How to breed chironomid larvae? Detailed explanation of breeding methods and techniques
How to breed chironomid larvae?
(1) Artificial egg collection This is done using a dedicated artificial egg-collection box. The size of the artificial egg-collection box, the biological density and sex ratio of the chironomids, the suitable temperature, humidity, lighting and adult food are all conditions that must be considered during artificial egg collection.
① Egg-collection box The size of the egg-collection box is 1 m x 1 m x 2 m, with a box frame made of 4-5 cm square fir wood, with insect netting for mosquito prevention hung on the outside, covered with transparent plastic sheeting on top, so as to maintain humidity inside the box and allow observation from outside.
② Individual density and sex ratio of chironomids Collect adult chironomids or larvae and place them in the egg-collection box. Their individual density is one of the main factors affecting the fertilization rate. At a density of 2,000 individuals per m, a fertilization rate of more than 80% can be obtained. As the density increases, the fertilization rate also increases. When the density is 4,000 individuals per m, the fertilization rate reaches 90%. The sex ratio is one of the important biological conditions; the optimal condition is an equal number of female and male chironomids, or slightly more males than females. Therefore, during the egg-collection process, attention should be paid to supplementing male individuals.
③ Temperature The optimum temperature range is 23°C-25°C. When the temperature is lower than 20°C or higher than 28°C, the fertilization rate drops sharply. When the temperature is low, it can be solved by artificial heating. Generally, 2 40-watt light bulbs are placed inside the egg-collection box to dissipate heat, controlled by a constant-temperature relay.
④ Humidity Suitable relative air humidity is a necessary condition for male and female chironomids to mate. At a relative humidity above 90%, a fertilization rate of 80%-85% can be obtained; when the relative humidity is less than 80%, the fertilization rate drops below 20%. Humidity can be regulated by a sprayer in the egg-collection box, and evaporation is prevented by the plastic sheeting outside the box.
⑤ Lighting Research results show that the optimal condition for intermittent lighting is 4 intermittent lighting periods within 24 hours. With each light-off period of 30 minutes and each light-on period of 5.5 hours of intermittent lighting, the fertilization rate is above 80%. Egg laying begins when the light is on, and the number of eggs laid within 2 hours of lighting is 60% of the total number of eggs laid.
⑥ Food The food is placed in a basin in the egg-collection box, or sprayed on a cloth curtain hung in the egg-collection box. The adult food is 2% sucrose, 2% honey, or a mixture of both, all of which can achieve a relatively high fertilization rate.
With the above conditions of the egg-collection box, the duration of fertilized egg masses is 12-15 days, and a maximum of 400-750 egg masses can be obtained in 1 day (average 100-120 egg masses). Assuming that the average number of eggs in 1 egg mass is 500, then 100,000 individuals can be collected per day, and after 2 weeks 1.4 million individuals can be obtained, equivalent to about 7 kg of larvae.
(2) Culture medium
① Agar culture medium Dissolve agar in hot water to prepare a 0.8% agar solution, cool to 50°C, and then add milk. According to the amount of milk added, increase or decrease the amount of distilled water added, so that the agar concentration is finally adjusted to 0.75%. Then pour 25 mL of the culture medium solution into a glass dish with a diameter of 90 mm and cool it to solidify the agar, then add 10 mL of distilled water on top.
⑦ Clay-milk culture medium Take a certain amount of clay used for firing tiles, add 10 times its weight of distilled water, grind it in a large mortar to make it into a dispersed colloidal state, remove the sandy matter, sterilize it in an autoclave at 117.68 kPa for 30 minutes, and after cooling take a certain amount, add milk, and it will quickly begin to coagulate; the clay particles and milk together form a block-like precipitate, which can be used as a culture medium for larvae.
⑧ Clay-plant leaf culture medium Take weeds or mulberry leaves or marine eelgrass, add an appropriate amount of sea sand and water, grind the plant leaves in a mortar, filter and squeeze out the plant juice through a 50-mesh sieve silk net, and after standing take out the fine sand from the plant juice. Then add an appropriate amount of calcium chloride to the clay solution, then add the plant juice, and it will coagulate just like milk, until the supernatant is not colored and not turbid; after waiting 10-20 minutes, pour off the supernatant. Add distilled water and shake, then let stand for another 10-20 minutes, remove the supernatant, and after repeating this 2-3 times, the precipitated part can be appropriately diluted and used as a culture medium.
