CHAPTER II MOSQUITO FAUNAL DIVERSITY

15 CHAPTER II MOSQUITO FAUNAL DIVERSITY

16 CONTENT Chapter II. MOSQUITO FAUNAL DIVERSITY 2.1 INTRODUCTION 17-29 2.1.1 Classification and Taxonomy 17 2.1.2 General Morphology of Adult Mosquito 19 2.1.3 General Biology of Mosquitoes 26 2.1.4 Breeding Ecology of Mosquitoes 28 2.2 REVIEW OF LITERATURE 30-38 2.2.1 Diversity studies 30 2.2.2 Mosquito studies in Kerala 34 2.2.3 Objectives 38 2.3 MATERIALS AND METHODS 39-47 2.3.1 Design of study 39 2.3.2 Study area 39 2.3.3 Selection of Study sites 43 2.3.4 Sites selected for sampling 45 2.3.5 Mosquito collection method 46 2.3.6 Analysis of Data 47 2.4 OBSERVATIONS 48-111 2.4.1 Diversity and Composition of Mosquito Fauna 48 2.4.2 Medically important vector mosquitoes 98 2.4.3 Seasonal distribution of mosquito fauna in the study area 98 2.4.4 Spatial distribution of mosquito fauna 106 2.4.5 Altitudinal distribution of mosquitoes 107 2.4.6 Breeding ecology of mosquitoes 109 2.5 DISCUSION AND CONCLUSION 112-123 2.5.1 Diversity and Composition of 112 2.5.2 Medical importance of mosquitoes collected 115 2.5.3 Seasonal Distribution 117 2.5.4 Spatial distribution 119 2.5.5 Altitudinal distribution 119 2.5.6 Breeding ecology of mosquitoes 121 2.5.7 Conclusion 123

17 2.1 INTRODUCTION For variety and abundance, insects rank among the most successful animals on earth. The taxonomic investigations of both fossil and living species show that insects are diversified groups and have outnumbered all other organisms (Labanderia and Sepkoski, 1993; Ananthakrishnan, 1994). Among the various insect groups, mosquitoes always get special attention as vector and nuisance pests. Mosquito-borne diseases are among the world's leading causes of illness and death. Despite great strides over the last 50 years, the World Health Organization estimates that more than 300 million clinical cases of mosquito-borne illnesses occur each year (Kalluri et al., 2007). The epoch-making discoveries in mosquito life histories and the part played by the adults as disease carriers, have given a new impetus to the study of mosquito. 2.1.1 Classification and Taxonomy Mosquitoes are found throughout the world except in places that are permanently frozen. Over 3150 species of mosquitoes have been reported globally (Service, 2004; Walker, 1994; Knight and Stone, 1977). All mosquitoes belong to the Class: Insecta, Order: Diptera and Suborder: Nematocera. Insects are mainly characterized by three pairs of legs and three body regions-head, thorax and abdomen. Diptera are readily separated from all other insects in that the hind wing is modified into club shaped halteres. Suborder: Nematocera is characterised by delicate body, long and filamentous antennae, and elongate and flexible maxillary palpi. All mosquitoes are placed in a single family, Culicidae. They are characterized by a

18 slender body, long and slender proboscis, presence of scales on the body, legs and wings. (Roy and Brown, 1970). The family is divided into three subfamilies- Anophelinae, Culicinae, and Toxorhynchitinae (Knight and Stone, 1977). Adult Anophelines are readily recognized by their resting or standing posture. Most adults of subfamily Anophelinae stand with the body inclined at an angle of 30-45 to the surface. They have dark and pale spots of scales on the veins of the wings. Some species have the wing veins entirely covered with dark scales. The maxillary palpi of both sexes are about as long as the proboscis. The palpi of some species have semi-erect scales that give them a rather shaggy appearance. The scutellum is evenly rounded in most Anophelines. The abdominal sterna, and usually the terga, are completely or nearly devoid of scales. Subfamily Anophelinae include three genera such as Bironella, Chagasia and Anopheles. Of the three genera, only the genes Anopheles is of medical importance. There are all about 365 recognised species of Anopheles in the world and classified under six subgenera (Knight and Stone, 1977; Rao, 1984). Members of the subfamily Culicinae have maxillary palpi that are usually much shorter than the proboscis. The scutellum has three lobes with setae confined to each lobe. The wing veins are usually entirely dark-scaled, but speckles or patches of white or yellow scales are present in some species. The abdominal terga and sterna are densely covered with scales. Adult culicines stand with the body parallel to the surface on which they are resting. Subfamily Culicinae includes 34 genera and about 2700 species (Knight and Stone, 1977).

19 Mosquitoes of Subfamily Toxorhynchitinae are characterised by the long and curved proboscis. Adults are large; usually brightly coloured with green, yellow, purple and white scales which have a metallic lustre. Subfamily Toxorhynchitinae contains only one genus, Toxorhynchites. The genus contains about 65 species (Knight and Stone, 1977) 2.1.2 General Morphology of Adult Mosquito Mosquitoes are slender, small to large sized Dipteran fly. Mosquitoes are distinguished from all other flies of somewhat similar shape and size by the possession of a conspicuous forwardly projecting proboscis, the presence of numerous scales on the thorax, legs, abdomen and wing veins, a fringe of scales along the posterior margin of the wings. The body of mosquito is distinctly divided into a head, thorax and abdomen. a. Head The head is the anterior section of the body bearing the compound eyes, antennae and proboscis. The head is largely composed of a pair of prominent compound eyes. The dorsal surface of the head behind the compound eyes consists of anterior vertex and posterior occiput, and is more or less covered by erect and/or decumbent scales. The erect scales are usually truncate or forked apically and often differ in colour from the decumbent scales. They may be numerous, occurring on the vertex and the occiput, or they may be restricted to the occiput, usually in a transverse row. The decumbent scales may be narrow and curved or broad and flat. A row of anteriorly directed, ocular setae arise along the margin of each eye. One or more pairs of interocular setae located at the junction of the eyes (interocular space) are usually

