General Rules and Considerations, Alys Cheatle Jarvela
-Avoid release of adults and disposal of live eggs or larvae.
- Cups and pans containing viable eggs or larvae should be bleached before washing components down the drain. I allow the bleach to sit overnight or over a weekend. Large larvae (L3/L4) may be removed by pouring the culture through a mesh strainer and tapping them into the garbage. The strainer should be cleaned with dish soap with care to not wash live larvae down the drain that may still be stuck on.
- Adult cages should be frozen in the cold room (again overnight or for several days) before disposing of insects and cleaning cages. Individual escapees should be squatted with a bug zapper. Empty cages are washed with soap and water and dried before reuse.
-Anopheles are housed in the 29 degree C chamber with 50-80% humidity. The humidity in that chamber often fails. When this happens, check with Todd to avoid duplicate reporting and alert FMS and all groups housing insects in that chamber. To maintain humidity while waiting for service, place many trays of tap water on the shelves of the room. Anopheles adults will die if kept in low humidity for too long.
Schedule (optimized to avoid need for weekend care)
Week 1
Thursday or Friday (Wednesday if need be)
- bloodfeed (note 1) cages of adults that are 3 days post-eclosion or older.
- provide adult cages with cotton balls soaked in 10% w/v sucrose
Week 2
Monday or Tuesday
- Fill a plastic dixie cup approx halfway with water and line the cup with filter paper or paper towels such that the paper is half submerged. Repeat for each cage that requires egg collection. *We generally get many more eggs than needed for culture. I also stage and collect eggs for my experiments and will co-ordinate with you.
- fill 2 pans with tap water and allow to sit in chamber for several days to warm and dechlorinate
- Rewet sugar feeders on adult cages
Wednesday or Thursday (two days after providing oviposition substrate)
- Using the prewarmed water, fill several pans to 0.5-1 inch.
- Divide hatched L1 larvae among the pans by pouring from the cup. Sterilize cup with 10% bleach to kill remaining eggs and larvae before rinsing materials down the sink.
- The number of pans needed depends on the number of eggs obtained, but typically 3-4 is good for a dense cup of larvae.
- Feed each pan 1/32 teaspoon of tetramin fish food (see note 2)
- Set out more water to prewarm.
- Rewet sugar feeders on adult cages
Friday
- Split each pan into two by adding prewarmed water and dumping half into an empty pan.Try to distribute the larvae evenly.
- If the initial pans are very densely crowded, it may make sense to transfer some to a kill pan and split the remaining cultures into three instead of two.
- Feed each pan 1/16 teaspoon of fish food to last the weekend
- Replace sugar feeders on adult cages with new cotton
Week 3
Monday
- Feed each pan 1/16 teaspoon of tetramin fish food
- If larvae seem very dense, dump some into kill pan to allow remaining to be split again
- Rewet sugar feeders on adult cages
Tuesday
- Check pans for beginning of pupation*
- If larvae are searching the bottom of the pan for food and the water is clear, add 1/32 teaspoon of fish food
Wednesday
- Check pans for beginning of pupation*, but ideally this will be the main pupation day
- If larvae are searching the bottom of the pan for food and the water is clear, add 1/32 teaspoon of fish food
- Rewet sugar feeders on adult cages
Thursday
- This will likely be day 2 or 3 of pupation.
- When most pupae have been picked, strain the water to catch any remaining larvae and sterlize the empty pan with a small amount of bleach.
- Rinse pans thoroughly with water and allow to dry if time allows
*whenever pupation starts, collect pupae into a cup and either start a new cage, or if none are clean, add the cup to the youngest adult cage and prepare a cage for tomorrow (note 3). Expect the day after pupae are first spotted to be a big day for picking pupae. Pupation will likely continue for another way, but it should really peak on day 2 of 3. Extended days of pupation is an indicator of overcrowded and/or underfed cultures.