④ Sewage ditch mud culture medium Collect fresh mud from sewage ditches or fish ponds, remove large pieces of debris, add an equal amount of tap water and stir, let stand for 30 minutes, then pour off the supernatant; repeat this 1-2 times to remove suspended matter from the sewage ditch mud, sterilize under high pressure in a pressure cooker for 30 minutes, and after cooling pour off the supernatant, add an appropriate amount of distilled water, and it can be used as a culture medium.
(3) Culture methods
① Inoculation For chironomid larvae reared by artificial egg collection and artificial culture medium, select larvae with a body length of 3-4 mm through a 60-mesh sieve into a basin, add distilled water for 1-2 days, then transfer to a sieve net, rinse clean with distilled water, drain off the water, and inoculate the larvae onto the culture medium.
⑦ Static water culture method The common feature of the above 4 culture media is that they are two-phase culture media, that is, the base of the culture medium is the precipitate of solid substances such as clay, milk, broken plant leaves or sewage ditch mud, and the upper part of the culture medium is a water base of distilled water. When using a culture dish with a diameter of 90 mm to hold the culture medium, inoculate chironomid larvae larger than 3 mm into the vessel for culture; this is static water culture. This static water culture can be continued until just before pupation, when they can be harvested. It has the advantage of being easy to operate. However, because this culture method cannot ensure sufficient oxygen supply, the culture medium is prone to deterioration, and the yield is far inferior to the flowing water culture method.
⑧ Flowing water culture method Use a plastic container of 33 cm x 37 cm x 7 cm or a round basin with a diameter of 45 cm, place a sand layer with a thickness of 10 mm at the bottom, then spread the clay-milk culture medium on top, add it once every 3 days, inject a slight flow of water from one end and discharge it from the other end, then use larvae 24 hours after hatching for flowing water culture. The flowing water can play a role in removing waste and increasing oxygen, and the culture results are better than static water culture.
④ Culture of larvae with a body length of less than 3 mm The mouthparts of larvae with a body length of less than 3 mm are not yet fully developed, their resistance to various external environments is weak, and they are even less able to resist a flowing water speed of 0.1 m/s, so another culture method is needed. This method is: in a 500 mL triangular flask, inject half a flask of water, add 50 mL of culture medium, add the egg masses about to hatch into the flask, aerate with an air stone, introducing 800-1,000 cm of gas per minute, with the temperature preferably 23°C-25°C. Under these conditions, the egg masses will hatch smoothly, and after 4 days the body length can reach 3 mm, then they are transferred to flowing water culture for continued culture.
① Egg-collection box The size of the egg-collection box is 1 m x 1 m x 2 m, with a box frame made of 4-5 cm square fir wood, with insect netting for mosquito prevention hung on the outside, covered with transparent plastic sheeting on top, so as to maintain humidity inside the box and allow observation from outside.
② Individual density and sex ratio of chironomids Collect adult chironomids or larvae and place them in the egg-collection box. Their individual density is one of the main factors affecting the fertilization rate. At a density of 2,000 individuals per m, a fertilization rate of more than 80% can be obtained. As the density increases, the fertilization rate also increases. When the density is 4,000 individuals per m, the fertilization rate reaches 90%. The sex ratio is one of the important biological conditions; the optimal condition is an equal number of female and male chironomids, or slightly more males than females. Therefore, during the egg-collection process, attention should be paid to supplementing male individuals.
③ Temperature The optimum temperature range is 23°C-25°C. When the temperature is lower than 20°C or higher than 28°C, the fertilization rate drops sharply. When the temperature is low, it can be solved by artificial heating. Generally, 2 40-watt light bulbs are placed inside the egg-collection box to dissipate heat, controlled by a constant-temperature relay.
④ Humidity Suitable relative air humidity is a necessary condition for male and female chironomids to mate. At a relative humidity above 90%, a fertilization rate of 80%-85% can be obtained; when the relative humidity is less than 80%, the fertilization rate drops below 20%. Humidity can be regulated by a sprayer in the egg-collection box, and evaporation is prevented by the plastic sheeting outside the box.
⑤ Lighting Research results show that the optimal condition for intermittent lighting is 4 intermittent lighting periods within 24 hours. With each light-off period of 30 minutes and each light-on period of 5.5 hours of intermittent lighting, the fertilization rate is above 80%. Egg laying begins when the light is on, and the number of eggs laid within 2 hours of lighting is 60% of the total number of eggs laid.
⑥ Food The food is placed in a basin in the egg-collection box, or sprayed on a cloth curtain hung in the egg-collection box. The adult food is 2% sucrose, 2% honey, or a mixture of both, all of which can achieve a relatively high fertilization rate.
