Protocol for Mosquito Rearing (A. gambiae)

Published 7/04/2007
10 Comments
  CITE THIS  SHARE 
Biology

You must be subscribed to JoVE to access this content.

Fill out the form below to receive a free trial:

Welcome!

Enter your email below to get your free 10 minute trial to JoVE!





By clicking "Submit," you agree to our policies.

 

Summary

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Cite this Article

Copy Citation

Das, S., Garver, L., Dimopoulos, G. Protocol for Mosquito Rearing (A. gambiae). J. Vis. Exp. (5), e221, doi:10.3791/221 (2007).

Abstract

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28°C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol

Mosquito food:

  1. Food A: grounded fish food (Aquaricare). A small pinch needs to be added.
  2. Food B: grounded CAT food (Purina). A small pinch needs to be added.
  3. Food C: cat food (Purina). Two tablets needs to be added.

Blood:

Animal or human blood can be used to rear mosquitoes. Fresh blood is collected with a syringe and put in a sterile 15 ml Falcon tube containing 1ml of CPD (anticoagulant) for each 10 ml of blood, then mixed gently and centrifuged at room temperature at 2000 rpm for 5 min. The supernatant is discarded, taking care to remove the buffy coat which comprises other blood cells (e.g. WBC). The RBC pellet is then suspended in an equal volume of RPMI medium by pipetting, and further washed in this medium three times. After the final wash, the pelleted RBCs are resuspended in an equal volume of RPMI medium and stored at 4°C (can be kept for 8-10 days). Just before feeding, the RBC (in RPMI) is centrifuged (2000 rpm for 5 min) to pellet down the cells, and the packed RBC is resuspended in serum (O+ human serum) to obtain a 40% haematocrit. The blood must be kept at 37°C always for the feeding.

DAY 1:

  1. The 3-5 day old adult female mosquitoes are fed on blood to lay eggs.
  2. For blood feeding, an artificial membrane (parafilm) feeding method is used as follows:

    1. Red blood cells (see above for blood preparation) are mixed with heat inactivated serum to obtain a ~40% haematocrit (packed RBC 40% and serum 60%). This is added to the glass feeder.
    2. The feeder is connected to a warm water-jacket (37°C), and placed on the cage to allow the mosquitoes access to the membrane surface.
    3. The mosquitoes are allowed to feed for ~30 minutes.

DAY 3:

  1. The females will lay eggs two days after they feed on blood. A small filter paper wrapped in a conical shape is put in a small beaker containing distilled water, making sure that filter paper gets moist. The beaker is kept inside the cage overnight for the mosquitoes to lay eggs.

DAY 4:

  1. The filter paper containing the mosquito eggs is placed in a plastic tray with ~300 ml distilled water. A pinch of food A is added to the tray and eggs are allowed to hatch to larvae during the next days.

DAYS 5 - 8:

  1. Growing larvae are fed every day with two tablets of food C, and monitored for density and population. On the eighth day (5 day old larvae), the larvae population is diluted from 1 tray to ~10 trays, with a pinch of food A and two tablets of food C in each tray. (~30 mins)

DAYS 9 - 12:

  1. The larvae are fed every day with food C. On the 12th day (9 day old larvae), the water is changed with fresh water, and food is added (a pinch of food B and two tablets of food C). The pupae starts developing at this stage. The trays are covered with nets to avoid escape of adults. (~ 8-10 mins)

DAY 13 - 15:

The pupae are allowed to emerge to adults for the next 2 - 3 days. Food is given every day to the larvae/pupae by carefully removing the net to avoid escaping of adults.

DAY 16:

  1. The adults are collected into a cage through an aspirator connected to vacuum. The cage consists of a small 100 ml bottle with a cotton wick that is soaked with 10% sucrose (autoclaved) and a paper towel lining on the bottom to soak any potential sugar spills which may occur during cage handling. (~ 30-40 mins)

DAY 17 - 21:

  1. The adults (both males and females) are then kept in the insectary room for 4-5 days, fed on 10% sucrose before they are again blood-fed to begin the next cycle. The same mosquitoes can be used to lay eggs more than once.
  2. The mosquitoes that are not needed for experiments or rearing can be killed by placing the cage in a freezer. The used trays and cages need to be cleaned and dried before they can be used again.

NOTE: There can be some variations in the mosquito rearing method and different labs may have different techniques.

Subscription Required. Please recommend JoVE to your librarian.

Materials

Name Type Company Catalog Number Comments
Food A Aquaricare Grounded fish food. A small pinch needs to be added.
Food B Purina Grounded cat food. A small pinch needs to be added.
Food C Purina Cat food. Two tablets needs to be added
RBC prepared red blood cells: 40% hematocrit in human serum
RPMI Medium
CPD anticoagulant
serum O+ human serum
Mosquito feeder
parafilm
Anopheles gambiae mosquitos
10% sucrose mosquito food
insectary

