BASIC FACTS ABOUT HONEYBEE LARVAE

 

Honey bees belong to the class insects. They have a complete lifecycle (complete metamorphosis), that is, they develop through four stages: egg, larva, pupa and adult. This compares with insects such as aphids that have an incomplete lifecycle and have only three stages: egg, nymph, and adult.

 

(i) The egg (ovum), develops in a tubule called the ovariole, inside one of two ovaries of the queen bee. When the egg is laid, sperm may be released from the spermatheca onto the egg. The outer shell of egg is called the chorion, with tiny perforations at one end called the micropyle. The perforations allow sperm to enter the egg for fertilisation to occur. If the egg is fertilised, it will develop into a female bee (worker or queen), but if sperm is not released by the queen (the egg is not fertilised), it will develop by parthenogenesis into a drone. As the queen lays an egg she glues it to the bottom of the cell at one end so the egg stands upright. The egg is 1.3-1.8 mm long, weighing 0.12-0.22 mg and is white and sausage shaped. Over three days the egg gradually keels over and the egg shell gradually dissolves (it appears to be unique to honey bees).

(ii) When the embryo inside the egg is fully formed the egg develops into the second stage, larva, which is white and grows very rapidly, developing through five stages or instars and moulting each time. The outer skeleton is shed as the larva outgrows it. The larva has no eyes, legs, wings or sting but has a well-developed mouth. The internal organs are made up of a well-developed stomach (ventriculus), and excretory tubules (malpighian) that are cut off from the hind gut so that the stomach contents are only voided after the cell is capped. At this stage feeding by nurse bees is finished.

(iii) The larva stretches out along the cell and spins a silk cocoon with the aid of the spinneret and silk gland - this is the prepupal stage. The larva then moults and pupates. The final larva weight of a worker (140 mg), queen (250 mg) and drone (350 mg) is about 900, 1700 and 2300 times the egg weight, respectively.

This is the pupal stage and the change in form from the larva to the adult is called metamorphosis (the larva in the cocoon radically changes its form). Antennae, proboscis, compound eyes, wings and legs develop and the head, thorax and abdomen become finally defined. The eyes change in colour from white, to pink, to purple, to black. When the adult is fully developed it moults for a sixth and final time and chews its way out of the tip of the cocoon.

(iv) The newly emerged adult, is white and fluffy and over the next 24 hours the cuticle (exoskeleton) hardens, providing protection and reducing water loss, as well as providing an attachment for muscles. A dense coating of branched hairs (important in gathering pollen) cover the exoskeleton - especially on the thorax of workers.

 

Caste developmental stages

Development times from egg to adult may vary considerably depending on temperature and nutrition. For example, the workers normally maintain the brood temperature at 35°C; a lower brood temperature will delay development.

The queen bee develops through the egg stage in three days and then hatches into a larva which remains in the unsealed stage, on average, for only 4 and 1/2 days. Once the cell is sealed the larva spins a cocoon and develops into a prepupa and then pupates. The sealed stage lasts for 8 and 1/2 days. The queen bee emerges as an adult after 16 days. The reduced time for queen development ensures that a colony can quickly replace a queen that has been lost.

Worker development takes three days as an egg, the unsealed larval stage lasts 5 and 1/2 days and the sealed larva, prepupa and pupal stage lasts 12 and 1/2 days, with the adult emerging after 21 days.

Drone eggs develop in three days, spending 6 and 1/2 days as an unsealed larva, and 14 and 1/2 days as a sealed larva, prepupa and pupa, emerging as an adult after 24 days.

 

Caste determination

The honeybee forms two female castes: the queen and the worker, both can develop from the same type of egg. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Only eggs that are not fertilised with sperm by the queen develop into drones.

The queen bee determines whether the egg is to be laid in a worker or a drone cell by inspecting the cell. Inspection is done with her forelegs or the angle of her abdomen during the egg laying process, if the egg is going to be laid in a drone cell, the queen bee will not release spermatozoa from the spermatheca.

Under certain conditions, workers can lay eggs and as they never mate, laid eggs are unfertilised and develop into drones only. Unfertilised eggs laid by workers are smaller than eggs laid by queens and are placed on the side of the cell rather than at the bottom of the cell as the worker abdomen is too short to reach the bottom of the cell.

The larva that develops from the egg is fed by nurse bees on either 'royal jelly' or 'worker jelly', depending on the type of cell the larvae develops in. Both jellies consist of secretions of the hypopharyngeal and mandibular glands in the head of worker nurse bees.

During the first 24 to 36 hours of larval development workers and queen bees are fed similar amounts of food, after that it differs. Worker larvae receive a light feeding of worker jelly for the first three days of their development, produced from secretions of the mandibular and hypopharyngeal glands of nurse bees. Mandibular gland secretions are white and made up of lipids (fats) whereas hypopharyngeal gland secretions are clear and made up of protein. Worker jelly consists of only 12% sugar, with the predominant sugar being glucose. During the fourth and fifth days, worker larvae receive light feeding of worker jelly made up of secretions from the hypopharyngeal glands only. Honey and pollen are added to the diet increasing the sugar content of the jelly to 47% and the predominant sugar changes to fructose.

By contrast, larvae developing in queen cells are fed royal jelly in copious amounts. During the first three days, the royal jelly fed to these larvae is produced from nurse bee secretions of the mandibular glands only. The mandibular gland secretions in royal jelly contain 18 times higher levels of biopterin and ten times more pantothenic acid than worker jelly. The royal jelly consists of 34% sugar and is predominantly glucose. During the fourth and fifth day larvae in queen cells continue to receive copious royal jelly made up of 1:1 mandibular and hypopharyngeal gland secretions with glucose remaining the predominant sugar.

The nurse bees feed the queen bee larva royal jelly for an average of 4 and 1/2 days before the queen cell is sealed over with wax and feeding ceases.

The quality and quantity of jelly fed to larvae on the third day is critical in determining development of worker or queen characteristics. Research, although not conclusive, suggests the following possible pathway for honeybee female caste determination. Larvae feeding on royal jelly with 34% sugar content, compared to 12% in worker jelly, have a higher food intake. The higher food intake provides a greater stimulation of stretch receptors in the midgut and this provides greater stimulation of the corpora allata, a large globular organ found on the sides of the oesophagus. Greater stimulation of the corpora allata results in higher levels of juvenile hormone produced and this in turn results in queen-like characteristics developing. Worker larvae, feeding on worker jelly with lower sugar content, have a lower food intake on the third day so there is less stimulation of the stretch receptors and corpora allata. This results in less juvenile hormone produced, and hence worker-like characteristics developing.

Another scientific view is that a 57-kDa protein in royal jelly (previously designated as royalactin) induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions.

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References

Kamakura M (2011) Royalactin induces queen differentiation in honeybees. Nature 473, doi:10.1038/nature10093, p. 478–483

 

Woodward D R (2014) Queen Bee: Biology, Rearing and Breeding. Oxford, United Kingdom, ISBN13 9781904846352, pp. 140

 

Yang W, Tian Y, Han M, Miao X. (2017) Longevity extension of worker honey bees (Apis mellifera) by royal jelly: optimal dose and active ingredient. PeerJ 5:e3118https://doi.org/10.7717/peerj.3118