20 longer than the other ocular setae. The narrow anterior part of the vertex between the eyes bears the frontal tuft characteristic of most Anopheles species. (Roy and Brown, 1970; Service, 2004). The antennae, maxillary palpi and the proboscis arise from a frontal area more or less transcribed by the compound eyes. In females the antennae is pilose type (whorls of short hairs) but in males it is plumose (long hairs). Each antenna is divided into three segments. The basal segment, the scape, is hidden behind the greatly enlarged second segment, the pedicel. The pedicel often bears a group of minute setae or a small patch of scales on its mesal surface. The third segment, the flagellum, is divided into 13 or 14 segments called flagellomeres. The first flagellomere usually also bears a mesal patch of scales. The remaining flagellomeres are usually without scales. In females, the flagellomeres are about equal in size, but in males of most species the apical two flagellomeres are longer. (Roy and Brown, 1970). The maxillary palpi are composed primitively of five segments called palpomeres. In both sexes of Anophelines, and in the males of nearly all other genera, the palpus is elongate and all five palpomeres are distinct. Because of reduction of the apical palpomeres, the palpus of females appears to be composed of two or three palpomeres. The basal half of the first palpomere in both sexes lacks scales and setae. The rest of the palpus is covered with scales, and the three distal palpomeres (3-5) of males usually have setae. (Roy and Brown, 1970). Arising between the palpi is the long, slender, scaled proboscis, which contains the piercing mouthparts of the mosquito. The mouth parts of a mosquito

21 consist of labium, labarum, hypopharynx, paired mandible and maxilla. The largest component of the mouth part is the long and flexible labium which terminates in a pair of small flap like structure called the labella. The labium is seen to almost encircle all the other component of the mouth parts and cover as a protective sheath. It forms the lower floor of proboscis. The labrum is slender, pointed and grooved along its ventral surface. It forms the supper roof of the proboscis. Mandibles and maxilla are paired and needle like with piercing tip. When a female mosquito bites an animal the labella are applied on the skin and the labium bends like an elbow. This allows the paired mandibles, paired maxilla, labrum and hyproharynx to penetrate the host s skin. Although male mosquitoes have a proboscis, maxilla and mandibles are usually reduced in size or the mandibles are absent, so males cannot bite animals; but feeds on nectar and water. (Roy and Brown, 1970; Service, 2004). b. Thorax The thorax is the middle part of the body and comprised of three segments. Most of the thorax belongs to the second segment, the mesothorax. The prothorax and metathorax are much reduced, especially dorsally. The dorsum of the prothorax is represented by laterally displaced halves, which are divided transversely into a lobe-like antepronotum and a flattened postpronotum. Dorsally the mesothorax occupies almost the entire thorax in the form of the large sclerite, the scutum, which is covered with scales and usually bears rows of setae. Those along the midline are called the acrostichal setae while the rows on either side are referred to as the dorsocentral setae. The arrangement of the scales and the patterns they form on the scutum are important in species identification. A small oblong or triangular sclerite

22 called the paratergite lies at the edge of the scutum between the mesothoracic spiracle and the base of the wing. The presence or absence of scales and setae on the paratergite are often important in generic and species identification. Behind the scutum is the crescent-shaped scutellum. The scutellum is usually tri-lobed (except in Anopheles, Bironella and Toxorhynchites), covered with scales and bears groups of setae along the posterior edge. A dome-shaped mesopostnotum lies below and behind the scutellum. This area is usually bare, but scales and/or setae are present in some species and taxa. The dorsum of the metathorax is represented by an inconspicuous sclerite behind the mesopostnotum. (Roy and Brown, 1970). The lateral side of each thoracic segment is known as a pleuron. (In winged insects, a vertical ridge divides the pleuron into an anterior episternum and a posterior epimeron, and each of these may be divided longitudinally into a dorsal anepisternum, a ventral katepisternum and dorsal anepimeron,a ventral katepimeron respectively. In mosquitoes, only the mesopleuron has the structure of a more generalized thoracic segment). The propleuron is represented by the partially fused, V-shaped proepisterna located between the forecoxae and the neck (cervix). Scales and setae borne on the proepisterna are often of taxonomic importance. The upper proepisternal scales and upper proepisternal setae are located above the base of each forecoxa while the lower proepisternal scales and lower proepisternal setae are borne in a mesal position below the neck and between the coxae. The upper and lower patches of scales may be contiguous. The anteprocoxal membrane situated between the forecoxa and the proepisternum and the postprocoxal membrane borne between the forecoxa and the mesothorax may also bear scales. (Roy and Brown, 1970).

23 The mesopleuron is divided into unequal anterior and posterior sclerites by a mesopleural suture. The larger anterior sclerite, the mesepisternum is subdivided by a longitudinal anapleural suture into a dorsal mesanepisternum and a ventral mesokatepisternum. The anepisternum is further divided by a diagonal anepisternal cleft into anterior and posterior sections. The anapleural suture is indistinct in most mosquitoes, except Uranotaenia, so that the posterior section of the anepisternum (prealar area), which bears the prealar setae, appears to be a dorsal extension of the mesokatepisternum. The anterior portion of the anepisternum bears the mesothoracic spiracle. An important group of setae, the prespiracular setae, arise from a small sclerite (prespiracular area) on the anterior side of the spiracle. These setae are usually small and inconspicuous and arise immediately posterior to the postpronotal setae, which can be mistaken for the prespiracular setae. The presence or absence of prespiracular setae in combination with other characters is important in distinguishing mosquito genera. The remainder of the anterior section of the anepisternum is arbitrarily divided into three areas, the hypostigmal area, subspiracular area; and postspiracular area, which may bear taxonomically important setae and/or scales. The mesokatepisternum is well developed and bears two important groups of setae, the upper mesokatepisternal setae and lower mesokatepisternal setae. Scales are also associated with these groups of setae, termed the upper mesokatepisternal scales and lower mesokatepisternal scales by association. The mesepimeron, a rectangular sclerite located behind the mesopleural suture, is also divided transversely. The dorsal mesanepimeron, or simply the mesepimeron, is quite large and bears patches of setae and scales of taxonomic