With the above conditions of the egg-collection box, the duration of fertilized egg masses is 12-15 days, and a maximum of 400-750 egg masses can be obtained in 1 day (average 100-120 egg masses). Assuming that the average number of eggs in 1 egg mass is 500, then 100,000 individuals can be collected per day, and after 2 weeks 1.4 million individuals can be obtained, equivalent to about 7 kg of larvae.
(2) Culture medium
① Agar culture medium Dissolve agar in hot water to prepare a 0.8% agar solution, cool to 50°C, and then add milk. According to the amount of milk added, increase or decrease the amount of distilled water added, so that the agar concentration is finally adjusted to 0.75%. Then pour 25 mL of the culture medium solution into a glass dish with a diameter of 90 mm and cool it to solidify the agar, then add 10 mL of distilled water on top.
⑦ Clay-milk culture medium Take a certain amount of clay used for firing tiles, add 10 times its weight of distilled water, grind it in a large mortar to make it into a dispersed colloidal state, remove the sandy matter, sterilize it in an autoclave at 117.68 kPa for 30 minutes, and after cooling take a certain amount, add milk, and it will quickly begin to coagulate; the clay particles and milk together form a block-like precipitate, which can be used as a culture medium for larvae.
⑧ Clay-plant leaf culture medium Take weeds or mulberry leaves or marine eelgrass, add an appropriate amount of sea sand and water, grind the plant leaves in a mortar, filter and squeeze out the plant juice through a 50-mesh sieve silk net, and after standing take out the fine sand from the plant juice. Then add an appropriate amount of calcium chloride to the clay solution, then add the plant juice, and it will coagulate just like milk, until the supernatant is not colored and not turbid; after waiting 10-20 minutes, pour off the supernatant. Add distilled water and shake, then let stand for another 10-20 minutes, remove the supernatant, and after repeating this 2-3 times, the precipitated part can be appropriately diluted and used as a culture medium.
④ Sewage ditch mud culture medium Collect fresh mud from sewage ditches or fish ponds, remove large pieces of debris, add an equal amount of tap water and stir, let stand for 30 minutes, then pour off the supernatant; repeat this 1-2 times to remove suspended matter from the sewage ditch mud, sterilize under high pressure in a pressure cooker for 30 minutes, and after cooling pour off the supernatant, add an appropriate amount of distilled water, and it can be used as a culture medium.
(3) Culture methods
① Inoculation For chironomid larvae reared by artificial egg collection and artificial culture medium, select larvae with a body length of 3-4 mm through a 60-mesh sieve into a basin, add distilled water for 1-2 days, then transfer to a sieve net, rinse clean with distilled water, drain off the water, and inoculate the larvae onto the culture medium.
⑦ Static water culture method The common feature of the above 4 culture media is that they are two-phase culture media, that is, the base of the culture medium is the precipitate of solid substances such as clay, milk, broken plant leaves or sewage ditch mud, and the upper part of the culture medium is a water base of distilled water. When using a culture dish with a diameter of 90 mm to hold the culture medium, inoculate chironomid larvae larger than 3 mm into the vessel for culture; this is static water culture. This static water culture can be continued until just before pupation, when they can be harvested. It has the advantage of being easy to operate. However, because this culture method cannot ensure sufficient oxygen supply, the culture medium is prone to deterioration, and the yield is far inferior to the flowing water culture method.
⑧ Flowing water culture method Use a plastic container of 33 cm x 37 cm x 7 cm or a round basin with a diameter of 45 cm, place a sand layer with a thickness of 10 mm at the bottom, then spread the clay-milk culture medium on top, add it once every 3 days, inject a slight flow of water from one end and discharge it from the other end, then use larvae 24 hours after hatching for flowing water culture. The flowing water can play a role in removing waste and increasing oxygen, and the culture results are better than static water culture.
④ Culture of larvae with a body length of less than 3 mm The mouthparts of larvae with a body length of less than 3 mm are not yet fully developed, their resistance to various external environments is weak, and they are even less able to resist a flowing water speed of 0.1 m/s, so another culture method is needed. This method is: in a 500 mL triangular flask, inject half a flask of water, add 50 mL of culture medium, add the egg masses about to hatch into the flask, aerate with an air stone, introducing 800-1,000 cm of gas per minute, with the temperature preferably 23°C-25°C. Under these conditions, the egg masses will hatch smoothly, and after 4 days the body length can reach 3 mm, then they are transferred to flowing water culture for continued culture.