LITERATURE REVIEW

Overview of Mosquito Species

There are some 3300 species of mosquitoes belonging to 41 genera, all belongingin the family Culicidae. This family is divided into three subfamilies: Toxorhynchitinae, Anophelinae (anophelines) and Culicinae (culicines). Mosquitoes have a worldwide distribution. They occur throughout thetropical and temperate regions and extend their range northwards intothe Arctic Circle. The only areas from which they are absent are Antarctica,and a few islands. They are found at elevations of 5500mand down minesat depths of 1250m below sea level.The most important pest and vector species belong to the genera Anopheles,Culex, Aedes, Mansonia, Ochlerotatus, Psorophora,Haemagogus and Sabethes.Anopheles species, as well as transmitting malaria, are vectors of filariasis(caused by Wuchereria bancrofti, Brugia malayi and Brugia timori) and a fewarboviruses. Certain Culex species transmit W. bancrofti and a varietyof arboviruses. Aedes species are important vectors of yellow fever,dengue, encephalitis viruses and many other arboviruses, and in a fewrestricted areas they are also vectors of

W. bancrofti and B. malayi. Species in the very closely related genus Ochlerotatus also transmit filariasis and encephalitis viruses. Mansonia species transmit B. malayi an d sometimes W. bancrofti and a few arboviruses. Haemagogus and Sabethes mosquitoe s are vectors of yellow fever and a few other arboviruses in Central and South America, while the genus Psorophora contains some troublesome pest species in North and Sout h Americaas well as a few transmitting arboviruses (Service, 1997).

External morphology of mosquitoes

Mosquitoes possess only one pair of functional wings, the fore-wings.The hind-wings are represented by a pair of small, knob-like halteres.Mosquitoes are distinguished from

other flies of a somewhat similar shapeand size by: (1) the possession of a conspicuous forward projecting proboscis;(2) the presence of numerous appressed scales on the thorax, legs,abdomen and wing veins; and (3) a fringe of scales along the posteriormargin of the wings.Mosquitoes are slender and relatively small insects, usually measuringabout 3–6 mm in length. Some species, however, can be as small as 2 mmwhile others may be as long as 19 mm. The body is distinctly divided intoa head, thorax and abdomen.The head has a conspicuous pair of kidney-shaped compound eyes.Between the eyes arises a pair of filamentous and segmented antennae. Infemales, the antennae have whorls of short hairs (that is pilose antennae),but in males, with a few exceptions in genera of no medical importance,the antennae have many long hairs giving them a feathery or plumoseappearance. Mosquitoes can thus be conveniently sexed by examinationof the antennae: individuals with feathery antennae are males, whereasthose with only short and rather inconspicuous antennal hairs are females (Service, 1997).

Just below the antennae is a pair of palps which maybe longor short and dilated or pointed at their tips, depending on the sex of theadults and whether they are anophelines or culicines. Arisingbetween the palps is the single elogated proboscis, which contains the piercing mouthparts of the mosquito. In mosquitoes the proboscis ch aracteristically projects forwards. The thorax is covered, dorsally and laterally, with scales which may bedull or shiny, white, brown, black or almost any colour. It is the arrangementof black and white, or coloured scales on the dorsal surface of thethorax that gives many species (especially those of the genera Aedes andOchlerotatus) distinctive patterns.The wings of mosquitoes are long and relatively narrow, and the number and arrangementof wing veins is virtually the same for all mosquito species.

The veins are covered with scales which are usually brown, black, white or creamy yell

ow, but more brightly coloured scales may occasionally be present. The shape of the sc ales and the pattern they form differconsiderably between both genera and species of mosquitoes. Scales alsoproject as a fringe along the posterior border of the wings. In life the wingsof resting mosquitoes are placed across each other over the abdomen inthe fashion of a closed pair of scissors. The legs of mosquitoes are longand slender and are covered with scales which are usually brown, blackor white and may be arranged in patterns, often in the form of rings (Service, 1997).

