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Malaria is transmitted by Anopheles mosquitoes. This applies to the malaria of all mammals. Avian malaria on the other hand is chiefly transmitted by Culicinae. There are some 400 Anopheles species, 40 of which are good vectors while 28 are poor vectors. Anopheles mosquitoes are relatively small (8 mm), two-winged insects (Diptera; Gr. di = two and pteryx = wing) sometimes with a typical posture while feeding: head down and the lower body upwards. [There are exceptions to this such as Anopheles culicifacies, which, as its name suggests, is similar in posture to Culex.] As with many mosquitoes there are countless scales on the body and wings. In Anopheles there are darker and lighter coloured scales arranged in groups, which produces a distinctive marking on the wing (a speckled pattern). Culex mosquitoes on the other hand are of an even colour.
Anopheles mosquitoes undergo induced color change based on perception of the background against which they are cultured. When larvae are reared on either a black or white background, they become pigmented dark or pale. The degree of darkening depends in part on the length of time the larvae have been cultured on a black background and the degree of fat body development. This color change phenomenon is called homochromy. Anopheles mosquitoes are active at night. They do not buzz much and are not easily noticed. Every species of mosquito has its own characteristics as to behaviour, reproduction, biting habits, etc. This is of course important for mosquito control.
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Many invertebrates have an open circulation, in which blood pumped by the heart empties via an artery into an open, fluid-filled space, the haemocoel, which lies between the ectoderm and the endoderm. The fluid contained within the haemocoel is referred to as haemolymph or blood. The blood is not circulated through capillaries but bathes the tissues directly. This blood circulation is of minor importance for oxygen transport. Instead they have a tracheal system in which respiratory gases are transported directly to tissues through air-filled tubes. Most insects have a large capacity for aerobic metabolism.
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Genomic DNA from both males and females of the PEST strain of Anopheles gambiae was sequenced. The PEST strain was derived from a cross between a laboratory strain and a field-collected isolate of A. gambiae. This strain was choosen for several practical reasons.
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At tropical temperatures the first oviposition occurs 2 to 3 days after the first blood meal. Each time the females lay about 100 eggs in water (they are not attached to one another, unlike the eggs of Culex mosquitoes which lie in groups). The eggs cannot survive drying out, unlike those of Aedes. After they have laid their eggs the females will once more be searching for a blood meal. The interval between sucking blood and laying eggs is called the gonotrophic cycle. The period of this cycle is important in determining vector capacity. The more often the mosquito feeds on blood, the more risk there is of becoming infected with parasites. At lower temperatures the gonotrophic cycle is longer, some 4 or 5 days. This reduces the risk of disease transmission because:
(1) the number of times that the mosquito can take up or inject parasites is smaller, (2) the speed of development of the parasite is dependent on temperature and is slower in a cold climate, and (3) the mosquitoes have a greater chance of dying before they become infectious. If the lifetime of the mosquito is shorter than the development time of the parasite (extrinsic incubation period) there will be no transmission of the disease. If vector control is by means of insecticides, an attempt is being made to make the average life span of the mosquitoes shorter than the extrinsic incubation period of the parasite. Thus the majority of female mosquitoes are killed before they become infectious. How can this be measured? One way to ascertain the age structure of an insect population is to determine the proportion of females who have laid eggs at least once. This can be done by examining the ovaries. Specific attention is paid to the shape of the trachea (breathing tubes). In nulliparous insects these have a different shape to those of insects which have already laid eggs. They exhibit a scar, caused by the swelling of the eggs.
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After 2-3 days legless larvae emerge from the eggs. There are four larval stages.
The larvae of Anopheles lie parallel with the surface of the water, unlike Culex larvae. They breath air via small caudal openings (spiracles) [the larvae also have dorsal "gills", but these are actually osmoregulatory organs]. This surface position makes them susceptible to chemicals which float on water, e.g. oil with special dispersion detergents to use less product per hectare). Yet larvae can also to a limited extent take up oxygen dissolved in the water, so an oil film with mechanical protection is only of limited benefit. The larvae are filter feeders and have oral tufts of hair ("mouth brushes"). They feed on all kinds of microscopic organisms. For example they can take up dead Bacillus thuringiensis var. israelensis. The spore of this bacterium contains toxins which kills the larva and this is used for vector control (see also onchocerciasis). The entomopathogenic Bacillus sphaericus may also be taken up.After 7-20 days a larva will form a pupa. This pupa is quite active (motile) but does not feed.
