Appendix 16 Determination of dry weight and ash-free dry weight

1. Number (with a pencil) and ash 4.7 cm GF/C filter in a muffle furnace at 450 °C until constant weight.

2. Weigh filters on an electronic microbalance (±1 μg). Record weights for each numbered filter.
3. Set-up a Millipore filtering apparatus and connect to a vacuum pump. 4. Place one filter on filtering apparatus.
5. Count larvae using a Sedgewick-Rafter cell.
6. Calculate number of larvae per ml.
7. Using a 7 mm Tygon tube, siphon required number of ml into a graduated cylinder to obtain total number of larvae. For Day 2 larvae collect minimum of 150 000 larvae. For pediveligers collect a minimum of 5 000 larvae.
8. Pour collected larvae through a 20 μm sieve.
9. Wash larvae with isotonic ammonium formate (3 percent) to remove mineral salts.
10. Wash down larvae with ammonium formate onto filter fitted to filtering apparatus.
11. Ensure that all larvae from sieve and sides of filtering apparatus are collected by thoroughly washing with isoosmotic ammonium formate.

12. Remove filter from filtering apparatus and place in a foil disc.
13. Record number of larvae collected and number of filter.
14. Dry filter in an over at 60 °C until constant weight (approximately 48 hours). 15. Transfer filters to a dessicator to avoid moisture affecting weight.
16. Record weight after drying using same electronic balance.
17. Place filters in a muffle furnace at 450 °C until constant weight.
18. Remove from furnace, transfer into a dessicator, and weigh with electronic balance. 19. Record weight and filter.
20. To calculate larval dry weight (DW):
a) Dry weight (μg) of filter + Larvae - Initial filter weight
21. To calculate Ash-free dry weight (AFDW):
a) Larval ashed weight (inorganic matter)= Ash weight of filter + Larvae - Initial filter weight
b) Larval ash-free dry weight (organic matter)= Larval ash weight - Larval dry weight
22. To calculate Condition Index:
a) Condition Index= Organic matter/Larval dry weight