Journal Archive

Royal jelly collected from A. mellifera colonies at the apiary of NAAS in 2013 stored at temperature about -20°C in a freezer was thawed by placing it at room temperature for 2 hours. 50 grams of royal jelly (50%) were weighed and put in the beaker (100ml). Distilled water was boiled for 5 minutes, then the water was cooled. A component including 6 grams of D-glucose (6%), 6 grams of Dfructose (6%), and 1 gram of yeast (1%) extract were weighed and put in the same beaker (50ml). When the water was cooled to about 45°C, 37ml of water was measured, and added to the beaker (50ml) containing sugars and yeast above. This mixture was dissolved by using a spatula to stir. The dissolved sugars and yeast extract was decanted into the beaker (100ml) containing royal jelly and mixing them thoroughly using a spatula. The larval diet is now ready for use. The beaker was labeled with date kept in refrigerator at 4°C to use only for 3 days.

Worker larvae were obtained from the honey bee A. cerana colonies collected in Cheon An province. These colonies were placed at the apiary of NAAS. A marked worker comb of empty cells was placed in a colony. On the fourth day, the bees were shaken off the marked comb and the comb was brought into the grafting room. Before grafting larvae, diet was pre-warmed (34°C, 15 minutes). Each well of 48-well cell culture plate was added 100μl of diet. First instar larvae (hatched within 24 hrs) were transferred gently into cells plates with one larva per well using the queen grafting tool. About every 10 larvae the grafting tool was sanitized with 70% ethyl alcohol to reduce cross contamination by pathogenic microorganisms. The larval rearing plates was transferred into a desiccator where the relative humidity was maintained at 95% (10% sulfuric acid solution in water, v/v). The desiccator was kept in a dark incubator maintained at 34°C. Each day, excessive diet was removed using a vacuum. Next, pre-warmed fresh diet was added to the larval rearing plate. The amount of food to add each day was showed in Table 1. Larvae will defecate when they are ready to pupate. Defecation is characterized by the presence of uric acid crystals and stringy material in the well. When a larva defecates, it is ready to be transferred to a pupation plate. Larval mortality is recorded each day.

Pupating plates are 24-well plates matted with 2 layers of dust-free Kimwipes. The Kimwipes were previously rinsed with 70% ethyl alcohol and dried before being cut into pieces of 3×1.4cm. The mature larvae were dried on 2 layers of pre-cleaned Kimwipes and transferred to the pupation plates using a modified grafting tool (with the plunger removed). The pupation plates were kept in an incubator at 34°C and 75% relative humidity (maintained by a RH regulated incubator), until adult bees emerge. After 3 days the bees were inspected for survival to pupation plates as the pre-pupal stage is the most sensitive stage to movement and manipulations.

When larvae developed, their body gradually increased in size and often moved (Fig. 1). The surviving young larvae were characterized by the movement of larvae under a stereomicroscope for young larvae on day 3, and day 4 from grafting. From day 5, larval movement could be seen with the naked eye.

Fig. 1. 
The development of A. cerana larvae rearing in vitro. A-I: day 1, 3, 4, 5, 6, 7, 12, 14 and 18.

On the contrary dead larvae could be recognized by absence of movement, lack of turgidity and flattened body. Larvae defecated by day 6 or day 7 after grafting. In our experiment defecation were indicated by the presence of uric acid crystals and the appearance of light yellow feces (Fig. 2). When a larva defecates it is important to clean excessive diets and feces off the larvae and transferring to pupating plate.

Fig. 2. 
Defecated larvae with transparent uric acid crystal and feces.

A. mellifera larvae reared in vitro would defecate on day 8 or day 9 (Chan, 2012). Peng et al. (1992) showed that A. mellifera larval defecation began on the fifth day (9.5%), 30% did on sixth day, peaked on seventh day (55%) and declined thereafter. According to Peng et al. (1992) A. mellifera larvae that defecated on day 8 and day 9 probably did exhibit a delay growth and development.

Fig. 3 demonstrates the emerge time of A. cerana worker bee reared in vitro. The observation on the emerge of 68 worker bees showed that 17.6% worker bees emerged on day 17 after grafting, worker bees emerged peak on day 18 (57.4%) and then declined.

Fig. 3. 
The on set of adult emerge rearing in vitro

In an A. cerana hive total of developmental time for worker caste is 19 days including 3 days egg stage; 5 days larvae stage and 11 days prepupae and pupae stage (Oldroyd and Wongsiri, 2006). in vitro condition our data demonstrated that the onset of adult emergence started on day 17 from grafting. In the A. mellifera hive average emergence time for the worker caste is 18 days after egg hatching (Winston, 1987). A. mellifera reared in vitro adult worker emerged after 17-18 days from grafting (Jay, 1963; Peng et al., 1992). This was rather similar to those in the hive. However, Chan (2012) reported that in vitro condition A. mellifera adult worker emerged after 21 days from grafting. According to Peng et al. (1992) there is much of the variation in developmental time in the hive and in vitro condition can be attributed to temperature, nutrition, and genetic differences.

In Table 2 showed the survival rates of A. cerana larvae reared in vitro for two trials with a total of 224 larvae grafted. The average survival rate from larvae to prepupae was 74.6%. While survival rates from prepupae to adults was 40.7%. Consequently survival rate from grafting larvae to adults was 30.4%.

This is considerably lower than the survival rates of larvae to adults on A. mellifera reported 50% up to 80% (Peng et al., 1992; Aupinel et al., 2005; Huang, 2009; Kaftanoglu et al., 2011; Chan, 2012). Our data demonstrated that most of them died in prepupae to adult stage. There are probably two main factors affecting the survival rates of larvae reared in vitro. Firstly, the quantity and quality of diets that larvae ingested was not enough to support normal growth. In our experiment royal jelly in the diet to feed A. cerana larvae were from A. mellifera colonies. The amount of diets was modified from Huang’s protocol of rearing A. mellifera larvae in vitro; 600μl of diets was fed to an A. cerana larva instead of 900μl of diets recommended for A. mellifera larva. Secondly, that is defecation of larvae as well as cleaning feces and excessive diets from larvae before transferring them to pupation plates. In the natural hive, worker larvae were barely fed with amount of diets by adult worker bee before the cells to be sealed (Chinh, 2012). In the cells larvae turned, defecated, and deposited feces at the bottom of the cells. While in vitro larvae remained in contact with the feces all over the body (Chan, 2012). Therefore, the process of cleaning feces and excessive diets may increase mortality. in vitro incomplete defecation of larvae might contribute to higher mortality, as defecation was an important way to excrete wastes and feces remaining inside larvae might be harmful for development (Peng et al., 2009; Chan, 2012).

In summary, A. cerana larvae can be reared in vitro at 34°C and 95% RH by using artificial diets including royal jelly, glucose, fructose, yeast extract and distilled water. However, in the present experiment the survival rate of grafted larvae to adult stage was still low (below 50%). This was probably due to diet quantity, royal jelly used in the experiment, and transferring methods of larvae to pupation plate. Therefore next experiments are needed to improve the survival rate and complete a standard method for rearing A. cerana in vitro that will be useful tools for further research on honey bee biology, pathology and the effect of pesticide to honey bee.