Life cycle

Blood-feeding and the gonotrophic cycle

Most mosquitoes mate shortly after emergence from the pupa. After mating,sperm passed by the male into the female enters her spermatheca.This sperm, in the spermatheca, usually serves to fertilize all eggs laidduring her lifetime; thus only one mating and insemination per female isrequired. With a few exceptions, a female mosquito must bite a host andtake a blood-meal to obtain the necessary nutrients for the development ofthe eggs in the ovaries. This is the normal procedure and is referred to asanautogenous development. A few species, however, can develop the firstbatch of eggs without a blood-meal, and more rarely subsequent batches. This process is called autogenous development. The speed of digestion ofthe blood-meal depends on temperature. In most tropical species it takesonly 2–3 days, but in colder, temperate countries blood digestion may takeas long as 7–14 days (Service, 1997).

Each gonotrophic cycle begins with an unfed adult which passes through blood-fed, half-gravid and gravid conditions. After oviposition the female is again unfed and seeks another blood-meal. After a blood-meal the mosquito‟s abdomen is dilated and bright red incolour, but some hours later the abdomen becomes a much darker red. Asthe

blood is digested and the white eggs in the ovaries enlarge, the abdomenbecomes whitish posteriorly and dark reddish anteriorly. This conditionrepresents a mid-point in blood digestion and ovarian development, andthe mosquito is referred to as being half- gravid. Eventually allblood is digested and the abdomen becomes dilated and whitish due tothe formation of fully developed eggs. The female is now saidto be gravid and she searches for suitable larval habitats in which to layher eggs. After oviposition the female mosquito takes another blood-mealand after 2–3 days (in the tropics) a further batch of eggs is matured. Thisprocess of blood-feeding and egg maturation, followed by oviposition, isrepeated several times throughout the female‟s life and is referred to as thegonotrophic cycle.Male mosquitoes cannot bite but instead feed on the nectar of flowersand other naturally occurring sugary secretions. Males are consequentlyunable to transmit any disease. Sugar feeding is not, however, restrictedto males; females may also feed on sugary substances to obtain energy forflight and dispersal, but only in a few species (the autogenous ones) is thistype of food sufficient for egg development (Service, 1997).

Biology of mosquito eggs

Depending on the species, female mosquitoes lay about 30–300 eggs at anyone oviposition. Eggs are brown or blackish and 1 mmor less long. In manyCulicinae, eggs are elongate or approximately ovoid in shape, but eggs ofMansonia are drawn out into a terminal filament. In the Anophelinae,eggs are usually boat-shaped. Many mosquitoes, such asspecies of Anopheles and Culex, lay their eggs directly on the water surface (Figure 2.1).In Anopheles the eggs are laid singly and float on the water, whereas thoseof Culex are laid vertically in several rows held together by surface tensionto form an egg raft which floats on the water(Service, 1997).Mansonia

specieslay their eggs in a sticky mass that is glued to the underside of floatingplants.

None of the eggs of these mosquitoes can survive desiccation andconsequently they die if they become dry (Figure 2.2). In the tropics, eggs hatch within2–3 days, but in cooler temperate countries they may not hatch until after7–14 days, or longer.Other mosquitoes, such as those belonging to the genera Aedes, Ochlerotatus,Psorophora and Haemagogus, do not lay eggs on the water surface butdeposit them just above the water line on damp substrates, such as mudand leaf litter, or on the inside walls of tree-holes and clay water-storagepots. Eggs of these genera can withstand desiccation, especially those ofAedes, Ochlerotatus and Psorophora which can remain dry for months oreven years but still remain viable and hatch when soaked in water (Figure 2.3). Becausesuch eggs are laid above the water line of breeding places, it may be manyweeks or months before they become flooded with water, and thus have theopportunity to hatch (Macdonald, 1960).

Figure 2.1:Generalized life cycle of mosquitoes (OME, 2008).

Figure 2.2:Life cycle of direct-hatching mosquitoes whereeggs are laid on top of shallow standing water and hatch within 2-3 days (Darsie and Ward,2005).