24 importance, while the ventral mesokatepimeron is an insignificant strip of bare cuticle. A small triangular sclerite known as the mesomeron is located below the mesepimeron and between the mid- and hind coxae. The positional relationship of the mesomeron to the base of the hind coxa is important in the higher classification and generic recognition of mosquitoes. The metathorax has limited value in mosquito taxonomy (Roy and Brown, 1970). Each thoracic segment bears a pair of long, slender legs, which are almost entirely clothed in scales. Individual legs and leg segments are denoted by prefixing fore-, mid- or hind- as appropriate. Each leg is composed of six segments, the coxa, trochanter, femur, tibia, tarsus and post-tarsus. The tarsus consists of five false segments termed tarsomeres. The terminal post-tarsus bears a pair of ungues, a padlike or spiculose empodium, and sometimes a conspicuous pair of pulvilli. The various parts of the legs often bear patterns of pale and dark scaling, especially bands and stripes, that are taxonomically useful. The ungues are usually simple in females, but in some species they bear tooth-like basal processes (Roy and Brown, 1970). Mesothorax carries functional wings, one on each side and the metathorax bears a pair of knob-like halteres. The wings are long and relatively narrow, and the number and arrangement of the wing is virtually the same for all mosquito species. The veins are covered with scales which are usually brown, black, white or creamy yellow, but more brightly coloured scales may occasionally be present. The shape of the scales and the pattern they form differs considerably between both genera and species of mosquitoes. Scales are also project as a fringe along the posterior boarder of the wings (Roy and Brown, 1970; Service, 2004).

25 c. Abdomen The abdomen consists of 10 segments of which only eight are distinctly visible. Segments IX and X are reduced and more or less modified as part of the external structures involved in reproduction, the genitalia. In some species segment VIII is sexually differentiated from the preceding segments, and in such cases it is also treated as part of the genitalia. Each segment consists of a dorsal sclerite, the tergum, which is joined by an elastic pleural membrane to a ventral sclerite, the sternum. Intersegmental membrane connects the terga and sterna of adjacent segments. It is the elasticity of the pleural and intersegmental membranes that allows the abdomen of the female mosquito to become distended when she is taking a blood meal or when she is gravid and full of eggs. The terga are collectively referred to as the dorsum; likewise the sterna as the venter. In mosquitoes of the subfamily Culicinae and toxorhynchitinae, the abdomen is usually covered dorsally and ventrally with scales of varying colours. In the Anophelinae, however, the abdomen is almost or entirely devoid of scales. The terga may have basal, apical or lateral patches of pale scales which provide useful taxonomic characters. Scale patterns are generally not so evident on the sterna. Modifications of the genital parts are more or less complex and varied according to the genera and species, thus providing important taxonomic characters. The last abdominal segments of female mosquito terminate in a pair of small finger-like cerci, whereas in the males a pair of prominent claspers, comprising part of the male external genitalia, is present (Service, 2004).

26 2.1.3 General Biology of Mosquitoes Mosquitoes are holometabolous insects and their life cycle contains four stages, i.e. egg, larvae, pupa and adult. The length of the life cycle of mosquitoes is primarily dependent upon temperature. a. Egg Depending on the species, a female lays between 30 and 300 eggs at a time and they generally float on the surface of the water or found attached to leaves and stems of aquatic plants. The eggs are small dark bodies just visible to naked eye. In the case of Culex species, the eggs are stuck together in rafts of a hundred or more eggs. Anopheles and Aedes species do not make egg rafts and lay eggs separately. Eggs of Anopheles have a pair of lateral floats. Culex and Anopheles lay their eggs on water while Aedes lay their eggs just above the water level or damp soil that will be flooded by water. Mansonia species lay their eggs in a sticky mass that is glued to the aquatic plants. Eggs of most species hatch into larvae within 2-3 days (Rozendaal, 1997; Roy and Brown, 1970; Service, 2004). b. Larva Mosquito larvae, commonly called "wigglers" or "wrigglers", live in water from 7 to 14 days depending on water temperature. Larvae come to the surface at frequent intervals to obtain oxygen through a breathing tube called a siphon. During growth, the larva molts four times. At the 4 th instar, the larva reaches a length of almost 1 / 2 inch. When the 4 th instar larva moults it becomes a pupa. Anopheles are unlike Culex and Aedes larvae since they do not have a breathing tube, they must lie

27 parallel to the water surface in order to get a supply of oxygen through a breathing opening (Rozendaal, 1997; Roy and Brown, 1970). Mosquito larvae have a well developed mobile head, the eyes varying according to the age of the larvae. A pair of dense tufts of long hair or feeding brushes is present over the mouth on either side of their brushes a current is set in motion which wafts microscopic food particles towards the mouth. Nine abdominal segments are present and anal somites are surrounded at its apex by four tracheal gills. These organs are small in surface feeders such as Anopheles, but larger in Aedes which is a bottom feeder. The respiratory system is metaneustic. When at rest and during feeding, the larva of Anophelines float horizontally just beneath the surface film with palmate hairs and spiracular area in contact with the surface film. In this feeding habit, mosquito larvae may be phytophagous or carnivorous. They feed upon minute algae and other particles contained in the water. Certain forms, Toxorhynchites, however carnivorous, they may be readily recognized either by the mouth brushes being replaced by stout spines, which serve to sieve the prey or by the prehensile antennae. The organism most frequently preyed upon by this mosquito is other mosquito larvae (Roy and Brown, 1970; Service, 2004). c. Pupa All mosquito pupae are aquatic and comma-shaped. They are very active and respire by means of a pair of breathing trumpets communicating with the anterior spiracle. They float at the top of the water with their trumpets attached to the surface film. Mosquito pupae commonly called "tumblers" live in water from 1 to 4 days, depending upon species and temperature. The pupa is lighter than water and

