Aim: to serve as a general guide to the techniques used for production of disease-free PL during the larval rearing phases.

• Ensure water treatment protocols are effective at removing viral particles, bacteria and other pathogens and organic material from the inlet water.
• Do thorough dry-outs (minimum 7 d) between larval rearing cycles in each hatchery to prevent contamination from one cycle to the next within the hatchery.
• If possible, conduct rapid batch stocking of each hatchery unit so that they can be thoroughly dried and disinfected between cycles.
• Conduct thorough disinfection of eggs and nauplii prior to stocking larval rearing tanks to prevent vertical transmission of virus from broodstock to larvae.
• When available, utilize a PCR laboratory to check each individual broodstock and batch of nauplii for WSSV (and possibly other viruses) prior to stocking the larval rearing tanks.
• Where possible, attempt to stock each larval rearing tank with a separate batch of nauplii from a single spawning to maintain biosecurity and enable destruction of any WSSV-positive larvae. If the average spawn size is 540 000 eggs, giving 430 000 nauplii, then stocking at 100 nauplii/litre would mean ideal larval rearing tank volume would be just 4–5 tonnes.
• Where this is not possible, attempt to separate broodstock testing positive and negative for WSSV, then preferably discard the positive broodstock or at a minimum, stock each category into separate larval rearing tanks.
• Carry out periodic checks of shrimp, water and feeds with simple viral detection tests to ensure that the hatchery remains free from viruses.
• Utilize probiotics and non-banned antibiotics during all hatchery phases to control pathogenic bacteria numbers and reduce stress on the larvae.
• Apply immunostimulants (i.e. ß-glucan at 2–10 g/kg, lipopolysaccharides or peptidoglycan at 1 g/kg or fucoidan at 3 g/kg and probiotics at 10 g/kg) by mixing first with water or fish oil and then mixing this paste with the formulated diets during larviculture to protect larvae from increasing viral loads – helping to improve survival rates in the hatchery and especially during pond on-growing. All these immunostimulants have been shown to protect shrimp from laboratory challenges with WSSV. It is also possible to bioencapsulate these immunostimulants into rotifers/Artemia for use during larval rearing or as dip before transport.
• Investigate the use of permissible chemicals such as chloramine-T, formalin, povidone-iodine and Virkon-S during larval rearing to reduce prevalence and/or infectivity of WSSV during the larval stages.
• Maintain water quality through aeration, regular water exchange, monitoring using test kits (NH₃, NO₂, pH, alkalinity, salinity and temperature), siphoning and the use of probiotics.
• Feed larvae with appropriate feeds and monitor dose rates to avoid stress, overfeeding and reductions in water quality.
• Disposal of diseased larvae is currently conducted by disinfecting/killing (usually with bleach) then disposal to the sea. It may be preferable to disinfect then discharge to an effluent treatment plant before disposal in the sea to avoid possibilities for disease transfer (see Section on Wastewater treatment).
• Some laboratory-based data on the concentration of various chemicals to inactivate free WSSV in water are shown in Table 11.

	Table 11. Inactivation of free WSSV in water.
	Sodium hypochlorite: 1 ppm for 10 min Povidone-iodine: 2.5 ppm for 10 min (0.25 ml/10 litres of 10  percent liquid) Formalin 1–5 ppm for 10 min UV 1–3 x 104 µW/sec/cm2 for 5 min Ethanol (30  percent) for 1–10 min Temperature –  constant 33 °C (50 °C for 50 min or 60 °C for 1 min)