2.5.2. Obtaining pupae of known age

Instead of caging the queen and waiting until pupation to obtain pupae of desired age, freshly capped cells can be identified. This saves time since larval development time can be ‘spared’ and one need only wait the desired time after capping before obtaining pupae for experiments (see Table 5 for a timeline for worker pupae).

  1. Remove frames containing many mature (L5) larvae from the colony.
  2. Place an acetate sheet over each frame.
    Be sure to label the sheet and mark it according to its position on each frame to be able to place it accurately when using it later and thus avoid confusion.
  3. Mark the position of all sealed brood on the sheets of acetate (Fig. 6).
  4. Remove the acetate sheets.
  5. Replace the frames in the hives.
  6. Remove and re-examine frames at regular intervals (as needed for the experiment, usually a minimum of two hours).
  7. At each interval, mark the position of cells which have been capped since the last check.
    To do this, the acetate sheet is returned to the surface of the frame and aligned with the original point of reference (Fig. 6).
  8. Remove the acetate sheet.
  9. Replace the comb in the colony.
  10. Remove the relevant combs from colonies at pre-determined times and collect pupae of desired age, as indicated by the transparent sheets.

The average duration of the sealed brood stage is 12 days (288 hours) for workers and 14-15 days (340-360 h) for drones in A. mellifera in the U.K. (Martin, 1994, 1995). Relatively high variations are reported for different localities and subspecies (up to 19 hours for worker development, Milum, 1930; Le Conte and Cornuet, 1989; 40 hours for duration of capped stage in A. mellifera capensis workers, Allsopp, 2006). The expected durations should be verified before starting an experiment since these vary from subspecies to subspecies.

The same principle can be used to obtain drone pupae of known age when open drone brood is available. Table 6 gives the timeline for drone pupae development. For queens, follow the procedures described in the BEEBOOK paper on queen rearing and selection for queen rearing (Büchler et al., 2013). Table 7 gives the timeline for queen pupae development

Table 5. Colour changes in worker pupae, modified from Jay (1962). Days are counted from cell capping to correspond to Fig. 7. Body parts mentioned in the table are annotated on Fig. 8.

Number of days from capping

Colour

Body parts

5

slightly marked light pink

eyes, ocelli

6

light pink-purple

eyes

dark pink

ocelli

7

dark pink-purple

eyes, ocelli

8

slightly marked light brown

head, thorax

light brown

tibio-tarsal joints, sutures outlining mesonotum, wing bases

9

 

 

light yellow

abdomen, legs

light brown

head, thorax

light to medium brown

leg joints, claws, mandibles, antennae, sting, spurs, spines, hair

medium brown

tibio-tarsal joints, wing bases, sutures outlining mesonotum

dark purple

eyes, ocelli

10

light grey

wing pads

medium brown

flagellar segments, leg joints, wing bases, mandibles, claws, sting, spines, spurs, hair, sutures outlining mesonotum

dark yellow

abdomen, scapes, pedicel, tongue, legs

dark brown

head, thorax

brownish-purple

ocelli

black

eyes

11

medium grey

wing pads

dark yellow to light brown

abdomen, scapes, pedicel, tongue, legs

dark brown

leg joints, wing bases, claws, sting, spines, spurs, hair, sutures outlining mesonotum

dark grey

head, thorax

dark brownish-black

ocelli

black

eyes, flagellar segments

12

 

pupal moult complete


Fig. 6.
Marking freshly capped cells on an acetate sheet fixed to the frame. Photo: V Dietemann.

figure06


Table 6.
Colour changes in drone pupae according to Jay (1962). Days are counted from pupation.

Number of days from pupation

Colour

Body parts

2

slightly marked light pink

eyes, ocelli

3

light pink-purple

lower parts of eyes, ocelli

dark pink

eyes, ocelli

4

dark pink

eyes, ocelli

dark pink-purple

lower parts of eyes

5

light pink-purple

eyes, ocelli

dark purple

lower parts of eyes

6

light yellow

wing base

dark pink-purple

eyes, ocelli

dark purple

lower parts of eyes

7

slightly marked light yellow

abdomen, tongue, antennae, wing pads, head, thorax, legs, wing bases

light brown

tibio-tarsal joints, claws, mandibles, sutures outlining mesonotum

dark purple

eyes, ocelli

8

light yellow

abdomen, tongue, scapes, pedicel, legs

light brown

head, thorax, spurs, spines, hair, flagellar segments

light grey

wing pads, tip of abdomen

medium brown

leg joints, wing bases, claws, mandibles, sutures outlining mesonotum

dark purple

eyes, ocelli

9

light brown

scapes, pedicel, tongue

light grey

wing pads

medium brown

head, thorax, spines, spurs, hair

dark yellow

abdomen, legs

dark brown

leg joints, wing bases, claws, mandibles, sutures outlining mesonotum, tip of abdomen

purple-black

eyes, ocelli, flagellar segments

10

medium to dark grey

wing pads

dark yellow to light brown

abdomen, scapes, pedicel, tongue, legs

dark brown

leg joints, wing bases, mandibles, claws, spines, spurs, hair, sutures outlining mesonotum

dark grey to dark brown

head, thorax

black

eyes, ocelli, flagellar segments, tip of abdomen

11

 

pupal moult complete

 

Table 7. Colour changes in queen pupae according to Jay (1962). Days are counted from pupation.

Number of days from pupation

Colour

Body parts

1

light pink

eyes

2

light pink

ocelli

medium pink

eyes

3

light pink-purple

eyes

light yellow

head, thorax, mandibles

dark pink

ocelli

4

light yellow

abdomen, legs, antennae

light brown

head, thorax, leg joints, claws, sting, sutures outlining mesonotum

dark pink-purple

eyes, ocelli

dark brown

mandibles

5

light grey

wing pads

medium grey

head, thorax

dark yellow to light brown

abdomen, legs, frons, clypeus, tongue, scapes, pedicel

dark brown

leg joints, claws, sting, mandibles, spines, spurs, hair, sutures outlining mesonotum

black

eyes, ocelli, flagellar segments

6

 

pupal moult complete