4.3.3. How to measure reproductive success?
The reproductive success of a mite is assessed by reconstructing the mite family in infested cells (Martin, 1994, 1995a). See section 4.3.2. ’When to measure reproductive success’ for the optimal time when to measure this parameter.
It is important to examine cells infested by single mother mites as opposed to multiply infested cells since multiple infestations add an additional effect on the success of mite reproduction. Whether only one ‘mother’ mite reproduces can be tricky to ascertain in cells with multiple red mites, but using the mite development guide (Figs 11 & 12) will indicate what is expected and give a good idea of the number of mother mites present. For statistical significance, at least 30 single mite infested cells per colony should be examined.
Different aspects of mite reproduction can be measured. Within each pupal cell, the following information can be collected: sex, developmental stage and vitality. Live or dead mites are easily identified if fresh material is used. If frozen material is used, mite appearance must be relied on. A shrivelled individual or abnormal appearance means it is dead. This characteristic can also be used to distinguish a dead individual from an individual in an immobile developmental stage in live samples. In addition, death rate can be estimated indirectly by comparing the developmental stages obtained with the reference: the development of any dead individual is prematurely arrested and can therefore be identified with the help of Figs 11 and 12.
1. Using forceps, carefully open each capped cell by pealing back its top, papery seal and push away the walls of the cell.
2. Remove the pupa from the cell.
This is best done by sliding the forceps each side of the neck between head and thorax and gently lifting up. When getting pupa out of cell, place it on a microscope slide next to the cell to avoid mites dropping in the comb.
3. Record its developmental stage based on the appearance description given in Fig. 11.
It is important to examine the pupae under a stereomicroscope once it is removed from the cell to make sure that mite progeny are not discarded with the pupae. Cell walls should also be inspected meticulously for mites and exuviae. The use of an optic fibre light source is particularly suitable to direct the beam to the bottom of the cell and inspecting it after the removal of the pupa.
4. Remove complete mite families together with exuviae from their cells and pupae using a fine brush.
5. Examine under a stereomicroscope.
Protonymphs can be distinguished from similar looking young deutonymphs by the number of hair in the intercoxal region (between the 4 pairs of legs on the ventral side). Male and female protonymph have 3 and 4 pairs of hair, respectively, whereas deutonymphs have 5-6 pairs (see drawings pp55-57 in Fernandez and Coineau, 2007). This information allows the mite family to be reconstructed in birth order or to check for multiple infestation and normality of development. Protonymphs are usually not sexed, but the number of hairs on the intercoxal region and the patchy hair pattern on the dorsum of males compared to the homogeneous and dense pattern of females provide recognition traits (Fernandez and Coineau, 2007). For the number of eggs laid by mites and to allow a more accurate comparison between studies, only mites that laid two or more eggs are compared. Mites that produce no eggs (non-reproductive) or one egg (single male) are considered as distinct reproductive categories (see b and c below). The mortality of the mite offspring in worker and drone cells is calculated by comparing the number of live and dead offspring at each position in birth order, i.e. first offspring, second offspring, etc. Then the average number of surviving females (unfertilised and fertilised) is calculated using only the levels of offspring mortality.
7. All infested cells are analysed by placing the mother mites into one of the following six categories:
a) mother dead
b) mother (alive), no offspring
c) mother plus only male offspring
d) mother plus (live) mature male and female offspring so mating is assumed
e) mother plus (live) female offspring and dead male offspring
These female offspring might remain unfertilised since the male might have died before they were mated (Harris and Harbo, 1999). Dissection of the spermatheca and microscopic examination of its content can help determine their mating status (see Steiner et al., 1994 for pictures of reproductive tract with spermatheca).
f) mother without (live) female offspring.
When working with frozen material, beware of repeated freezing and thawing cycles since this damages the samples. Only take out of the freezer the amount of material can be dealt with at a given time.
Pros: if this type of data is collected it can be compared with previous studies (e.g. Martin, 1994, 1995a, b; Medina and Martin, 1999; Martin and Kryger, 2002), where the same data for reproductive parameters of susceptible mites were obtained by using the exact same methodologies.
Cons: tedious, time consuming.