost solid material that could not be collected and did not readily dissolve in water.

“Queens never use their stings for defense of the colony. Instead queen stinging is reserved for fighting rival queens just prior to emergence or that emerge during the same time period”

The worker’s poison sac contains no venom at the time of emergence, whereas, newly emerged queens have already produced venom (Roat et al. 2006b). Queens exhibit maximal synthesis from zero to seven days, the process of degeneration occurs immediately after mating and they essentially stop venom production around day 30 (Owen and Bridges 1976). Queen venom contains much more histamine than worker venom, but lacks MCD-peptide (mast cell degranulating peptide); and by one year of age, queen venoms have little phospholipase A2 and half the melittin of workers, and possess several other proteins.

Melittin is a major protein component of bee venom, comprising 50% of its dry weight (Habermann 1972). The biosynthesis of melittin was studied in vivo by feeding radioactive amimo acids to honey bees. Extracts from venom glands were analyzed for the presence of labeled melittin and other components. Radioactivity was first incorporated into another peptide which is considered to be a precursor of melittin (Kreil and Bachmayer 1971). After feeding labeled leucine to worker and queen bees of different ages, the synthesis of melittin and its precursor promelittin in the venom gland was analyzed. Marked changes in the synthesis of promelittin and the rate of its conversion to melittin occur during the maturation of the insects. In queen bees, both processes operate already close to full capacity in the newly emerged insects. On the other hand, in worker bees the production of promelittin increases slowly to reach a maximum at the 8th to 10th day and then decreases. During the first two days only promelittin synthesis was observed, whereas conversion to melittin was detectable only later on (Bachmayer et al. 1972).

The amount of melittin increases from the time of eclosion to an age of about four weeks when about 500 µg of melittin is present. In older bees (five to six weeks) the melittin level falls to about 250 µg. Melittin synthesis is most active in bees aged between one and two weeks after eclosion. The melittin content of the venom system changes as the Summer progresses. Melittin levels in a bee of any age greater than one week are lower in mid-August than in a bee of the same age in early June (Owen and Pfaff 1995).

Phospholipase A2 is the most lethal of the honey bee venom peptides and melittin which is slightly less lethal, is the most abundant. Concurrent analyses of melittin, phospholipase and the combination of the two at their natural 3:1 mixture in bee venom revealed that the lethal activity of the mixture was about the same as native venom. This value was less than that for either melittin or phospholipase alone and indicates that synergism of the two peptides is not occurring. The results are consistent with independent lethal activities for the venom components, and show that melittin is not only the dominant, but also the main lethal component of honey bee venom (Schmidt 1995).

Owen et al. (1990) measured phospholipase A2 activity in venom of worker bees of known ages. Low levels of phospholipase A2 is present in the venom system at the time of eclosion (emergence from the pupal case). Phospholipase A2 activity in the venom increases steadily through the 10 days after eclosion. Maximal phospholipase A2 levels (about 40 µg phospholipase A2/venom sac) are maintained through the rest of the life of a worker bee in Summer.

Histamine is about 50 times as lethal to honey bees as to mammals (Owen et al. 1977). The amounts of histamine and histidine in honey bee venom glands, venom-reservoir tissue and venom taken from single worker bees were measured (Owen and Braidwood 1974). Neither of these venom components is present in newly emerged bees Histamine and histidine were first detected in the venom of week-old bees, and the amounts present increased for three to four weeks, reached maxima, and then fell off again in six-week-old bees. Owen and Bridges (1982) analyzed venom of honey bees of known ages for dopamine (DA) and noradrenaline (NA). They found both age dependent and seasonal variation in DA and NA levels in the venom.

Roat et al. (2006a) analyzed the influence of juvenile hormone treatments on the ultrastructure of the worker’s venom glands. Newly emerged workers received topical application of one µl of juvenile hormone diluted in hexane, in the concentration of 2µg/µl. Two types of controls were used; one control group received no treatment and the other group received a topical application of one µl of hexane. The glandular cells of the group of newly emerged workers that received no treatments showed that the glandular cells are not yet secreting actively. Changes in the glandular cells happened according to the worker age and to the area of the gland. The most active phase of the gland occurred from the time of emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic with the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

Honey bee venom contains a multitude of enzymes, peptides and active amines. The main lethal factors for mammals have been considered to be phospholipase A2, melittin and apamin, which are present in the venom in quantities of about 15-20%, 40-60% and 2%, respectively (Schmidt 1995).
The major component in honey bee venom is the peptide melittin, which upon injection releases histamine from mast cells and ruptures red blood corpuscles, causing pain and swelling. Two other peptides are present: apamine and mast cell degranulating peptide which also releases histamine from the mast cells, which contributes to the swelling. Two enzymes are present: phospholipase A2 and hyaluronidase; the former causes the disintegration of red blood cells and the later acts as a spreading agent (Morse and Hooper 1985).

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Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.