Bee Development
Development includes a pre-capping period (with the cell open) and a post-capping one with the cell sealed.
By: Clarence Collison
Honey bees are holometabolus insects displaying complete metamorphosis; having a four stage life cycle: egg, larva, pupa and adult. The three developmental stages (egg, larva, pupa) collectively known as brood are similar in all three castes but differ in length of time to complete development. Unfertilized eggs become drones and fertilized eggs become either workers or queens. The timing of bee development depends on the caste of the developing individual as well as environmental and genetic factors. Total developmental times average 16, 21, and 24 days for queens, workers and drones, respectively.
The three castes are reared in distinctive cells. New worker and drone cells are horizontal with a slight upward slant, and are hexagonal in cross-section with pyramidal bases; the angles of the cells become rounded when successive generations of larvae have lined them with cocoons and excrement. Queen cells are vertical, circular in internal cross-section, and taper slightly from the base towards the open end. Unlike worker and drone cells, a queen cell is extended, as the larva in it grows, and is reduced again after the queen has emerged (Jay 1963a). Cappings on all three types are porous and rough, consisting of an aggregate of particles, whereas the internal cell walls and bases are smooth and non-porous. Worker cell cappings are slightly convex at first but get somewhat flatter after a time, whereas drone and queen cell cappings are highly convex.
Development includes a pre-capping period (with the cell open) and a post-capping one with the cell sealed. The pre-capping period includes an egg stage, followed by a feeding and growing larval stage. The post-capping period has a ‘spinning’ larval stage during which the cocoon is made, a quiescent prepupal stage, followed by a molt and an imaginal (having the form of an adult insect) stage within the capped cell before emergence (Jay 1963a).
The queen bee attaches each egg to the base of an empty cell in combs that have been cleaned by workers. The honey bee egg is a smooth, white, sausage-shaped object about 1.5 mm in length. Drone eggs are longer and wider than worker eggs (Bishop 1961). During the first day, the egg nucleus divides – if the egg is unfertilized; or if the egg is fertilized, the fusion nucleus or zygote divides. It is not until the third day that the embryo forms, in which head and body segments can be seen within the egg. The head is present at the larger unattached end and the back (dorsum) is on the in-curved (concave) side (Waller 1980). After three days the egg hatches into the feeding stage called the larva.
The first sign of hatching occurs when an egg is 72 to 84 hours old. Muscular contractions by the embryo cause a gentle, weaving motion that apparently results in a tiny hole being torn in the outer membrane (chorion). Fluid from within the egg soon emerges and covers the external surface. The embryo with its “tail” attached to the base of the cell continues to move about until its head also touches the base and an arch is formed. In this “croquet wicket” stage, the chorion evidently is dissolved. The larva then eases itself over against the bottom of the cell into the familiar C-shaped position (Waller 1980). Hatching of eggs is similar in worker, drone and queen cells (Jay 1963a).
“Developing bees undergo six molts during which the outer skeleton is shed; five of these take place during the larval stage, and the last occurs when the bee emerges as an adult.”
The larva is a whitish wormlike grub with no legs, eyes, antennae, possessing simple mouthparts which need only lap up the copious quantities of food placed in the larval containing cells by nurse bees. The food is placed close to or even on top of the larvae. The larvae are able to rotate within the cells to get to food not placed directly next to their mouths (Winston 1987). Larvae usually move forwards around their cell bases while feeding. Worker larvae which lie curled in the bases of their cells, dorsal surface outwards use body folds on their sides and back as locomotory appendages. These folds are retracted into the body and then protruded in a more advanced position, resulting in crawling movements. After the cell is capped queen larvae make a complete turn in their cells once every 50-70 minutes. At three days old, worker larvae make up to two complete turns in their cells every 1.75 hours (Jay 1963a).
“During cocoon construction the predominant movement made by larvae of the three honey bee castes is a forward somersault which is made dorsal side outermost. Worker, drone, and queen larvae complete one somersault in 52, 46 or 67 (at two different times), and 32 minutes, respectively with the total number of somersaults made during cocoon construction being 27-37, 40-50, and 40-80, respectively; the larvae take 37, 54 and 30 hours, respectively to complete their cocoons.”
All three castes gain an enormous amount of weight during the larval stage, about 900, 1700 and 2300 times the egg weights for workers, queens and drones, respectively. Worker weights at capping are approximately 140 mg; queens and drones weigh about 250 and 346 mg, respectively (Winston 1987).
