From Essential Malariology,  4^th Edition, 2002

www.malaria.blogfa.com  by: Abbas Poudat 

ADULT BIOLOGY

۱. Blood feeding and the gonotrophic cycle

Because egg production and blood feeding are intimately linked and blood feeding is also essential to the acquisition and transfer of malaria infection, malaria entomologists have paid special attention to this physiological phenomenon termed the gonotrophic cycle. If the duration of this cycle is known, the number of gonotrophic cycles can serve as a proxy for the physiological age of the mosquito, an important determinant in malaria epidemiology.

Adults usually mate within 1-2 days after their emergence. Mating normally occurs in the evenings and in many species is preceded by the formation of swarms of males. Females entering the swarm are apparently recognized by their lower wing-beat frequency. During copulation, a male passes spermatozoa into the female, which in turn passes them to her spermatheca. Usually, these spermatozoa are sufficient for the fertilization of all egg batches laid during the life of the female, although females sometimes mate more than once. Most female anophelines are anautogenous, i.e. a female must obtain a blood meal to provide the proteins and amino acids required for the maturation of the eggs. However, females of a few species are autogenous, i.e. they can mature and lay their first batch of eggs without a blood meal, although blood is essential for all subsequent ovipositions.

After feeding, the swollen abdomen appears bright red and the mosquito is referred to as blood-fed. As digestion occurs, the abdomen darkens and the ovaries enlarge and appear whitish through the abdomen. At the halfway stage, when much of the blood has been digested and ovarian development is half completed, the mosquito is termed half-gravid or semi-gravid. When all blood has been digested and the eggs in the ovaries have matured, much of the dilated abdomen appears whitish and the mosquito is now gravid. After the female lays her eggs, the abdomen is again thin and appears empty and the female is classified as unfed. It is this cycle from unfed to blood-fed to half-gravid to gravid to unfed again that is called the gonotrophic cycle and it is repeated several times (often four to five times) until the female dies.

The duration of the gonotrophic cycle is dependent on temperature and, in the tropics, at temperatures above 23 °C, it usually lasts 2-3 days, but in the colder temperate climates it may take many days or even weeks. Females of some species, including the important African malaria vector A. gambiae, may require two blood meals before the first batch of eggs can develop. Rarely, a species may require three to four blood meals. After her first blood meal, if ovarian development is not complete, the female is referred to as pre-gravid. In subsequent gonotrophic cycles, a batch of eggs is produced after each blood meal. It is possible to estimate the number of gonotrophic cycles from dissection of the ovaries and this technique provides a comparative measure of the survival of mosquito populations.

Plant sugars are a major energy resource for mosquitoes and both males and females will feed on nectar, sugary exudates from fruit, honeydew and even damaged or intact plant tissues. Although male mosquitoes have a conspicuous proboscis, the mandibles are reduced or absent in many species and the stylets are unable to pierce the skin of vertebrates. Thus, plant juices are the only food resource for males.

 2. Blood feeding behaviour

The females of most species of Anopheles feed on warm-blooded animals, predominantly mammals. Some species prefer to feed on humans and are termed anthropophagic or anthropophilic, whereas others favour animals, such as cattle, and are termed zoophagic or zoophilic. However, these traits are not absolute, for whereas most species may be predominantly anthropophagic or zoophagic, they are not usually exclusively so. Anopheles are attracted to their hosts by a range of stimuli, including exhaled carbon dioxide, lactic acid, other host odours, warmth and moisture. The distance over which a mosquito is attracted varies with species, meteorological conditions and topography, but is usually between 7 m and 20 m.

People are more attractive to mosquitoes than others, but the reasons remain largely unknown, although odour substances from the skin of different humans differ in their attractiveness to Anopheles. Adults are bitten more often than infants and children, perhaps because of their larger size. Having located a suitable host, the female mosquito alights and makes a minute incision in the skin with her mandibles and maxillae, before inserting her labrum. Once this has located and penetrated a capillary vessel, blood feeding commences and is usually completed within 1 minute.

