Open Access

Table 3

Description of steps to be followed to assess egg hatch.

Egg collection Step 1. Egg collection (oviposition substrates) from ovitraps and preserving them wet (use wet kitchen paper to wrap them).
Important note: Avoid egg dryness during the trip from the field to the laboratory (avoid leaving the eggs in a car, in the sun).
Storage Step 2. Oviposition substrates should be left in the wet paper under standard laboratory conditions (25 ± 1 °C, 80% RH, 14:10 L) for 1 day to dry.
Step 3. The next day, the oviposition substrates are placed in a sealed plastic container with vapours of potassium sulphate (K2SO4) under standard laboratory conditions. In each plastic container, 100 mL of a saturated solution (potassium sulphate: 120 g/L) was used. The oviposition substrates are left to embryonate under K2SO4 vapours for one week.
Hatching solution Step 4. Preparation of hatching solution. The maternal solution is prepared using 12.5 g broth (Nutrient Broth OXOID) + 2.5 g yeast powder/100 mL of deionised water.
Important note: The hatching solution must be used immediately and cannot be stored.
Hatching Step 5a. 1st hatching. For egg hatching, 1 L volume glass jars with caps are used. 700 mL of deionised water and 2 mL of the hatching solution are added in each glass jar. Five oviposition substrates are put in one glass jar. The jars must be hermetically closed and opened 20–24 h later (not before 16 h). Then, hatched and unwatched eggs are counted. For the evaluation of the hatching, it is recommended to examine the eggs under the microscope rather than counting larvae. Sterile eggs are collapsed or exploded.
Step 5b. If the hatching rate in control is less than 80%, a second hatching assessment must be applied for the substrates collected both in control and in SIT areas. After Step 5a, the eggs are left to dry for 3–4 days and then repeat the hatching protocol.

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