Notes:
i. Be sure that bovine blood with Na-heparin anticoagulant is available and less than approx. 1 month old. Donor animal should be free from ivermectin treatment
ii. Fill a small cup halfway with water and microwave for 30 seconds. Fill a 50 mL conical tube with 10-20 mL of bovine blood and place in warm water. Each bloodmeal dish requires ~5-10 mL of blood depending on diameter, so scale depending on the number of cages that must be fed.
iii. Cut several squares of parafilm
iv. Collect several reusable handwarmers that are in liquid state. Solid state/used handwarmers can be reset by placing into boiling water until salts dissolve completely.
b. Make a bloodfeeder
i. Pour prewarmed blood into a clean metal jar lid
ii. Pre-stretch parafilm so that the middle is uniformly thin and has no rips or holes over an area large enough to cover the lid. Fit onto the lid. Press and fold down the sides to seal.
iii. Snap the metal disc in a handwarmer to trigger the reaction. Place on table top so that as it becomes solid, it is nice and flat.
iv. Place the lid parafilm-side-down onto a mesh face of an Anopheles cage. Place a warm handwarmer on top so that it is centered and the edges aren’t touching the cage. Anopheles will be more attracted to the handwarmer if they can reach it.
c. Allow feeding to occur for approx 30 min or until many fully fed females are resting on the sides and bottom of the cage.
d. Females seem more inclined to feed when you are working nearby (cleaning pans, picking pupae) than if they are alone in the room
e. This strain has been domesticated for a long time and doesn’t seem to require particular lighting or time of day to feed anymore.
f. Very young cages sometimes are not interested in feeding. Some may try as early as 3 days post eclosion, but realistically 5-7 days post-eclosion is a better time to first offer blood.
g. It is normal for small drops of blood to fall while Anopheles feed, but keep an eye out for leaks and drips from the feeder. Tears can form when feeders are moved between cages.
h. When all desired cages are fed, sterilize the remaining blood by adding bleach (freshly diluted to 10%) for several minutes. The sample should turn from red to black. Then it can be rinsed thoroughly.
i. Note the feed date on the cage label. This will help us keep track of days when eggs can be obtained and when the culture is “done”. Cages will only lay eggs after the first three bloodmeals and should be discarded after that.
2. Fish food- we buy tetramin fish food in a large tub and store in the freezer. As needed, Flakes are pulverized to a fine powder using a coffee grinder, aliquoted in plastic tubes and stored in the freezer to prevent mold/bacterial growth.
3. New adult cages
- add a label to the cage noting the strain/species (Anopheles stephensi India wild type, or Aste Ind WT for short) and the eclosion date (i.e. est days 2-3 of population pupation) Leave space to record the dates of future bloodmeals.
- line the bottom of the cage with papertowels to catch blood drips and leaks
-provide sugar water within one day of first adults eclosing
-Avoid release of adults and disposal of live eggs or larvae.
- Cups and pans containing viable eggs or larvae should be bleached before washing components down the drain. I allow the bleach to sit overnight or over a weekend. Large larvae (L3/L4) may be removed by pouring the culture through a mesh strainer and tapping them into the garbage. The strainer should be cleaned with dish soap with care to not wash live larvae down the drain that may still be stuck on.
- Adult cages should be frozen in the cold room (again overnight or for several days) before disposing of insects and cleaning cages. Individual escapees should be squatted with a bug zapper. Empty cages are washed with soap and water and dried before reuse.
-Anopheles are housed in the 29 degree C chamber with 50-80% humidity. The humidity in that chamber often fails. When this happens, check with Todd to avoid duplicate reporting and alert FMS and all groups housing insects in that chamber. To maintain humidity while waiting for service, place many trays of tap water on the shelves of the room. Anopheles adults will die if kept in low humidity for too long.
Schedule (optimized to avoid need for weekend care)
Week 1
Thursday or Friday (Wednesday if need be)
- bloodfeed (note 1) cages of adults that are 3 days post-eclosion or older.
- provide adult cages with cotton balls soaked in 10% w/v sucrose
Week 2
Monday or Tuesday
- Fill a plastic dixie cup approx halfway with water and line the cup with filter paper or paper towels such that the paper is half submerged. Repeat for each cage that requires egg collection. *We generally get many more eggs than needed for culture. I also stage and collect eggs for my experiments and will co-ordinate with you.
- fill 2 pans with tap water and allow to sit in chamber for several days to warm and dechlorinate
- Rewet sugar feeders on adult cages
Wednesday or Thursday (two days after providing oviposition substrate)
- Using the prewarmed water, fill several pans to 0.5-1 inch.
- Divide hatched L1 larvae among the pans by pouring from the cup. Sterilize cup with 10% bleach to kill remaining eggs and larvae before rinsing materials down the sink.
- The number of pans needed depends on the number of eggs obtained, but typically 3-4 is good for a dense cup of larvae.
- Feed each pan 1/32 teaspoon of tetramin fish food (see note 2)
- Set out more water to prewarm.