Throughout the penetration and feeding process, the mosquito pumps saliva down the hypopharynx into the wound. This saliva contains a complex mixture of anti-haemostatic and vasodilatory compounds to aid feeding. These salivary constituents also help produce the characteristic skin reactions to mosquito bites.

In anophelines, feeding takes place, almost without exception, between dusk and dawn, but some species may feed during the day in densely shaded woods and forests. Some have early peaks of biting, as with A. albimanus in Central America (19.00-21.00 hours), whereas others, such as A. gambiae in Africa, are late feeders (24.00-03.00 hours). The readiness of mosquitoes to feed at a particular time of night depends to some extent on environmental conditions, such as temperature, wind speed and moonlight, but the times of biting (biting cycles) are determined mainly by in-built circadian rhythms.

Some malaria vectors feed mainly outside and are termed exophagic; other species may feed readily, or even predominantly, inside houses and are called endophagic. After engorging on blood, the female mosquito usually seeks shelter in which to rest and digest her blood meal, and to develop her eggs. Some anophelines rest inside houses, both before and after feeding, and are termed endophilic. Others rest in various outdoor sites, in vegetation, in rodent holes, on earthen banks, between buttress roots of trees, in cracks in the ground, in excavation pits, in granaries or cow sheds, on fencing around cattle enclosures or in culverts, and are termed exophilic. Few Anopheles exhibit exclusively one set of behaviour patterns. For example, although in Africa A. gambiae may be predominantly anthropophagic, endophagic and endophilic, in some areas cattle may be much more numerous than humans and so the mosquitoes will become zoophagic, exophagic and, perhaps, exophilic.

The time and place of biting can be of epidemiological importance and influence control measures. A vector that feeds early in the evenings, outside, can transmit malaria to young children as well as to adults, but one that bites outside later at night when young children are predominantly indoors will be less important in this respect. It is therefore important to study the customs of the people and their sleeping habits as well as the behaviour of the vectors. For example, in the hot, dry seasons, substantial numbers of people may sleep outside and, as a consequence, be more frequently bitten by exophagic vectors. Furthermore, the use of insecticide-impregnated bed-nets indoors will be of little value if the vector bites predominantly outdoors. The resting behaviour of adults is also an important consideration in planning control measures. In many malaria control operations, the interior surfaces of houses, such as walls and ceilings, are sprayed with a residual insecticide to kill mosquitoes resting on them. However, this strategy would not be effective in killing the vectors if they were predominantly exophilic.

The quantity of blood ingested at a single feed depends largely on the mosquito’s size, but ranges from about 1 mg to 2.5 mg, which approximates to the weight of the unfed female. However, anophelines are able to increase the volume of blood they take in through diuresis. Thus, the blood ingested is concentrated by the discharge of a few drops of clear liquid from the anus. In nature, the volumes of blood ingested may be markedly limited by the defensive behaviour of the host.

The blood ingested by female mosquitoes is primarily used for egg production, but it may also be used as an energy resource. Nectar meals are less frequently sought when a mosquito is digesting its blood meal. Typically, each blood meal leads to the development of a batch of eggs. The process of blood feeding followed by full development of the ovaries is termed gonotrophic concordance, to distinguish it from gonotrophic dissociation. This latter term is applied when the ovaries fail to mature after a blood meal, such as in A. atroparvus of Europe, which hibernate during the cooler months, a strategy found mainly in temperate species. Another condition, known as gonotrophic discordance, describes the situation in which blood feeding leads to the maturation of eggs but the gravid female delays oviposition, with or without subsequent re-feeding. This condition is seen in those tropical species that undergo a process of aestivation during periods of drought, such as A. arabiensis in the northern Sudan, or A. culicifacies and A. stephensi in the Indian subcontinent.