28 therefore floats at the surface. It takes oxygen through two breathing tubes called "trumpets". When it is disturbed it dives in a jerking, tumbling motion and then floats back to the surface. The pupa does not eat. The metamorphosis of the mosquito into an adult is completed within the pupal case. The adult mosquito splits the pupal case and emerges to the surface of the water where it rests until its body can dry and harden (Roy and Brown, 1970; Service, 2004). d. Adult The adult mosquitoes have long and slender body which is divided into head, thorax and abdomen. Morphology of each division is explained in the previous section. Mosquito generally does not feed within 12-24 hours after emergence. Though both sexes can live on plant saps, a blood meal is necessary for the maturation of eggs (Roy and Brown, 1970). 2.1.4 Breeding Ecology of Mosquitoes Each species has itsown breeding ecology. In fact, the habitat of mosquitoes is extremely varied, from a spoon of water to a lake. They can breed in natural water bodies and artificial collections. Larger water bodies can only produce mosquitoes when they dry up and leave shallow, stagnant puddles in the stream bed. Each species has its own breeding preference. Mansonia breed in habitat with floating vegetation such as pond/ground pool, drains and paddy fields (Iyengar, 1938). Anopheles species prefer to breed in paddy field and shallow ground pool/pond (Kumar and Vijayan, 2005). Many of the Aedes species are container breeders, breeds in natural habitat such as tree holes, rock-pools, bamboo stumps, leaf axils, split coconut shells, etc and man-made habitat such as clay pot, water storage

29 containers, tyre, discards, etc. The immature stages of many Culex mosquitoes have been reported from a variety of aquatic habitats e.g., ponds, streams, ditches, swamps, marshes, temporary and permanent pools, rock holes, tree holes, crab holes, lake margins, plant containers (leaves, husks of fruits, tree holes, bamboo stumps), artificial containers (tires, tin cans, flower vases, bird feeders), and other habitats (Laird 1988; Rueda et al. 2005, 2006). A few mosquitoes breed almost exclusively in brackish or salt water, such as saltwater marshes and mangrove swamps, and are consequently restricted to mostly coastal areas (Service, 2004). Larvae of Toxorhynchites are found in a wide variety of both artificial and natural habitat such as containers (Steffan and Evenhuis,1981).

30 2.2 REVIEW OF LITERATURE History of medical entomology dates back to 1877 when Patrick Manson made the historic discovery that mosquitoes could transmit human filariasis. This was the first real evidence for the involvement of any Arthropods in the transmission of human diseases. Not long after the 1877 discovery, the involvement of mosquitoes in the transmission of Malaria (1898), Yellow fever (1900) and Dengue (1903) were also proved (Philip and Rozenboom, 1973). Ever since the important discoveries in that mosquitoes play the role of vectors of human diseases, these insects have received special attention of entomologists and health workers all over the world. As an immediate consequence of this, basic research, particularly in the field of mosquito taxonomy, received a great impetus and entomologists set about the task of describing and naming the different species that hitherto lay in obscurity. These studies have generated immense literature dealing with taxonomy, biology, ecology, disease relationship, etc. of mosquito, which constitute an integral part of vital component in the epidemiology and control of mosquito borne diseases. 2.2.1 Diversity studies Literature on Indian mosquito is less compared with that of the world. Series of papers dealing with Indian mosquitoes were published during the early part of the 20 th century. The first series of papers dealing with the taxonomy of Indian Anopheles commenced with that by Grassi (1899) and was followed by

31 those of Giles (1901), Theobald (1901, 1902, 1910), Liston (1901), James (1902), Cogill (1903) and many others. In the early part of the 20 th century, James and Liston (1911) made a notable contribution on Indian Anopheles through a monograph, which was largely used as the guide to the identification of species. The larvae of Indian anopheles were intensely studied by Puri (1931) resulting in his comprehensive monograph on the subject. A comprehensive work on the classification and systemetics of the Culicidae was published by Edwards (1932) in Genera Insectorum. The major revolutionary work in the early part of the 20 th century has been the publication of Fauna of British India by Christophers (1933) on Tribe Anophelini. This volume included extensive information on the species names and systematics, breeding habitat, adult bionomics, distribution and relation to diseases. Christopher recognised 4 subgenera and described then known 43 species of mosquitoes under the genus Anopheles. While much emphasis was placed on Anopheles, Culicine mosquitoes were completely neglected. However, Baurraud (1934) has done an excellent work on Culicines of British India. This investigation unearthed such a large number of culicine mosquitoes new to science that Barraud started publishing a series of paper entitled a Revision of Culicine Mosquitoes of India in the Indian Journal of Medical Research and continued to do so for even a decade from 1923 onwards until the publication of his Fauna of British India in 1934. Barraud recognised 16 genera and describes 245 species of mosquitoes under subfamily Culicinae. The genus Aedes contains 110 species and 12 subgenera. As far as Indian culicinae is concerned, Barraud s Fauna of British India still holds its place as the most

32 comprehensive monograph. The publication of these two great works of the fauna of British India marked a land-mark in the history of mosquito studies in the Indian sub-continent. The year 1934 marks the end of an era of very active taxonomic research on Culicidae by virtue of which mosquitoes became one of the best known groups of insects in the area. By this time as many as 43 Anopheline and 245 Culicine mosquitoes had come to be known from the area. After 1934, mosquito taxonomic works proceeded in a dead slow fashion. The number of new species identified was extremely low during the period between 1934 and 1960 (Qutubuddin,1960). In 1961 there was an excellent publication entitled The Vectors of Malaria in India by National Society of India for malaria and other mosquito born diseases. This publication dealt with ecology, relation to disease and control of few vectors of malaria in India. Even though very few species have been added since 1933, several changes in the taxonomic status of some species have taken place. A series of catalogues and revisions of genus or subgenus were published after 1960s to update the knowledge. The world catalogue of Stone et al., (1959) includes 31 genera and 2401 species of mosquitoes while world catalogue of Knight and Stone (1977) includes 34 genera and 2960 species of mosquitoes worldwide. A revision of the subgenus Culex in the oriental region (Sirivanakarn, 1976) has contributed substantially to better knowledge of several common oriental species. In this revision, 42 species of subgenus Culex are recognised and of these, 5 are new and 37 are revalidated and re-described. The revision of subgenus Stegomyia of Aedes