Figure 2.3:Life cycle of delayed-hatching mosquitoes where eggs are laid on moist substrates in sites where standing water existed previously (Darsie and Ward,2005).

Larval biology

Mosquito larvae can be distinguished from other aquatic insects by beinglegless and having a bulbous thorax that is wider than both the head and theabdomen. There are four active larval instars. All mosquito larvae requirewater in which to develop; no mosquito has larvae that can withstand desiccationalthough they may be able to survive short periods, for example,in wet mud.Mosquito larvae have a well-developed head, bearing a pair of antennaeand a pair of compound eyes. Prominent mouthbrushes are presentin most species and serve to sweep water containing minute food particlesinto the mouth. The thorax is roundish in outline and has varioussimple and branched hairs which are usually long and conspicuous. The10-segmented abdomen has nine visible segments, most of which havesimple or branched hairs. The last segment, which differsin shape from the preceding eight segments, has two paired groups of longhairs forming the caudal setae, and a larger fan-like group comprising theventral brush. This last segment ends in two pairs of transparent,sausage-shaped anal papillae, which although often called gills arenot concerned with respiration but with osmoregulation (Service, 1997).Mosquito larvae, with the exception of Mansonia and Coquillettidiaspecies (and a few other species), must come to the water surface to breathe. Atmospheric air is taken in through a pair of spiracles situated dorsally on the t enth abdominal segment. In the subfamilies Toxorhynchitinae andCulicinae these spiracles are situated at the end of a single dark colouredand heavily sclerotized tube termed the siphon. Mansonia andCoquillettidia larvae possess a specialized siphon that is more or less conical,pointed at the tip and supplied with prehensile hairs and serrated cutting structures. These enable the siphon to be inserted into the roots orstems of aquatic plants and thus oxygen for larval respiration is obtainedfrom the plants (Service, 1997). In contrast, larvae of the Anophelinae do not have siphon.

Mosquito larvae feed on yeasts, bacteria, protozoans and numerousother micro organisms, as well as on decaying plant and animal material found in the water. Some, such as Anopheles species, are surfacefeeders,whereas many others browse over the bottoms of habitats. A fewmosquitoes are carnivorous or cannibalistic. There are four larval instarsand in tropical countries larval development, that is the time from egghatching to pupation, can be as short as 5–7 days, but many species requireabout 7– 14 days. In temperate areas the larval period may last severalweeks or months, and several species overwinter as larvae (Service, 1997).

Larval habitats

Mosquito larval habitats vary from large and usually permanent collectionsof water, such as freshwater swamps, marshes, ricefields and borrowpits, to smaller collections of temporary water such as pools, puddles,water-filled car tracks, ditches, drains and gulleys(Clements, 1963). A variety of „naturalcontainer-habitats‟ also provide breeding places, such as water-filled treeholes,rock pools, water-filled bamboo stumps, bromeliads, pitcher plants,leaf axils in banana, pineapple and other plants, water-filled split coconuthusks and snail shells. Larvae also occur in wells and „man-made containerhabitats‟,such as clay pots, water storage jars, tin cans, discarded kitchenutensils and motor vehicle tyres (Clements, 1963). Some species prefer shaded larval habitatswhereas others like sunlit habitats. Many species cannot survive in waterpolluted with organic debris whereas others can breed prolifically in watercontaminated with excreta or rotting vegetation.A few mosquitoes breedalmost exclusively in brackish or salt waters, such as saltwater marshes andmangrove swamps, and are consequently restricted to mostly coastal areas.Some species are less specific in their requirements and can tolerate a widerange of different types of breeding

places.Almost any collection of permanent or temporary water can constitute

amosquito larval habitat, but larvae are usually absent from large expansesof uninterrupted water such as lakes, especially if they have large numbersof fish and other predators. They are also usually absent from large riversand fast-flowing waters, except that they may occur in marshy areas andisolated pools and puddles formed at the edges of flowing water(Clements, 1963).