 3. Oviposition

 Depending on the species, and on the quality and size of the blood meal, a female Anopheles lays 50-200 eggs during a single oviposition, usually at night. Successive egg batches tend to decrease in size and there may be seasonal variations in the numbers of eggs laid. Anopheles eggs cannot withstand desiccation and usually hatch in 2-3 days, but in some species eggs remain alive for 16 days or longer on wet mud, and then hatch within seconds when flooded.

 4. Adult Longevity

The survival and longevity of an adult female depend to some extent on environmental factors such as temperature and relative humidity, but also vary among species. Although innate mortality rises with age, predators and disease are probably the most important causes of death. When the mean temperature exceeds 35 °C, or the humidity is less than 50 per cent, the longevity of Anopheles is drastically reduced, unless the adults can find a more favorable microclimate within their resting sites. The average duration of life of a female Anopheles in the tropics is about 10-14 days. Occasionally, the average life will be nearer 3 weeks and, in temperate regions, anopheline adults may live many weeks, or even months in species that hibernate during the winter. Males live for a shorter time than females.

Adult longevity is of paramount importance in malaria epidemiology. For example, if the mean daily survival fraction of a vector population is 0.65, then 0.65¹⁰ (only around 1 per cent) will survive the 10 days needed for the development of P. falciparurn to the infective sporozoites in the salivary glands.

 5. Flight Range

 Anopheles adults are not usually found more than 2-3 km from their breeding sites, at least in large numbers. Distances flown are largely determined by the environment: if suitable larval habitats and hosts are nearby, females have little need to fly far. However, strong winds may carry Anopheles up to 30 km more. In Egypt, A. pharoensis has been found 72 km from the nearest breeding place, and swarms A. pulcherrimus have invaded a ship 25 km off the Arabian coast.

 Adults may also be transported long distances by hitching rides on aeroplanes, ships, trains or vehicles. The carriage of exotic, malaria-infected Anopheles by aircraft to temperate areas has been responsible for so-called airport malaria in several European countries. Generally, the control of anophelines within a radius of 2 km from airports, docks and other transportation systems where immigrant mosquitoes may be resting or breeding should provide adequate protection against their further dispersal.

For the study of anopheline dispersal or flight range, adult mosquitoes may be marked with different-coloured paints or fluorescent powders, or made radioactive by dosing larval rearing waters with radionuclides of phosphorus or strontium, and then released. Recaptured marked adults can be visibly recognized by their coloured marks, and radioactive ones detected by    Geiger-Muller or scintillation counters, or by their ‘fogging’ of photographic film.

 6. Seasonal Fluctuations In Population Size

Seasonal fluctuations in environmental factors such as rainfall, temperature and humidity affect the survival rate of anophelines and their population size. In most tropical countries, breeding continues throughout the year, albeit at a greatly reduced rate in the dry season due to the paucity of larval habitats. The onset of the rains or monsoon season usually creates a proliferation of potential breeding places and the numbers of adult Anopheles can increase explosively. Large populations are usually maintained throughout the rains and for some weeks afterwards, then anopheline numbers gradually decrease. In contrast, for species breeding in the margins of streams, population sizes may decrease with the onset of the main rains.

Atypical weather can have dire consequences. For example, excessive rainfall and high temperatures in Ethiopia in 1958 resulted in exceptionally large numbers of A. arabiensis, followed by a disastrous malaria epidemic involving an estimated 3 million cases and 150,000 deaths. In contrast, a severe drought in south-western Sri-Lanka in 1934-5 created numerous pools in partially dried streams that were colonized by A. culicifacies. Their excessive numbers resulted in a severe malaria epidemic, causing about 80,000 deaths among 2-3 million cases. The El Nino southern oscillation has recently been shown to be associated with epidemic malaria in Africa.

In hot regions, during the dry season when larval habitats are scarce, females of some species may seek shelter in cool, damp places, such as on the walls of wells. Although continuing to blood feed, they do not lay eggs until the beginning of the rains, although fully developed eggs may be present in their ovaries. Such seasonal behaviour is termed aestivation. Anopheles arabiensis is thought to survive the dry season in Khartoum and Omdurman in the Sudan by aestivating.