33 (Huang, 1979) clarifies some of the taxonomic problems and also provides a guide for the identification of 37 species occurring in the oriental region. In 1984 Rao prepared a monograph which provided a balanced and comprehensive account of the Anophelines of India. He incorporated all major taxonomic advances since the publication of Christopher s book (1933). During the latter part of the 20 th century and the early part of 21 st century individual research workers made their contribution. They were mainly concentrated on a region wise survey of mosquito. Nagpal and Sharma (1987) conducted an extensive survey in the eastern region on India and reported 61 species of mosquitoes belonging to 8 genera viz Anopheles, Aedes, Armigeres, Coquillettidia, Culex, Mansonia and Toxorhynchites. Dutta and Khan, (2003) identified 54 species of mosquitoes from Mizoram state while Chandra, (2002) recorded different species of Anopheles from West Bengal. Kaur and Kirti (2003) reported 21 species of mosquitoes from Haryana state. Many workers reported different species of mosquitoes from south India. A very rich and diversified mosquito fauna consisting of 60 species were reported from Rajiv Gandhi National Park Karnataka state (Kumar et al., 2004). Another survey conducted in two districts of Karnataka showed the presence of 29 species of mosquitoes (Kumar and Vijayan, 2005). Rajagopalan et al., (1979) recorded 27 species of mosquitoes from Thanjavur district of Tamil Nadu. Amala and Anuradha (2012) observed 13 species of mosquitoes in Sirumalai hills of Tamil Nadu.

34 2.2.2. Mosquito studies in Kerala Several species of mosquitoes belonging to different genera have been reported from different parts of Kerala (Table 2.1). Since Kerala was notorious for Brugian Filariasis and Malaria, attention of researchers was drawn to this area as early as 1900s. Early reference regarding the prevalence and distribution of mosquitoes in Kerala was given by James (1902), Giles (1902), Theobald (1901, 1902, 1905, 1910), James and Liston (1911), Horne (1914), Kamath (1917), Cruickshank and Wright (1914), Brunetti (1920) and Covell (1927,1931). A notable contribution was given by Iyengar (1938). Studies undertaken by Iyengar on epidemiology of filariasis in Travancore brought out many species of mosquitoes belonging to different genera. Covell and Harbhagwan (1939) made a survey in the mountainous and thickly forested regions of the Wayanad in 1938-39 and reported 19 species of Anopheles. Mathew (1939) also reported different species of malaria vectors erstwhile Travancore state. The prevalence of three species of mansonoids, viz, Ma. annulifera, Ma. uniformis and Ma. indiana and their role in the transmission of filariasis in Kerala was established by many workers (Iyengar,1938; Sing et al., 1956; Nair and Roy, 1958; Nair, 1962; Chandrasekhar, et al., 1976; Pradeepkumar et al.,1989). Daniel et al., (1986) reported 13 species from Trivandrum city.

35 Table 2.1 Major mosquito species already reported from different parts of Kerala state Sl No Daniel et al., (1986) Rajendran and pasad, (1992, 1994) Mariappan et al., (1992, 1996, 1997) Hiryan et al.,(2003) CRME (2003) Rajavel at al., (2006) Dilipkumar (2006) Sabu Subramanian, (2007) species 1. Ae. aegypti + + + + + + + 2. Ae. albopictus + + + + + + + + + + 3. Ae. chrysolineatus + + 4. Ae. niveus + + + 5. Ae. vexans + + + + + + 6. Ae. vittatus + + + + + + 7. An. barbirostris + + + + + + + + 8. An. culicifacies + + 9. An. gigas + + 10. An. jamessi + + + + + + + + + 11. An.jeyporensis + 12. An. nigerrimus + + + + + + 13. An. pallidus + + 14. An. peditaeniatus + + + + 15. An. stephensi + + + 16. An. subpictus + + + + + + + 17. An. tessellatus + + + + 18. An. vagus + + + + + + + + 19. An. varuna + + 20. An.aconitus + + 21. An.roperi + + 22. Ar. sabalbatus + + + + + + + + + + 23. Cq. crassipes + + + + 24. Cx. bitaeniorhynchus + + + + + + + 25. Cx. brevipalpis + + + + + + 26. Cx. fuscanus + + + + + + + 27. Cx. fuscocephala + + + 28. Cx. gelidus + + + + + + + + 29. Cx. infula + + 30. Cx. minutissimus + + + + 31. Cx. pallidothorax + + + Thenmozhi et al., (2007) Sudharmini (2009) Present study

36 32. Cx. pseudovishnui + + + + + 33. Cx. quinquefasciatus + + + + + + + + 34. Cx. sitiens + + + + + + + + 35. Cx. tritaeniorhynchus + + + + + + + + 36. Cx. uniformis + + + 37. Cx. univittatus + + 38. Cx. vishnui + + + + + + 39. Cx. whitmorei + + + 40. Hz. chandi + + 41. Ma. annulifera + + + + + + + + 42. Ma. indiana + + + + + 43. Ma. uniformis + + + + + + + + + 44. Mi. hybrida + + + + + 45. Mi. chamberlaini + + + + 46. Ml. genurostris + + 47. Tx. splendens + + + + + + 48. Ur. atra + + 49. Ur. novobscura + + 50. Ur.habes + +sign indicates the presence There is a renewed interest in the study of mosquitoes of the state due to the emergence of various mosquito borne diseases. 35 species of mosquitoes viz, Aedes (3), Anopheles (7), Armigeres (1), Culex (14), Mimomyia (2), Uranotaenia (4) and Orthopodous (1) were reported from Kochi and adjoining island by Mariappan et al., (1992, 1996, 1997). One recent contribution is given by Hiriyan et al., (2003) who studied the mosquito fauna in Kuttanad region of Alappuzha. Altogether 26 species belonging to 6 genera of mosquito viz., Aedes, Anopheles, Armigers, Culex, Coquillettidia and Mansonia were identified from Kuttanad area. Sabesan et al., (1991) studied the seasonal abundance, biting behaviour of Mansonia species and their relative role in the transmission of Burgian Filariasis in