The rate of development of the larva and pupa is highly dependent on temperature. Malaria transmission quickly increases if the average temperature rises. A feature of the pupa is the two trumpets with which the animal breathes. Anopheles sexes can be distinguished easily in the pupal stage. This is often desirable in the laboratory, so that sexes can be separated before adulthood to avoid mating of adults intended for genetic crosses and otherwise to determine sex before adulthood. Claspers are present only in the male (visible on the rear of the pupa). After eclosion, the adult insect appears. Metamorphosis can be disturbed by various pesticides with a hormonal action. The metamorphosis is affected to an important extent by the corpora allata, small lobes near the insect brain. These corpora release what is called the juvenile hormone, a sesquiterpene. Methoprene (Altosid®) is a juvenile hormone analogue which prevents metamorphosis (the actual hormone is too unstable to be used). The shedding of the old cuticula is also under hormonal control. The brain excretes an activation hormone, called the prothoracicotropic hormone. This acts upon the prothoracic gland, which in turn excretes ecdysone, a steroid hormone. Due to the effect of ecdysone the old cuticula is separates enzymatically from the new cuticula which is forming underneath (apolysis). After the old cuticula has been discarded (ecdysis) the new one, which is still soft, quickly hardens under influence of bursicon, a hormone.
The ecological habitat which larvae need, varies greatly from one species to another, from permanent water surfaces to temporary puddles, fresh or brackish water, edges of streams or still water, in the open sun or in deep shadow, from pure water to polluted water, marshes or small water collections in plants, trees, rocks, hoof-prints or rubbish. Larvae are generally rare on large water surfaces, lakes or fast rivers (except at the edges). No larvae will be found in flowing water. The characteristics of the breeding grounds are precisely defined for each Anopheles species, so that if the local vector is known, the breeding grounds can be targetted for selective control.
Like all two-winged insects mosquitoes can only feed on liquid food. They suck plant juices and nectar, but females need blood to make eggs. Without blood no or few eggs can be laid. Only the females suck blood. The life of a male mosquito consists of a constant search for sex and nectar. The mouth parts must not be confused with the palpi and antennae. The long palpi lie on either side of the proboscis. The antennae have a distinctive structure (plume shaped and hairy in the male).
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Mouth parts
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The structure and action of the mouth parts of a female are important for the transmission of malaria. The mouth (proboscis) consists of 7 parts. Above is the upper lip (labrum) and below is the larger U-shaped lower lip (labium) which ends in a blunt thickening (labellum). The labrum and labium enclose a space containing 2 awl-shaped serrated mandibulae, 2 similar maxillae and 1 hollow hypopharynx. The cavity in the hypopharynx is the extended outlet from the salivary glands. When a female bites, the blunt labellum is placed on the skin. This cannot penetrate the skin and the labium bends backwards. This allows the other 6 mouth parts to penetrate the skin. Saliva is injected to prevent the blood from coagulating and blocking the mouth parts. It is in this saliva that the sporozoites are to be found.
Some mosquitoes are sensitive to one species of malaria parasites and resistant to another. Thus for example P. vivax can develop successfully in A. atroparvus, but P. falciparum from tropical regions is not capable of forming oocysts in this mosquito. P. falciparum from Southern Europe which has now been eradicated could develop in this mosquito. Penetration into the mosquito salivary gland cells is receptor-ligand dependant. This specificity explains why certain parasites can only be transmitted by certain Anopheles. For example, the complete sporogony of P. cynomolgi can be completed in Anopheles freeborni, but the parasites cannot penetrate the salivary glands of the insect. Some mosquito species can cause lysis of ookinetes in their intestines or encapsulate them in a melanotic capsule. There are certain peptides (defencins, similar to the cecropins of other insects) in the haemolymph of mosquitoes. These are being studied for their protective effect on the mosquito. Our knowledge of the protective mechanisms of Anopheles sp. against parasites is not complete.
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Different mosquito species have widely varying habits: some bite chiefly animals (zoophilic) and others humans (anthropophilic). They may live and bite outside (exophilic and exophagic respectively) or come into houses and bite there (endophilic and endophagic respectively). Anopheles mosquitoes are good flyers: they can cover several kilometres in one night. This is of course of great importance for their control. If there are lots of animals around the houses, malaria transmission by zoophilic vectors will be reduced. Contact with endophagic mosquitoes can be reduced by mosquito nets. Endophagic mosquitoes will often rest on walls after a blood meal. Residual insecticides which are applied there will kill the vector. There are also techniques for controlling adult mosquitoes outside houses. Vector control of malaria targets the larvae and the adult insects. Eggs and pupae are difficult to control.