In temperate areas, but also in some hot countries that experience seasonal cool periods, females may seek shelter from the cold in hibernation sites, such as caves, buildings or rodent burrows. Before hibernation, females of some species may undertake a pre-hibernation flight, which is longer than their normal ones. In Israel, 14-km pre-hibernation flights have been recorded in   A. sacharovi and, in California; A. freeborni may fly up to 42 km to seek out hibernation sites. Before hibernation, a last blood meal is taken from which abdominal fat reserves, not eggs, are formed. If there is complete hibernation, females remain inactive in their sheltered sites until warmer weather returns. However, in some species there is only partial hibernation and females need to emerge periodically from their shelters to take blood meals to renew their fat reserves. Such incomplete hibernation occurs in A. sacharovi at the beginning of hibernation in Israel, and in A. atroparvus, feeding of which in the winter has resulted n cases of malaria in the Netherlands, Germany and England. Only fertilized females successfully hibernate. In some species, such as the European A. claviger and A. plumbeus, adults die with the onset of the cold season and the population overwinters as larvae.

 LARVAL AND PUPAL BIOLOGY

 1. The duration of the larval and Pupal period

The duration of the larval period depends mainly on temperature and, in the hot tropics, may last some 7-11 days. In colder seasons and in the temperate regions, the larval period may extend to several weeks or months. The pupal stage typically lasts 2-4 days, although in colder climates this may extend to a week or more. The total duration of the aquatic cycle from egg to adult emergence varies from 7 days at 31 °C to 20 days at 20 °C.

 2. Larval Habitats

There is a great diversity in the types of water utilized by different anopheline species. Some larval habitats may be temporary, such as small ponds, pools and puddles, others may be more permanent, such as marshes and borrow pits (depressions created by excavating soil). Although most aquatic habitats are freshwater, some Anopheles species breed in saline waters. Most anophelines avoid organically polluted waters, such as those contaminated with human or animal faeces or rotting vegetation. Many species have a relatively narrow range of habitats, with some species preferring those fully exposed to the sun, whereas others are more or less restricted to shade.

Many anophelines are found in freshwater ponds and small collections of water such as pools and puddles that lack vegetation (e.g. A. gambiae in Africa and A. stephensi in India). Others are found in marshes or large ponds with floating or emergent vegetation (e.g. A. nili and A. funestus of Africa and A. albimanus in Central America). Mosquitoes are not found in fast-flowing streams, but a few anophelines, such as A. pseudopunictipennis in South America, A. superpictus in Europe, A. minimus in Asia and A. maculatus in Malaysia, breed in the shallow, relatively still waters at the edges of streams or in springs and seepages.

A few species (e.g. A. plumbeus in Europe) prefer rain-filled tree holes. Certain species of the subgenus Kerteszia (e.g. A. bellator and A.cruzii in Central and South America) select water-containing leaf axils of bromeliad plants growing on trees. Several species are found mainly in saline waters, including A. atroparvus in Europe, A. aquasalis in Latin America, A. sundaicus and A. litoralis in Southeast Asia, and A. melas and A. merus in West and East Africa. Artificial containers, such as water pots, are generally unsuitable for most anopheline species, although A. stephensi in India is often found in water tanks and cisterns sited on roofs of buildings, as well as in tin cans, pots and wells. For some species, larvae occur in a great diversity of habitats. In Africa, the larvae of A. gambiae can he found in roadside pools, small puddles and hoof prints as well as in borrow pits and rice fields and, very occasionally in water-filled village pots.

Man-made malaria is a term applied to many human activities that provide habitats suitable for malaria vectors, so favouring increased incidence of disease. Road building and track laying for trains, for example, create puddles and ditches, and gem mining results in pits that fill with rainwater. Land under agricultural irrigation, such as flooded rice fields, is colonized by several important malaria vectors, including A. gambiae and A. arabiensis in Africa,  A. culicifacies and A. subpictus in the Indian subcontinent, A. sinensis in China, A. aconitus in much of South-east Asia, A. darlingi in Central America and A. freeborni in the USA. Breeding in rice fields can result in increased intensity of malaria transmission and extend transmission into the dry season.

