Mosquitoes and malaria (1988)

[Music] malaria is transmitted by the bite of a mosquito not all mosquitoes can transmit malaria only one genus anopheles is responsible and only the female feeds on blood this female enophiles is feeding on the blood drawn from the capillaries in a man's wrist if she can't find blood she can feed on fruit juices but without a blood meal she can't make eggs and can't reproduce given the opportunity she'll feed on blood every 2 or 3 days and each time she takes in more than her own weight of blood her abdomen swells up and as she feeds she concentrates the blood and the clear fluid separated is excreted if a mosquito feeds on someone infected with malaria parasites will be taken up with the blood here is a mosquito's blood meal these are red cells and these are the sickle-shaped gyes of plasmodium falum falum means sickle-shaped a male and a female the male mettoy exf flagellates to produce the motile threadlike micro gametes when a male contacts a female the two fuse to produce an invasive motile zygote the okin this swims through the blood meal and penetrates the stomach wall where it grows rapidly into an uos cyst after 10 to 12 Days the osy is mature and pack with spores this mosquito is carrying a very heavy infection the uos cysts rupture into the body cavity and the sporozoites move through the body of the mosquito to enter its salivary glands when the mosquito feeds again the sporozoites are injected with the saliva into another person and the transmission cycle is complete to summarize the cycle the GTO sites are ingested with the blood meal the male ex flagellates fertilization takes place the okines form uos cysts on the stomach wall and sporozoites leave the uos cysts and migrate to the salivary glands to show that a mosquito is infected with malaria it's necessary to find uos cysts on its stomach wall or sporozoites in its salivary glands this can be done by dis action under a microscope and the following demonstration filmed in 1953 was carried out by PG shot and rs Bray if a recently fed mosquito is captured its stomach will be distended with blood it's best therefore to keep it for a day or two to allow the blood to digest and prevent the distended stomach from being broken during the dissection during this time the uros cysts will mature dissecting a mosquito is a quick and simple matter after a little practice in addition to the dissecting microscope the only instruments needed are two very sharp stainless steel dissecting needles stainless steel because the dissection is carried out in sine which is corrosive to ordinary steel the mosquito is placed in a narrow test tube shaken to the bottom of the tube and then stunned by tapping the tube sharply on the hand the insect is killed by pressing it lightly on the thorax not too hard or it'll be crushed the insect is then held by One Wing while the legs are pulled off one at a time the other Wing is pulled off and the wing that has been held is cut off the head is then cut off and the body of the mosquito is ready for Dissection the salivary glands are removed first a magnification of about 10 times is used the insect is placed on the microscope stage with its thorax to the right a drop of sine is put on the slide close to the thorax but not touching it holding the thorax firmly with one needle at a point just behind behind the glands they're pressed gently out into the saline drop with the other needle this is how the operator sees the process each gland has three loes usually they're about about a third of a millimeter long but of course they'll vary between insects if sporozoites are present they'll be found in the cells and ducts of the glands a microscope cover slip is placed over the glands dropping it just heavily enough to rupture without shattering them the material is examined under a standard microscope with a good quality high power objective and if sporozoites are present they'll be seen as relatively inactive threadlike organisms drifting slowly in the surrounding fluid the second stage in the dissection is to remove the stomach of the mosquito the insect's body is transferred to another slide into a new drop of saline the thorax is held with one needle and the other used to Nick the abdominal covering just above the tail at the level of the sixth or seventh abdominal segment these detached segments can then be gently teased away and the gut pulled away with them it needs a little care and patience this is the operator's view first the Malian tubules and hind gut now the stomach or midgut and finally the esophagus the slide is turned round and the maligi and tubs cut off a cover slip is lowered very gently onto the specimen just to flatten the stomach without breaking it the slide is transferred to the ordinary microscope and examined under the low power uo cysts are seen as Tiny translucent vesicles on the stomach wall the specimen is heavily infected the uos cysts are welldeveloped and easy to identify if oocysts are found on the wall of the stomach this only shows that the mosquito is susceptible to infection to confirm that it is acting as a vector sporozoites must be found the sporozoite rate is the percentage of mosquitoes with sporozoites in their salivary glands it's an important component of epidemiological studies it shows which spe Es are likely to transmit malaria and is essential for judging the effectiveness of control measures in highly endemic areas the sporo rate may be up to 4% in epidemic areas it may vary between 30% During a severe outbreak to almost zero when there's very little transmission but dissection is labor intensive a trained technician can process about a 100 mosquitoes a day and nowadays immunological techniques are helping to reduce the labor increase the output and to detect very light infections the protein on the surface of a sporo has a central region of the molecule made up of repeated amino acid sequences unique to each malaria species plasmodium ferum has a group of four amino acids repeated 41 times and plasmodium viax a group of nine amino acids repeated 19 times monoclonal antibodies that recognize these specific repeat regions have been prepared and are used in an enzymelinked immunosorbent assay Eliza to detect sporozoites using this technique as few as 15 parasites have been detected in a single mosquito and most infected mosquitoes contain more than 500 mosquitoes that have been collected in surveys weeks or months before can be used for the test with the aid of a miniature pestle and mortar mosquito heads and thores are ground up with a little detergent to extract the protein samples of the fluid are then distributed into the wells of a plastic plate already coated with monoclonal antibody after standing a while to allow the porzo protein if present to stick to the antibody the plate is washed monoclonal antibody labeled with an enzyme is then added and this is captured by any sporo protein in the wells the plate is washed again a color producing substrate for the enzyme is added and where the enzyme has stuck the color develops this is how it works here are two wells in the plate coated with specific monoclonal antibody protein extracts from mosquitoes are added the one on the left contains sporo protein and it sticks after washing antibody labeled with an enzyme is added and this also sticks to the sporo protein the plate is washed again and the enzyme substrate added the enzyme releases the color in the left hand well but nothing has stuck in the right hand well and no color develops the depth of color depends upon how much sporozoite protein and therefore how much enzyme has stuck by using the elizer technique our technician can process about 3,000 mosquitoes in batches of 20 every day you can also determine using appropriate plates whether plasmodium falum or plasmodium viac are present in our demonstration we have featured the simplest manual elizer equipment sophisticated automatic machines for processing and reading the plates are now available and are in everyday use in Laboratories around the world Eliza is used for a wide range of tests including AIDS but in areas where elizer equipment may not be available mosquito dissection and Keen eyesight will continue to play an important part in the epidemiology of malaria [Music]