37 this region. They also reported 15 other species belonging to genus Culex (7), Ficalbia (1), Armigers (1), Aedes (2), Anopheles (4). The recent outbreak of arboviral diseases such as CG & DF attracted the attention of researchers towards Aedes mosquitoes. Entomological study conducted by CRME (2003) in connection with dengue emergence in Kerala reported 22 species of mosquitoes. Though both species of Aedes aegypti and Aedes albopictus have been reported from Kerala (Sharma et al., 2004; Das et al., 2004), Aedes albopictus is indicated as the major species (Kalra and Prasittisuk, 2004). Sylvan environment of rubber plantation was detected as unique habitat of Aedes albopictus. Earlier studies in rubber plantation indicated that the dominant breeding site for Aedes albopictus was containers used for collecting rubber sap (Sumodan, 2003). Its breeding in plastic tea cups has also been established (Hiriyan et al., 2003). Detection of dengue virus in wild caught Ae. albopictus around Kozhikode airport indicated the role of this mosquito as vector of DF in Kerala (Das et al., 2004). Rajavel et al., (2006) identified 17 species belonging to 7 genera from mangrove forest of Kannur. 27 species under 7 genera of mosquitoes were reported from Trissure (Sabu and Subramanian, 2007). 14 species were recorded during a study conducted in different districts of Kerala (Thenmozhi et al., 2007). By compiling the various study reports from Kerala, it is found that more than 50 species have been recorded so far (Sabesan et al.,1991; Mariappan et

38 al.,1992, 1996, 1997; Hiriyan et al., 2003; CRME 2003; Rajavel et al.,2006; Thenmozhi et al., 2007). The review of literature showed that though a large amount of literature on Indian mosquitoes is available; our knowledge on systematics and distribution of mosquitoes of Kerala is far from complete. The reported studies are mainly confined to costal districts like Alappuzha, Ernakulum and Trivandrum where mosquito borne diseases were more prevalent. At this juncture, the present study was undertaken with the following objectives. 2.2.3 Objectives The general objective of the study was to understand the Diversity, Composition, Distribution and Breeding ecology of mosquitoes in Central Kerala. The specific objectives were:- 1.1.To understand the diversity and composition of 1.2. To list the medically important vectors 1.3. To study the seasonal distribution of mosquitoes 1.4. To report the spatial distribution of mosquitoes 1.5. To record the altitudinal distribution of mosquitoes 1.6. To understand some aspects of the breeding ecology of mosquitoes

39 2.3 METHOD AND MATERIALS 2.3.1 Design of study Present research work was conducted by sample survey for a period of three years, from February 2008 to January 2011. Two districts-kottayam and Idukki - of central Kerala were randomly selected. Each calendar year was divided into three seasons such as pre-monsoon (February to May), monsoon (June to September) and post-monsoon (October to January). 2.3.2 Study area Kottayam and Idukki districts are located towards the centre of Kerala state (Fig 2.1). Both the districts are heterogeneous in many respects and have their own peculiarities as detailed below (Kottayam, 2007; Idukki, 2007) a). Physiography- Kottayam district has a total area of 2208 sq.km and lies between latitude 9 15 and 10 21 and longitude 76 22; and 77 25. District is bordered on north by Ernakulum district, on the east by Idukki district and on south by Alappuzha and Pathanamthitta. Vembanad lake forms the western boundary. The district has no costal area. Idukki district has a total area of 4358 sq.km and lies between latitude 9 15 an 10 21 and longitude 76 37 and 77 25. The district bound on the east by Madurai district of Tamil Nadu state, west by Ernakulum and Kottayam, south by Pathanamthita, north by Trichure and Coimbatore district of Kerala and Tamil Nadu states respectively. More than 50% of the area is covered by forest and about 97% of the district is covered by rugged mountains.

40 b). Demography According to the census 2001 Kottayam district had a population of 19.5 lakhs with a literacy rate of 95.82%. Population density per sq.km was 885. Number of house hold was 434520. According to the census 2001 Idukki district had a population of 11.29 lakhs with a literacy rate of 88.69 %. Population density per sq.km was 259. Number of household was 265344. c). Climate- Kottayam district has a tropical climate. The district normally gets annual average rainfall of 3130.33 mm. The hot season from March to May, is followed by south- west monsoon from June to September. The month of October and November constitute the post monsoon season or north-east monsoon. Months of December to February form the winter. As far as Idukki is concerned climate shows variation from one area to another. The annual rainfall varies from 2500-4250 mm. East and north eastern regions get very low rainfall normally up to 1500 mm. d). Agriculture- Agriculture forms the livelihood of the majority in both the districts. Cash crops as well as food crops are cultivated. In Kottayam Rubber is the major cash crop, grown in 109582 hectares the largest area under rubber cultivation in the state. Paddy is the most important food crop cultivated in 25213 hectars. Other crops include tapioca, pine apple, plantain, ginger, tubes, vegetables etc.