- Rewet sugar feeders on adult cages
Friday
- Split each pan into two by adding prewarmed water and dumping half into an empty pan.Try to distribute the larvae evenly.
- If the initial pans are very densely crowded, it may make sense to transfer some to a kill pan and split the remaining cultures into three instead of two.
- Feed each pan 1/16 teaspoon of fish food to last the weekend
- Replace sugar feeders on adult cages with new cotton
Week 3
Monday
- Feed each pan 1/16 teaspoon of tetramin fish food
- If larvae seem very dense, dump some into kill pan to allow remaining to be split again
- Rewet sugar feeders on adult cages
Tuesday
- Check pans for beginning of pupation*
- If larvae are searching the bottom of the pan for food and the water is clear, add 1/32 teaspoon of fish food
Wednesday
- Check pans for beginning of pupation*, but ideally this will be the main pupation day
- If larvae are searching the bottom of the pan for food and the water is clear, add 1/32 teaspoon of fish food
- Rewet sugar feeders on adult cages
Thursday
- This will likely be day 2 or 3 of pupation.
- When most pupae have been picked, strain the water to catch any remaining larvae and sterlize the empty pan with a small amount of bleach.
- Rinse pans thoroughly with water and allow to dry if time allows
*whenever pupation starts, collect pupae into a cup and either start a new cage, or if none are clean, add the cup to the youngest adult cage and prepare a cage for tomorrow (note 3). Expect the day after pupae are first spotted to be a big day for picking pupae. Pupation will likely continue for another way, but it should really peak on day 2 of 3. Extended days of pupation is an indicator of overcrowded and/or underfed cultures.
Notes:
- Bloodfeeding
i. Be sure that bovine blood with Na-heparin anticoagulant is available and less than approx. 1 month old. Donor animal should be free from ivermectin treatment
ii. Fill a small cup halfway with water and microwave for 30 seconds. Fill a 50 mL conical tube with 10-20 mL of bovine blood and place in warm water. Each bloodmeal dish requires ~5-10 mL of blood depending on diameter, so scale depending on the number of cages that must be fed.
iii. Cut several squares of parafilm
iv. Collect several reusable handwarmers that are in liquid state. Solid state/used handwarmers can be reset by placing into boiling water until salts dissolve completely.
b. Make a bloodfeeder
i. Pour prewarmed blood into a clean metal jar lid
ii. Pre-stretch parafilm so that the middle is uniformly thin and has no rips or holes over an area large enough to cover the lid. Fit onto the lid. Press and fold down the sides to seal.
iii. Snap the metal disc in a handwarmer to trigger the reaction. Place on table top so that as it becomes solid, it is nice and flat.
iv. Place the lid parafilm-side-down onto a mesh face of an Anopheles cage. Place a warm handwarmer on top so that it is centered and the edges aren’t touching the cage. Anopheles will be more attracted to the handwarmer if they can reach it.
c. Allow feeding to occur for approx 30 min or until many fully fed females are resting on the sides and bottom of the cage.
d. Females seem more inclined to feed when you are working nearby (cleaning pans, picking pupae) than if they are alone in the room
e. This strain has been domesticated for a long time and doesn’t seem to require particular lighting or time of day to feed anymore.
f. Very young cages sometimes are not interested in feeding. Some may try as early as 3 days post eclosion, but realistically 5-7 days post-eclosion is a better time to first offer blood.
g. It is normal for small drops of blood to fall while Anopheles feed, but keep an eye out for leaks and drips from the feeder. Tears can form when feeders are moved between cages.
h. When all desired cages are fed, sterilize the remaining blood by adding bleach (freshly diluted to 10%) for several minutes. The sample should turn from red to black. Then it can be rinsed thoroughly.
i. Note the feed date on the cage label. This will help us keep track of days when eggs can be obtained and when the culture is “done”. Cages will only lay eggs after the first three bloodmeals and should be discarded after that.
2. Fish food- we buy tetramin fish food in a large tub and store in the freezer. As needed, Flakes are pulverized to a fine powder using a coffee grinder, aliquoted in plastic tubes and stored in the freezer to prevent mold/bacterial growth.
3. New adult cages
- add a label to the cage noting the strain/species (Anopheles stephensi India wild type, or Aste Ind WT for short) and the eclosion date (i.e. est days 2-3 of population pupation) Leave space to record the dates of future bloodmeals.
- line the bottom of the cage with papertowels to catch blood drips and leaks
-provide sugar water within one day of first adults eclosing