Developing bees undergo six molts during which the outer skeleton is shed; five of these take place during the larval stage, and the last occurs when the bee emerges as an adult (Winston 1987). All castes of honey bees molt almost every 24 hours during the first four days of larval life. After four molting episodes, the larva reaches the fifth larval instar without phenotypic changes, except for a considerable increase in size. The larva-to-pupa metamorphic molt takes place within the cuticle of the fifth larval instar.
When the ecdysis or molting occurs, the skin splits over the head and slips off the posterior end of the larva. This process normally takes less than 30 minutes (Waller 1980). Four activities, each increasing in vigor as ecdysis proceeds, seem to remove the skin rearwards: (1) retraction and extension of the abdomen; (2) gyration of the head and thorax; (3) ventral bending and unbending, and side-to-side movements, of the head and thorax; (4) expansion and contraction of a bulb-like structure at the tip of the abdomen dorsal to the anus (Jay 1962b). Each new larval stage (instar) is at first only slightly larger than the previous one, but it grows rapidly. The fifth worker instar gains about 40 percent of the total mature larval weight during days eight and nine. By the end of the eighth day after the egg was laid, the cell containing the worker larva is capped. During the 9th day, the larva spins a cocoon using silk produced by the silk glands. On the 10th day, the larva stretches out on its back with its head toward the cell opening and becomes quiescent inside its cocoon. This inactive and intermediate stage is called the prepupa. The 5th molt, which occurs during the 11th day, reveals the pupal form – white in color and motionless with three major body regions that superficially look like those of an adult bee (Zheng et al. 2011). Color develops gradually, first in the eyes (13th day), then in the abdomen (15th day), legs (16th day), wings (18th day), and finally in the antennae (20th day). Throughout this period, the pupa is encased in a thin outer skin which is shed in the sixth and final molt on the 20th day. Thus, legs, wings and mouthparts are freed and the pupa becomes an imago (adult) which soon begins to chew its way out of the cell (Jay 1962ab; Rembold 1980; Waller 1980).
Much of the larval activity is concerned with eating large amounts of food. Both the Malpighian tubules (analogous to human kidneys) and midgut are shut off from the intestine until a larva is nearly mature. In this way, body wastes are stored internally and the food surrounding each larva is protected from fecal contamination. The larva defecates just prior to spinning the cocoon and the feces therefore lie between the cocoon and the cell wall (Waller 1980). Fully fed worker and drone larvae are thought to be sealed in their cells with a small amount of food: a queen larva is certainly sealed with a large amount of brood food at the base of its cell (Jay 1963a).
The pupal stage is the longest post embryonic developmental period of the honey bee. The temperature at which pupae are raised influences the tasks and behavioral determination of the adult bees (Tautz et al. 2003). It is reported that pupal weight increases with honey production and pupal head weight increases with higher royal jelly production. Comparative biochemical analysis between worker and queen heads has revealed that adult workers raised under higher temperatures show higher probability to dance, forage earlier, and more often are involved in more activities (Tautz et al. 2003).
Honey bees grow and differentiate from the larval to the adult stage through periodic degradation of the exoskeleton (or cuticle) and replacement by a new one. Each episode of cuticle renewal, or molt, comprises a series of events mainly marked by the detachment of the cuticle from the subjacent epidermis (apolysis), the synthesis and secretion of the components of the new cuticle by the epidermal cells, and the ecdysis or shedding of the old cuticle (Soares et al. 2011). This cyclic re-construction of the cuticle is a complex task involving the expression of genes for extensive synthesis of structural cuticle proteins and enzymes with roles in cuticle pigmentation (darkening) and sclerotization (hardening).
The types of cell death in the midgut epithelium of the worker honey bee during the larva-to-pupa transformation were analyzed by light and electron microscopes (Pipan and Rakovec 1980). The metamorphosis begins with an increase in the number of autophagic vacuoles in larval epithelial cells and terminates with lytic destruction of the whole intestinal epithelium. Apoptosis (process of programmed cell death) seems to be independent of cell age, but important in fashioning of the new organ. Even in the cells in the regenerative nests of the larval epithelium, from which the pupal epithelium develops, apoptotic death occurs. Single apoptotic cells are eliminated gradually from the primary multilayer tissue until the monolayer pupal epithelium is formed. Some of the apoptotic cells are endocytosed (energy using process by which cells absorb molecules such as proteins by engulfing them) by sister epithelial cells.
The last few days of larval life are spent constructing a cocoon within the cell. Soon after the brood cell is capped, the larval honey bee spins a thin cocoon over the inside of the cell. To spin the cocoon, the larvae uncurl and stretch out fully within the cells with their heads toward the capped end (Jay 1963b) and begin weaving the cocoon with their spinnerets. The silk for the cocoon is produced in two large glands, each longer than the larva itself, situated in the ventral part of the body cavity. Silk glands are well developed in queen, worker and drone larvae.