DOWNLOAD MATERIALS LIST

References

  1. Bangs, M. J., Soelarto, T., Barodji,, Wicaksana, B. P., Boewono, D. T. Colonization of Anopheles maculatus from Central Java, Indonesia. J Am Mosq Control Assoc. 18, 359-363 (2002).
  2. Beier, M. S., Beier, J. C., Merdan, A. A., el Sawaf, B. M., Kadder, M. A. Laboratory rearing techniques and adult life table parameters for Anopheles sergentii from Egypt. J Am Mosq Control Assoc. 266, 266-270 (1987).
  3. Benedict, M. Q. Care and maintenance of anopheline mosquitoes, The molecular biology of disease vectors: A methods manual. Crampton, J. M., Beard, C. B., Louis, C. Champman & Hall. London. 3-12 (1997).
  4. Casanges, A. H., Mcgovran, E. R., Chiles, J. V. Rearing of Anopheles quadrimaculatus Say and Aedes aegypti (L.) in the laboratory. Mosq News. 9, 112-117 (1949).
  5. Merritt, R. W., Dadd, R. H., Walker, E. D. Feeding behavior, natural food and nutritional relation ships of larval mosquitoes. Annual Rev. Entomol. 37, 349-376 (1992).
  6. Nasirian, H., Ladonni, H. Artificial bloodfeeding of Anopheles stephensi on a membrane apparatus with human whole blood. J Am Mosq Control Assoc. 22, 54-56 (2006).
  7. Ng'habi, K. R., John, B., Nkwengulila, G., Knols, B. G., Killeen, G. F., Ferguson, H. M. Effect of larval crowding on mating competitiveness of Anopheles gambiae mosquitoes. Malar J. 30, (4), 49 (2005).
  8. Stobart, R. H. Factors affecting the control of body volume in the larvae of the mosquitoes Aedes aegypti and Aedes detritus. Edw. J. Exp. Boil. 54, 67-82 (1971).

Comments

10 Comments

  1. For your information, there are four larval instars/stages not three as mentioned in the section Late Stage Larvae.

    An alternative to collecting adult mosquitŒs by sucking is to pick the pupae into a cup with water and place the cup in the adult cage. This dŒs not require an advanced apparatus or the work of mouth aspirating. Also, there is no risk of the pupae escaping as with adults. The disadvantage of picking pupae is that it has to be done on a daily basis, but with good planning of the feeding it is possible to avoid picking the main bulk of the pupae on weekends.Olle Terenius, University of California, Irvine, USA

    Reply
    Posted by: Anonymous
    March 13, 2008 - 3:46 PM
  2. My name' Dui, I work in Institude of Malariology. Parasitology and Entomology in Ho Chi Minh city. I'm responsible for insectary. I feed mosquitŒs by alive mouse. Now I want to feed mosquitŒs by membrane feeder. I follow your instruction in video clips but the mosquitŒs couldn't suck blood through the membrane. I don't know The reason why The mosquitŒs didn't have blood inside their body. Can you help me ?

    Reply
    Posted by: Anonymous
    April 29, 2009 - 7:22 PM
  3. Hi, This is Suchismita from Johns Hopkins University. Membrane feeding usually works for many mosquito species, but there are several that somehow do not take blood by membrane-feeding-for example, several arabiensis species and many others. What is your mosquito species? Also, are you using fresh blood. We have noticed that sometimes blood older than 1 week are no good. Then which serum are you using, as all these citical steps help. I would also recommend that you starve your mosquitŒs for few hours (6-7) before you provide blood by membrane-feeding assays. These makes them really hungry and they go for blood. Also try to swithch off the light of that room, dark somehow initiates their feeding. I hope these tips help you. In case you have more questions, please send me an email at sudas@jhsph.edu, I will be happy to answer.  

    Reply
    Posted by: Anonymous
    April 30, 2009 - 12:30 PM
  4. Dear Das:

    Greetings. I am an entomology researcher based in Jakarta, Indonesia.
    Could you please provide information on where I could obtain apparatus glass membran feeding for mosquito?

    Thank you.

    Arik Farzeli, DVM
    Medical Entomology Department
    Naval Medical Research Unit ²
    Jakarta, Indonesia

    Reply
    Posted by: Anonymous
    July 8, 2009 - 12:14 AM
  5. Hi, thanks for your video guide it has been very useful but I would like to know are there anyways by which i can download or buy., since the link in my area is very very bad. It took me 4 hours to watch 10:²3 minutes of this clip. can you help?

    Reply
    Posted by: Anonymous
    July 9, 2009 - 1:29 PM
  6. FYI: Our lab uses mosquitŒs to maintain the life cycle of filarial nematode Brugia malayi and to generate larval stages of these parasites for other research activities. For membrane feeding, we routinely use "casing" (used in sausage making) or skin from chicken legs as membranes. "Casings" cost only few dimes and can be bought at any major grocery store. Casing is easy to attach to the glass feeders. Aedes mosquitŒs feed well on blood through casings than parafilm and should be good for other blood sucking critters.

    Reply
    Posted by: RamKrishna R.
    July 24, 2009 - 2:55 PM
  7. Hi RamKrishna,
    The technique you use is very practical, could you please give me more details about this, how you build
    the system feeding with skin from chicken legs. In the University insectary need to implement another
    technique different of menbrana parafim, becouse use a large population of mosquitŒs about of 1000 mosquitŒs simultaneously. We are raising the species: Aedes aegypti and Anopheles darlingi.

    What are the cage dimensions you use and how many mosquitŒs are in it?.

    thanks for share your experiences with us.
    if you could write to my email would be great, iram_8586@hotmail.com
    Maribel :D

    Reply
    Posted by: Maribel T.
    May 1, 2013 - 7:54 PM
  8. I work at K-state and maintain a small colony of A. gambiae. The adult feed on a homemade membrane feeder. The last ² feedings 1/² of the adults died. I ordered fresh blood citrated horse blood, this did not help. Do you have any thoughts as to what could be going on? I maintained the colony for several years, now I am having problems.
    The next question, is how to you clean the larva rearing trays?

    Reply
    Posted by: Anonymous
    October 20, 2010 - 12:21 PM
  9. thank you for your video guide,lot of thanks.

    Reply
    Posted by: Anonymous
    January 25, 2012 - 3:14 AM
  10. Hi everybody

    I am Carlos and I am working with Anopheles darlingi in an insectary in the Peruvian Amazon
    .
    I would live to have the video. I don´t look it in my computer.

    Greetings

    Reply
    Posted by: Carlos T.
    December 30, 2013 - 10:31 AM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Video Stats