SPECIES COMPLEXES

Many species of anopheline mosquito can be reliably identified by their morphology, but an increasing number of species defined on morphological criteria are found on closer inspection to consist of several, reproductively isolated entities that constitute good biological species. Such sibling species, despite their apparent similarity, may differ in important biological characteristics that influence their role in the transmission of malaria. A group of several such species, with a common line of descent, comprise a species complex. The discovery over 50 years ago that the European malaria vector A. maculipennis consisted of several biologically distinct species helped explain the puzzle of anophelism without malaria, and was a landmark in the study of malaria vectors.

The recognition of sibling species complexes is important because the constituent members may differ in their host preference, breeding sites, preferred sites for resting or feeding and even their capacity to transmit malaria. The A. gambiae complex, which includes the most important malaria vectors in Africa, has been studied more than any other vector species complex and well illustrates the practical importance of distinguishing sibling species. The complex consists of seven described species, six of them formally named, although it is believed that additional cryptic species occur in the savannah regions of West Africa. The two most important species in the complex are A. gambiae and A. arabiensis. The former predominates in humid areas, prefers feeding on humans and resting indoors and is a highly efficient malaria vector. In contrast, A. arabiensis, although found in the humid zones, is more tolerant of the drier savannah regions. Whereas it readily bites humans and may rest indoors> it often feeds on cattle and rests Outdoors, particularly in man-made structures. It, too, is an efficient vector> second only in importance to A. gambiae. Both these species are widespread over much of Africa, whereas A. quadriannulatus (now recognized as comprising two species, A and B) is restricted to north-eastern and southern Africa. In southern Africa, it is considered to be both exophilic and wholly zoophagic, and consequently not a vector.

 The two brackish-water, and mainly coastal, .species A. melas of West Africa and A. merus of East and southern Africa are more zoophagic and exophilic than A. gambiae and malaria vectors of lesser importance. The remaining species, A. bwambae, is known only from the Semliki forest on the Rift Valley escarpment in western Uganda, where it breeds in seepage water from geothermal springs. It is therefore a very localized and minor vector. In many parts of Africa, mosquitoes of the complex occur in mixed, sympatric populations, comprising two or even three species. Although the reproductive barriers between the species are strong, occasionally hybridization does occur where species occur in sympatry, and this leads to introgression, a limited exchange of genes.

The techniques used to recognize the existence of sibling species are diverse. The cross-mating of forms suspected to be separate species was the first method employed with malaria vectors and provides a sound basis for recognizing good biological species. Studies of the giant polytene chromosomes found in the larval salivary glands and ovarian nurse cells of the adult female often reveal species-specific karyotypes and this method has been widely used. A limitation is that adult females must be half-gravid for their chromosomes to be useful. Increasingly, molecular techniques are being applied and, in some cases, these have led to polymerase chain reaction (PCR)-based methods for identification. These can be readily used with dry specimens, even quite old museum specimens, as well as specimens preserved by other means. Because this is a rapidly advancing field of study, any attempt at a comprehensive summary of anopheline sibling species is likely to be quickly out of date. Table 4.1 illustrates selected examples from major regions of endemic malaria. The estimated number of species within a complex is provisional arid likely to be an underestimate. Furthermore, not all authorities define the boundaries to the complexes in the same manner.
Using a PCR reaction based on species differences in ribosomal DNA sequence. DNA extracted from individual dry mosquitoes is amplified with a cocktail of two species-specific primers and one primer homologous to both species. The PCR products are then run in agarose gels along with molecular weight markers, to reveal a DNA product with a species-characteristic number of base-pairs.

Examples of anopheline sibling species from major regions of endemic malaria