Cocoons consist of silk gland secretion (in thin sheets and threads), a colorless material, a light yellow material, and a more solid brown material (feces); the last three are discharged from the larval anus. Possibly some brood food is incorporated into the cocoon but skin secretions or larval blood are not.
During cocoon construction the predominant movement made by larvae of the three honey bee castes is a forward somersault which is made dorsal side outermost. Worker, drone, and queen larvae complete one somersault in 52, 46 or 67 (at two different times), and 32 minutes, respectively with the total number of somersaults made during cocoon construction being 27-37, 40-50, and 40-80, respectively; the larvae take 37, 54 and 30 hours, respectively to complete their cocoons (Jay 1964). About 72 hours after spinning the cocoon the silk glands disappear and no trace of them can be seen in the developing pupae. Soon thereafter, the adult thoracic salivary glands develop from the basement membranes of the silk glands.
The drone and worker larvae spin their cocoons over the whole of the inside of the cell (completely closed cocoons), but the queen larva always spins her cocoon on the lower half of the cell only. Queen cocoons do not cover the base of the cell where a supply of royal jelly remains. Queen larvae continue to feed on royal jelly and gain weight after capping is completed. Uneaten royal jelly that remains later becomes firm, dry and brown in the cell base. The mean weight of a queen larva is 192 mg. before spinning begins 213 mg. when spinning has just begun and 278 mg. when the cocoon is complete; maximum weights recorded for queen larvae range from 260 to 323 mg. (Jay 1963a). In addition, the queen cocoon does not touch the inside tip of the cell.
Following the final exoskeletal shedding, the teneral adult remains inside the cell for several hours as the new cuticle begins to harden (Winston 1987). Emerging worker bees begin by perforating the capping with small holes as they rotate within the cell. The antennae often protrude through these holes. Although some pieces of capping fall outside the cell, the emerging bee works most of them between the mandibles and fastens them to the wall just inside the cell entrance. Later these fragments will be collected by bees for re-use. Other bees often help the emerging bee, by thinning down the capping before emergence, or by removing pieces of it during emergence, putting it on the rims of uncapped cells, or on the cappings of sealed cells. After several unsuccessful attempts to emerge, the young bee enlarges the opening sufficiently to crawl out. Workers will also often chew away the tip of the queen cell a day or more before the queen emerges. It is believed that removing the tip of the cell facilitates the queen’s emergence. Drones and queens usually cut the capping, so it is in one large piece; queen cappings usually retain a small area of attachment to the cell (Jay 1963a).
After emerging, the teneral bee completes its development during the next eight to 10 days (Winston 1987). The emerged bee is still soft, and the cuticle finishes hardening during the next 12-24 hours. The workers, in particular, have a soft, fuzzy appearance at this time until their hairs stiffen and they cannot sting until the skeleton around the sting glands has hardened. Over the next few days internal development is completed, particularly glandular development and growth of fat bodies.
References
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Jay, S.C. 1964. The cocoon of the honey bee, Apis mellifera L. Can. Entomol. 96: 784-792.
Pipan, N. and V. Rakovec 1980. Cell death in the midgut epithelium of the worker honey bee (Apis mellifera carnica) during metamorphosis. Zoomorphologie 94: 217-224.
Rembold, H., J.-P. Kremer, and G.M. Ulrich 1980. Characterization of postembronic developmental stages of the female castes of the honey bee, Apis mellifera L. Apidologie 11: 29-38.
Soares, M.P.M., F.A. Silva-Torres, M. Elias-Neto, F.M.F. Nunes, Z.L.P. Simões and M.M.G. Bitondi 2011. Ecdysteroid-dependent expression of the Tweedle and Peroxidae genes during adult cuticle formation in the honey bee, Apis mellifera. PLoS One 6(5): e201513.
Tautz, J., S. Maier, C. Groh, W. Rossler and A. Brockmann 2003. Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development. Proc. Natl. Acad. Sci. USA 100: 7343-7347.
Waller, G.D. 1980. Honey bee life history. In: Beekeeping In The United States, USDA ARS Handbook 335, pp. 24-29.
Winston, M.L. 1987. The Biology Of The Honey Bee. Harvard University Press, Cambridge, MA, 281 pp.
Zheng, A., J. Li, D. Begna, Y. Fang, M. Feng and F. Song 2011. Proteomic analysis of honeybee (Apis mellifera L.) pupae head development. PLoS ONE 6(5): e20428 doi: 10.1371/journal.pone.0020428.
Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.