Mosquitoes are attracted by an increased CO2 concentration, or to put it better, by a CO2 gradient. Yet CO2 is an aspecific attractant, since all animals breath out CO2. This is therefore a problem for mosquitoes which have a preference for a specific host. Dilution in the atmosphere limits the range of this substance to approximately 20 metres. The warmth of the skin, lactic acid and moisture (breath) play a part over short distances. Every animal produces a number of volatile substances in its skin, breath, faeces and urine. A number of the substances (kairomones) are used by the mosquito to find its prey. The skin has various microhabitats (armpits, groin, soles of the feet, scalp, and so on) which are colonised by various bacteria (staphylococci, micrococci, coryneforms, etc.) These bacteria can convert some substances in sweat and sebum into attractants. The presence of Brevibacterium epidermidis plays a part in the typical smell of sweaty feet (comparable to the smell of Limburger cheese, in which Brevibacterium linens produces the typical odour). This odour is highly attractive to mosquitoes. The details are complex, however. Thus Anopheles gambiae prefers to land on the feet, while A. atroparvus prefers to bite the face.
Various methods have been developed to find attractants. The substances may be identified by the measuring the activity of receptors in the mosquitoes’ antennae when they are in a stream of air in which the attractants occur. In the laboratory the chemicals are separated in a gas chromatograph. With the purified chemicals, one can perform electro-antennography, or single-cell recordings with micro-electrodes on the olfactory receptor cells of the insect. Potential mosquito repellents may also be screened and researched in this way. Perhaps one day certain cocktails of attractants can be used to make mosquitotraps effective.
Malaria vector control is primarily based on the use of insecticides. Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide uses in malaria control programmes.
Pyrethroids and DDT, two important insecticides used for vector control, block the nerve-impuls conduction by preventing a para-type sodium channel from returning to the closed-gated configuration after an action potential. An important mechanism that confers resistance to pyrethroids and DDT, known as knockdown resistance or kdr, was first described in the housefly Musca domestica. It has been reported that a single mutation in the S6 transmembrane segment of domain II in the para-type sodium channel sequence is the molecular basis of kdr in Musca domestica. Recently, the para-type sodium channel gene has been characterized for different insects, including the African malaria vector Anopheles gambiae. Mutations in this gene have been linked to knockdown resistance. For A. gambiae, diagnostic PCR tests have been developed for the detection of the kdr-mutation.
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This is not a all-inclusive list. In bold, important species.
1. North American
2. Central American
3. South American
4. North Eurasian
5. Mediterranean
6. Afro-Arabian
7. Afrotropical
8. Indo-Iranian
9. Indo-Chinese hills
10. Malaysian
11. Chinese
12. Australasian
Note: Anopheles gambiae complex
Mosquitoes which are identical as regards size, colour, pattern, morphology, may nevertheless differ in behaviour, adjustment to the environment, and DNA such as the banding pattern on the polytene giant chromosomes of the larval salivary glands or the ovarian nurse cells of adult mosquitoes. Polytene chromosomes are found in salivary glands of Diptera. Their enormeous length (2 mm in Drosophila) is much longer than metaphase chromosomes. The polytene chromosomes consist of 100 or more copies of DNA, arranged side by side. The DNA is said to be endoreduplicated. Examination of these chromosomes by Feulgen staining and light microscopy reveals alternating highly and moderately dense regions, called bands and interbands. Polytene chromosomes can be also be analysed by staining with antibodies. The expanded length of polytene chromosomes, relative to metaphase chromosomes, allows a much higher resolution mapping of morphological chromosome features than is possible with G- or Q-banding (staining with Giemsa or Quinacrine) The high local DNA concentration of aligned DNA sequences make polytene chromosomes ideal targets for in situ hybridization with specific sequence probes. The lesser density of interbands suggests that the chromatin in these regions is less condensed and may correspond to euchromatin of normal interphase nuclei. Conversely, the bands probably correspond to the condensed heterochromatin. Electron micrographs allow the fine definition of banding patterns.
Morphological identical mosquito species were found to be in fact different species since genetic exchange between them is no longer possible or leads to sterile hybrids. Such mosquitoes may be found in the same region (sympatric mosquitoes). One can often differentiate sibling species by electrophoresis. Proof of speciation is based on the lack of heterozygosity of the diagnostic iso-enzymes in species which breed in the same environment, and also by culture experiments. Nowadays molecular markers are used on dried specimens. In this way six closely related species were identified in the most important African vector, Anopheles gambiae. These mosquitoes form the Anopheles gambiae species complex. They are mosquitoes which cannot be morphologically distinguished from each other.