Larval distribution in nature

A number of ecologist have analysed the water in which mosquito larvae are found and from which they are absent and have concluded that certain solutes are harmful to the larvae, preventing the breeding of mosquitoes where they occur. Harmful properties have been ascribed to ammonium salts, nitrites, nitrates and protein, but these results have generally been contradicted by other workers.The distribution of larvae in natural waters of varying pH shows that many species are able to live under both alkaline and acidic conditions. Anopheles culicifacies, for example, has been recorded in the pH range of 5.4-9.8 and the tree-hole species An. plumbens and Ae. geniculatus from pH

4.4 to pH 9.3. Aedes flavopictus larvae develop in the laboratory in media ranging from pH 2 to 9 and Armigeres subalbatus larvae in media ranging from pH 2 to 10. In the laboratory acid conditions are sometimes unfavourable to larvae but this may be due to the bacteria which develop under acid conditions or to the acid present, hydrochloric acid, for example, being more toxic than phosphoric acid.Observations such as these and the knowledge that female mosquitoes are highly selective in nature in choice of oviposition site have prompted the suggestion that the distribution of larvae is controlled not by survival in suitable and extinction in unsuitable habitats but by the discrimination of the ovipositing females. This view is now generally accepted but unfortunately the means by which gravid female select the oviposition sites are very

incompletely known. Although most mosquitoes are restricted to freshwater, a number

of species can develop in extremely high concentrations of salts. Larvae of Ae. natronius have been found in a crater lake in Uganda which had a salinity equivalent to

3.9 percent NaCL (0.67 M), an alkalinity of 0.7 N and a pH exceeding 10.5 (Clement, 1963).The larvae of brackish-water species are able to develop in the concentrated sea water which occurs in littoral pools. Aedes australis will develop in sea water containing 7.4 percent salts and Ae.detritus has been found in sea water containing 10 per cent salts. Certain fresh-water species can tolerate a fairly high salinity. Anopheles superpictus, for example, will develop normally in 30 per cent sea water. Larvae of Ae. aegypti reared in fresh water are killed by 0.19 M-NaCL (1.1 per cent NaCL) or by sea water isosmostic with 0.22-0.24 M-NaCL, but by gradually increasing the concentration the larvae can be adapted to 0.19 M-NaCL and to 50 per cent sea water (isosmotic with 0.3 M or 1.75 per cent NaCL). The restriction of species to particular larval habitats is probably due to discrimination by ovipositing females, an activity which will be affected in the long term by selective pressures operating during the larval stage as well as by those effective at oviposition (Clements, 1963).

Effect of temperature on mosquito larvae

The rate of postembryonic growth follows the familiar S-shaped curve with temperature. Below a developmental temperature threshold no growth occurs; above the threshold the rate of growth increases with increasing temperature, reaching a maximum at the so-called „optimum temperature‟ above which it declines. In Ae.aegypti, some growth takes place at 10°C but development is not completed. Above 14°C growth becomes increasingly rapid with rising temperature, reaching the

„optimum‟ at 32°C. The rate of growth decreases at still higher temperatures and above

36°C development is not completed. The temperature at which development proceeds fastest varies at different stages of development. When An. quadrimaculatus was reared

at constant temperatures throughout the life cycle, development of the egg was found to be most rapid at 33.3°C; of first instar larvae at 32.5°C; of fourth instar larvae at 30°C and of pupae at 30.5°C. Development of the larval and pupal stages considered as a whole was most rapid at 31°C. Similar variations were found in Ae.aegypti. The apparent drop in optimum temperature was possibly due to the cumulative injurious effects of high temperature (Clements, 1963).

When the immature of mosquito stages were subjected to fluctuating temperatures, development was faster than at constant temperature; equal to the mean of the fluctuating temperatures. When larvae and pupae of An. quadrimaculatus were exposed to a raised temperature for 9 hours each day, the velocity of development was accelerated by 13 per cent when the lower temperature was 19°C and by 4 percent when the lower temperature was 23°C, compared to the velocity of development at a constant temperature equal to the mean of the fluctuating temperatures. Variable temperature has been shown to accelerate development in Ae.aegypti also, but only over the middle portion of the curve relating developmental rates and temperature. At higher temperatures a fluctuating temperature does not increase the rate of development but at lower temperatures it slows development down. The accelerating effect has been explained by the assumption that the maximum rate of growth at a high temperature can only be sustained for a short time after which it declines, whereas too short a period at the lower temperature does not allow recovery from the deleterious effects of the higher temperature (Clements, 1963).