41 Fig 2.1 Map of Kerala showing study area

42 In Idukki agriculture is the main segment of the economy. Tea, cardamom, pepper coffee, rubber, coconut, etc are cultivated. e). Terrains- The study area is naturally divided into high land (HL) (above 100 meters from mean sea level), mid land (ML) (between 20 to 100 meters from mean sea level) and low land (HL) (below 20 meters from mean sea level) (Fig 2.2). Fig 2.2 Map showing the low land (LL), mid land (ML) and high Land (HL) terrains of study area

43 Kottayam district comprises high land, mid land and low land, the bulk being made up of midland (Fig 2.2). Of the five taluks Meenachil and Kanjirappally taluks have high land and midland areas while Kottayam, Changanacherry and Vaikom taluks have mid land and low land areas. Kanjirappally and Meenachil taluks have laterate soil, where as Vaikom taluk, part of Changanacherry and Kottayam taluk have alluvial soil. Idukki district lies mostly in the high land and low land is totally absent (Fig 2.2). High land was further subdivided into Mid-upland, Upland, Western Ghats High Range and Top Western Ghats High Range (Table 2.2). Terrain Table 2.2 Distribution of land pattern of study area Land Pattern Altitude(Above Mean Sea Level) Low land (LL) Low land 0-20 m Mid land (ML) Midland 20-100 m High land (HL) Mid-upland Upland Western Ghats High Range Top Western Ghats High Range 100-300 m 300-600 m 600-1200 m 1200m -above 2.3.3. Selection of sites For the convenience, number of localities for sample collection was fixed as 48. Multi-stage stratified random sampling was adopted for selecting localities for sampling. At the first stage study area was divided into two strata as lower

44 elevation (altitude between 0-100) and higher elevation (above 100). Localities (48) were equally distributed among these strata (24 each) (Table 2.3). In the second stage 24 localities were randomly selected from each stratum. Percentage of land lying in each terrain/land pattern was considered while fixing the number of localities for a terrain. Of the total 24 localities 12 were considered as fixed sites for regular sampling in each season and 12 as random sites (Table 2.3). Altitude of study locality was determined by topograph which is available at the Department of Environmental Science, Mahatma Gandhi University, Kottayam. Altitude of study localities was also verified by data provided by Centre for Earth Science Studies (CESS) Thiruvananthapuram. Table 2.3 Distribution of sampling localities in the study area. Strata Lower elevation Terrain/Land pattern Fixed sites Random sites Sub Total Low land 4 4 08 Midland 8 8 16 Mid-upland 3 4 07 Total 24 Higher elevation Upland 3 2 05 Western Ghats High Range Top Western Ghats High Range 4 4 08 2 2 04 TOTAL 24 24 48 48 24

45 2.3.4. Sites selected for sampling Of the 48 localities selected for the study, 24 belong to Kottayam district while the rest (24) belongs to Idukki district (Table 2.4). Table 2.4 District wise distribution of sampling localities Strata Lower elevation Higher elevation Terrain Fixed sites Kottayam District Rando m sites TOTA L Fixed sites Idukki District Rando m sites TOTA L Grant Total Lowland 4 4 8 0 0 0 8 Midland 6 7 13 2 1 3 Highland 2 1 3 10 11 21 24 16 TOTAL 12 12 24 12 12 24 48 Twelve localities selected from Kottayam district for regular sampling (fixed sites) were Chempu (LL), Vaikom (LL), Kumarakom (LL), Vazhappally (LL), Mulakulam (ML), Uzhavoor (ML), Mutholy (ML), Pallikathodu (ML), Manarcad (ML), Kangazha (ML), Parathode (HL) and Mundakayam (HL). Twelve localities (Random sites) were selected for random sampling in each season. Random sites varied from season to season. Twelve localities selected from Idukki district for regular sampling (fixed sites) were Thodupuzha (ML), Karimkunnam (ML), Peerumade (HL), Vandiperyar (HL), Kumily (HL), Upputhara (HL), Kattappana (HL), Vazhathope (HL), Nedumkandam (HL), Udumbanchola (HL), Munnar (HL) and Devikulam (HL) Twelve localities (Random sites) were selected for random sampling in each season. Random sites varied from season to season.

46 2.3.5. Mosquito collection Methods a. Adult collection Adult mosquitoes were collected from each selected localities once in a season using standard methods (WHO, 1975) for a period of three years, from February 2008 to January 2011. Samples were collected from indoor and outdoor of human dwellings and animal sheds using aspirator and flash light from 6 pm to 9 pm and 7 am to 11 am. Mosquitoes were collected for 40 minutes from each catching sites (6 sites/locality). Thus a total of 4 Man Hours were employed in each locality per season. A total of 1728 Man Hour was spent for the adult collection (4x48 locality =192 Man Hour/season, 192x3 season =576 Man Hour/year, 576x3 years =1728 Man Hour/study period). Details of collection such as district, locality, collection site, elevation, altitude, season, etc. were recorded on a data sheet (Appendix I) Collected specimens were narcotized with petroleum ether and identified using standard light microscopy and relevant taxonomic references (Christophers, 1933; Barraud, 1934; Sirivanakarn, 1976; Knight and Stone, 1977; Huang, 1979; Das et al., 1990; Reuben et al.,1994; Das and Kaul, 1998; Reinert, 2000). Abbreviation used for generic name follows Reinert (2001). Identified specimens were kept along with the entomological collections of the Department of Zoology, S. B College Changanacherry.

47 b. Larval collection Along with adult collection immature stages (larvae and pupae) were also collected from each locality. They were collected from their natural habitat using dippers in ground pools and other open sources and pipette in tree holes. Small containers were fully emptied into plastic bag/sample bottle (WHO, 1975; Service, 1993). Sample from each habitat were maintained separately in suitably labelled containers. Immature stages were allowed to emerge and were subsequently identified using keys. Details of collection such as district, locality, collection site, elevation, altitude, season, breeding habitat, etc. were recorded on a data sheet (Appendix 1). 2.3.6. Analysis of Data Collected data was entered in a computer and analysed by using Statistical Package for Social Science (SPSS) software version 17 and MATLAB. Descriptive statistics (frequency, percentage) were used to tabulate the composition of mosquito fauna, seasonal distribution, altitudinal distribution and breeding habitat. Density of mosquito was expressed as adult caught per hour, Man Hour Density (MHD). Analysis of variance (ANOVA) was used, wherever necessary. Summarised data is presented in suitable tables (Table 2.5 to 2.18) and graphs (Fig 2.3 to 2.7).