Note: Anopheles gambiae and Brazil
One could have the impression that the geographical distribution of mosquitoes is stable. However, sometimes insects are imported into new regions where they can flourish and become pest species. Just such a case occurred in 1930. In March of that year larvae of Anopheles gambiae were found in a very limited area in Natal, Brazil. This mosquito originated in Africa, however, and until then had never occurred in America. The local authorities were informed but showed no interest and refused to co-operate in control measures. In January 1931 there was a malaria epidemic with 10,000 cases. The size of the infested area increased. In 1938 the number of patients rose to 100,000 cases with 14,000 to 20,000 deaths. There was severe disturbance of everyday life. The cotton harvest was not picked due to the huge numbers of people who were sick, and the food supply was threatened. The President, Getubio Varga, intervened and appointed Fred Soper as leader of a huge eradication campaign. He was an energetic and extremely gifted man. After setting up laboratories, a training school, a cartography department and a surveillance system, the foci were charted and the region was divided into small units. Teams were trained and made repeated visits to every focus and individual dwelling. Spraying was carried out with copper aceto-arsenite (Paris Green) and drainage projects were completed. Oil was poured regularly on water surfaces to kill the larvae. Ships, aeroplanes, trains and cars on the roads were fumigated upon leaving the epidemic region. In one year he was able to drive the mosquito back to two small foci which were then brought under control. The mosquito was eradicated from the continent, a notable achievement in the time before DDT existed.
The development of plasmodia (from gametocytes to sporozoites) in a mosquito takes at least 9 days (sometimes as much as 30 days, depending on the temperature). After an infected blood meal a female mosquito will pass through at least 4 or 5 egg-laying periods (and blood meals) before it becomes infectious. There is thus ample time over several days to destroy the vector before she can transmit. Mosquitoes which are infectious are already "middle-aged". They may sometimes survive a month or more, but often the life span is much shorter. This long period before a mosquito becomes infectious is a weak point in the cycle of plasmodia. For example: at a temperature of 30°C at least 70% of mosquitoes need to survive every day if more than 1% of mosquitoes are to survive the 10-day development period.
A practical example can be seen in "airport malaria". Sometimes (due to lack of decontamination or resistance to insecticide) infected mosquitoes are brought to northern airports in aeroplanes. If such a mosquito can remain active due to high environmental temperatures (in a hot summer), bites someone and injects sporozoites, it will already be at least 10 days old. Adding the incubation period for P. falciparum and the time between the first fever and making the diagnosis (in northern regions one does not think quickly of the possibility of malaria), it can then be assumed that the mosquito which caused the disease will probably already be dead when the diagnosis is finally made. Patient's delay and doctor's delay are important factors in lethal malaria. One mosquito can infect several persons (for example if the blood meal is interrupted the mosquito will bite several times), also possibly outside the airport (carried by the wind, or flying). Also, several mosquitoes may be introduced. Due to these factors, clusters of airport malaria may occur in hot summers. The risk that descendants of an infected (or non-infected) mosquito would survive in Europe and find a gametocyte carrier and cause infections again after the incubation period, is very small.
Note on Diptera
The diversity of insects which can transmit pathogens can be overwhelming. Certain mosquitoes are of importance to malaria. To be able to place them taxonomically, the following explanation is given (for more details see entomology course). The order of Diptera (two-winged insects) is subdivided into two large suborders: Nematocera ("segmented antennae": antennae with many similar segments; 26 families all together, such as mosquitoes, simulids and ceratopogonids) and Brachycera ("short antennae", with 104 families all together). The Brachycera are divided in the Cyclorrapha ("round seams", which refers to a larval characteristic; 85 families, e.g. tsetse flies (Glossina sp), fruit flies (Drosophilidae), house flies (Muscidae); hover flies (Syrphidae), blow flies (Calliphoridae), Sarcophagidae and tachinids) and the Orthorrapha (19 families, exemplified by the horseflies; tabanids belong to this group, as do the robber flies (Asilidae) and long-legged flies (Dolichopodidae). In the higher flies (Cyclorrapha), pupation occurs inside the skin of the third last larval stage, which is known as the puparium. About 20% of all fly species, representing more than 20 families, are parasitoids. This means that their larvae develop inside the bodies of hosts, which are killed in the proces. Those insects (e.g. tachinids) are of immense importance in the control of natural populations of pest insects.
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Nematocera are subdivided into:
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Not biting humans and of no direct medical importance:
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Biting mosquitoes are divided into three large subfamilies :