Pupal biology

All mosquito pupae are aquatic and comma-shaped. The head and thoraxare combined to form the cephalothorax, which has a pair of respiratory trumpets dorsally. The

abdomen is 10-segmented although onlyeight segments are visible. Each segment has numerous short hairs and thelast segment terminates in a pair of oval and flattened structure termed paddles. Some of the developing structures of the adultmosquito can be seen through the integument of the cephalothorax; themost conspicuous features being a pair of dark compound eyes, foldedwings, legs and the proboscis.Pupae do not feed but spend most of their time at the water surfacetaking in air through the respiratory trumpets. If disturbed, they swim upand down in the water in a jerky fashion.Pupae of Mansonia and Coquillettidia differ in that they have relativelylong breathing trumpets, which are modified to enable them to pierceaquatic vegetation and obtain their oxygen in a fashion similar to the larvae. As a consequence their pupae remain submerged and rarely cometo the water surface (Service, 1997).

Moulting, pupation and emergence

Without any known exception, mosquitoes pass through four larval instars. At moulting, the epicuticle can be seen to separate from the cuticle and to secrete new cuticle and bristle beneath it. The tracheal epithelium similarly retracts from the tracheal linings and secretes new cuticular linings around them so that a wide space filled with fluid can be seen between the two.When the larva of Ae.aegypti is about to shed its skin, it rest at the surface and swallows water by contractions of the pharynx until the soft body wall becomes extremely taut. From time to time it flexes its head sharply until finally the head capsule splits. The ecdysial line along which the capsule splits follows the epicranial suture, starting in the dorsal midline of the collar and then diverging widely and passing forward on each side of the dorsum of the head to the bases of the antennae. Meanwhile, the old tracheal trunk have broken across just behind their connections to the non-functional spiracles in each segment and as the larva leaves

its old skin the fractured pieces of the tracheal system are withdrawn, the fragments in

each case passing out through the pair of spiracle immediately behind them. All the spiracles except the terminal pair are then sealed until the next ecdysis (Clement, 1963).During the process of pupation, the fourth instar larva sheds its skin while the respiratory trumpets and float hairs of the pupa appear as dark bodies beneath the cuticle. The larva comes to lie horizontally at the surface and shortly afterwards air appears in the short tracheae linking the respiratory trumpets to the longitudinal tracheal trunks, subsequently spreading between the larval and pupal cuticles.The larval cuticle and tracheal linings are shed much as at previous ecdyses and during this period the labial and maxillary-palp buds are extruded from their peripodial cavities (Clement, 1963).After pupation, practically the whole cuticle hardens and darkens, in contrast to the small area of cuticle which is sclerotized in the larva. The pupa usuallyrests at the surface, held in position against the surface film by the tips of the trumpets and the float hairs on the first abdominal segment, but it can swim well and when disturbed swims downwards by the beating of the abdomen with its large paddles, later floating to the surface again.Some mosquitoes show a strong tendency to pupate at a particular time of day.

The first sign of emergence is the appearance of a small amount of air beneath the pupal cuticle, at the bases of the respiratory siphons and elsewhere. The abdomen is slowly raised to a horizontal position and shortly after this the pupal cuticle splits along the mid-dorsal line of the thorax. A minute later the thorax of the adult, which is quite dry, protrudes through the split and slowly the whole body of the adult rises into view. It is most probable that the air which appears between the pupal and adult cuticles before the pupal cuticle splits penetrates through the respiratory trumpets when the tracheae from these organs fracture, for pressure on the pupal cuticle causes the bubbles

to escape through the trumpets(Clement, 1963).