48 2.4 OBSERVATIONS 2.4.1 Diversity and Composition of Mosquito Fauna Present study gives a clear idea on the composition of mosquito fauna in the study area. A total of 12196 mosquitoes belonging to 10 genera and 38 species were collected during 1728 man-hours of collection. a. Generic composition In the present study a total of 10 genera of mosquitoes were recorded from the study area (Table 2.5). They were Aedes, Anopheles, Armigeres, Culex, Coquilletidia, Heizmannia, Mansonia, Mimomyia, Toxorhynchites and Uranotaenia. Genus Culex was represented by 16 species, followed by genus Anopheles by seven species, Aedes by six species, Mansonia by three species and one species each by genus Armigeres, Coquilletidia, Heizmannia, Mimomyia, Toxorhynchites and Uranotaenia (Fig 2.3). Among the 10 genera, Culex was the predominant genus and was represented by 42.5 percentages of the total mosquitoes collected, followed by Aedes by 26.5 and Armigeres by 19.7 percentages (Fig 2.4). These three genera together constituted 88.7 percentage of the total collection.

49 Table 2.5 Generic composition of adult mosquitoes in study area Genus No. of species Total No. of specimens collected Percentage Aedes 06 3236 26.5 Anopheles 07 137 01.1 Armigeres 01 2404 19.7 Coquillettidia 01 36 00.3 Culex 16 5180 42.5 Heizmannia 01 43 00.4 Mansonia 03 1052 08.6 Mimomyia 01 2 0.02 Toxorhynchites 01 79 00.7 Uranotaenia 01 27 00.2 Total 38 12196 100.00

50 Fig 2.3 No. of species collected from each genus 18 16 16 14 No. of species 12 10 8 6 6 7 4 3 2 1 1 1 1 1 1 0 Fig 2.4 Percentage of mosquitoes collected from each genus 45.0 42.5 40.0 35.0 Percentage 30.0 25.0 20.0 15.0 10.0 26.5 19.7 8.6 5.0 0.0 1.1 Aedes Armigeres Culex Mansonia Anopheles 1.5 Others

51 b. Mosquito Species Diversity and Taxonomy i. Mosquito Species Diversity The area selected for study was rich in mosquito species diversity. Thirty eight species were identified from the study area (Table (2.6). Table 2.6 Species of mosquitoes identified from the study area FAMILY: CULICIDAE I. Sub Family: Anophelinae 1. GENUS : ANOPHELES a. Sub genus: Anopheles 01 Anopheles barbirostris Van der Wulp 1884 02 Anopheles nigerrimus Giles 1900 03 Anopheles peditaeniatus Leicester 1908 b. Sub genus: Cellia 04 Anopheles jamesii Theobald 1901 05 Anopheles pallidus Theobald 1901 06 Anopheles subpictus Grassi 1899 07 Anopheles vagus Doenitz 1902

52 II. Sub Family: Culicinae 2. GENUS : CULEX a. Sub genus: Culex 08 Culex bitaeniorhynchus Giles 1901 09 Culex fuscocephala Theobald 1907 10 Culex gelidus Theobald 1901 11 Culex infula Theobald 1901 12 Culex pseudovishnui Colless 1957 13 Culex quinquefasciatus Say 1823 14 Culex sitiens Wiedemann 1828 15 Culex tritaeniorhynchus Giles 1901 16 Culex vishnui Theobald 1901 17 Culex whitmorei Giles 1904 18 Culex univittatus Theobald 1901 b. Sub genus: Culiciomyia 19 Culex pallidothorax Theobald 1905 c. Sub genus: Eumelanomyia 20 Culex brevipalpis Giles 1902 d. Sub genus: Lophoceratomyia 21 Culex (Lophoceratomyia) minutissimus Theobald 1907 32 Culex (Lophoceratomyia) uniformis Theobald 1905 e. Sub genus: Lutzia 23 Culex (Lutzia) fuscanus Wiedemann 1820 3. GENUS : COQUILLITIDIA a. Sub genus: Coquillitidia 24 Coquillitidia crassipes Van der Wulp 1892

53 4. GENUS : ARMIGERES a. Sub genus: Armigeres 25 Armigeres sabalbatus Coquillett 1898 5. GENUS : AEDES a. Sub genus: Aedimorphus 26 Aedes vexans Meigen 1830 b. Sub genus: Finlaya 27 Aedes chrysolineatus Theobald 1907 28 Aedes niveus Ludlow 1903 c. Sub genus: Fredwardsius 29 Aedes vittatus Bigot 1861 d. Sub genus: Stegomyia 30 Aedes aegypti Linnaeus 1762 31 Aedes albopictus Skuse 1894 6. GENUS : HEIZMANNIA a. Sub genus: Heizmannia 32 Heizmannia chandi Edwards 1922 7. GENUS : MANSONIA a. Sub genus: Mansonioides 33 Mansonia annulifera Theobald 1901 34 Mansonia indiana Edwards 1930 35 Mansonia uniformis Theobald 1901

54 8. GENUS : MIMOMYIA a. Sub genus: Mimomyia 36 Mimomyia hybrida Leicester 1908 9. GENUS : URANOTAENIA a. Sub genus: Pseudoficalbia 37 Uranotaenia novobscura Barraud 1934 Ill. Sub Family: Toxorhynchitinae 10. GENUS : TOXORHYNCHITES a. Sub genus: Toxorhynchites 38 Toxorhynchites splendens Wiedemann 1819 ii. Taxonomy GENUS: ANOPHELES Meigen 1818 Distinguishing characteristics: The maxillary palpi of adult female are about as long as the proboscis; the posterior margin of the scutellum is evenly rounded; and the abdominal segments usually have only small patches of scales or none at all. Most members of genus Anopheles are immediately recognised by their overall appearance. The majority of the adults are slender insects with the head and abdomen oriented in a straight line at an angle to the surface when at rest. Following seven species of Anopheles have been identified.