Progress Report on “Survey and Management of Disease Carrier Insect (Mosquitoes- Anophales Sp., Culex Sp., Aedes Sp., Etc.,) B

PROGRESS REPORT ON

“SURVEY AND MANAGEMENT OF DISEASE CARRIER INSECT (MOSQUITOES- ANOPHALES SP., CULEX SP., AEDES SP., ETC.,) BY AQUATIC INSECT PREDATORS” (BT/IN/Indo-US/ Foldscope/39/2015 dt. 20/03/2018)

Submitted to Government of India Ministry of Science and Technology Department of Biotechnology Block-2, 7th Floor C.G.O. Complex Lodi Road, New Delhi-110003

Submitted by Dr. M. Bhubaneshwari Devi Associate Professor, P.G. Department of Zoology, D. M. College of Science, Imphal Manipur

2018

Contents

1. Introduction 3-5

2. Material methods 5-7

3. Results

a. Systematics 8-14

b. Life cycle Studies 15-17

c. Management (Biological Control) 18-20

4. Conclusion 20

5. Foldscopy up loaded in microcosmos 21-23

6. References 24-25

7. List of Publications Annexures I, II, III

8. List of Communicated papers Annexures IV

HALF YEARLY PROJECT REPORT INTRODUCTION Mankind has been plagued by mosquitoes as nuisances and as vectors of mosquito- borne diseases for centuries, resulting in inestimable economic losses and indeterminable human suffering. Mosquitoes transmit some of the deadliest diseases known to man malaria and yellow fever—as well as dengue, encephalitis, filariasis and a hundred or so other maladies. In spite of decades of mosquito control efforts throughout affected regions worldwide, this scourge has not left us and our present- day overpopulated, jet-linked world remains on the edge of resurgence and out- breaks of old and new mosquito-borne disease epidemics (Norbert et al., 2010, Shaalan and Canyon, 2009). The mosquitoes (family Culicidae) are at the centre of worldwide entomological research because of their importance as vectors of a wide range of debilitating viral and parasitic diseases affecting both humans and animals. More than half of the world’s population lives under the risk of becoming infected by mosquitoes that carry the causative agents of diseases such as malaria, dengue, Chikungunya, West Nile fever, Japanese encephalitis or lymphatic filariasis. Estimates made by the World Health Organisation (WHO) show that 247 million people became ill in 2006 and about one million people died (WHO 2008) from mosquito- borne diseases. Although approximately three quarters of all mosquito species occur in the humid tropics and subtropics, mosquitoes are a problem not only in these regions. Mosquito larvae colonise a wide range of water bodies, temporary and permanent, highly polluted as well as clean, large or small, stagnant or flowing, and even the smallest accumulations such as water-filled buckets, flower vases, old tyres, hoof prints or leaf axils. Adult mosquitoes vary greatly in their bionomics, e.g. in terms of the host-seeking, biting and dispersal behaviour, and strategy for reproduction. Their significant medical importance and their troublesome behaviour have historically attracted the interest of scientists. Their importance as vectors of malaria and yellow fever was suspected by Joseph Nott in 1848. In 1878 Sir Patrick Manson showed that the roundworm Wuchereria bancrofti is transmitted by Cx. p. quinquefasciatus. Only 3 years later, Carlos Finley postulated that yellow fever was transmitted by mosquitoes, which was later proved by Walter Reed and his co- workers in 1901. Sir Ronald Ross made a further pioneering discovery in Hyderabad, India in 1897, when he recognised the importance of the anophelines as vectors of malaria. The discovery of the transmission cycles of most vector-borne diseases led to the recognition that mosquitoes represented a major scourge to humans, which in turn triggered the development of mosquito control. The foundations for mosquito control were established at the beginning of the 20th Century. William C. Gorgas, a member of the Army Medical Corps, USA, dedicated most of his professional life to the control of yellow fever, with special focus on control of the vector mosquito Ae. aegypti [Stegomyia aegypti]. By the turn of the 20th Century, he succeeded in suppressing yellow fever in Panama, and set the basis for the construction of the Panama Canal. Gorgas was the first to recognise that only by the implementation of an integrated control programme, could the severe burden of vector-borne

diseases be reduced. His approach comprised draining the breeding sites, vegetation cutting to reduce preferred resting sites of adult mosquitoes, treatment of water bodies with oil derivates to suppress immature mosquito stages, screening and quarantine of infected people to interrupt the transmission, as well as killing the adult mosquitoes to reduce the vector- density and vector-human contact (Le Prince 1910; Le Prince and Orenstein 1916). Compared to Curculionidae, the largest family in the animal kingdom, with 35,000 known species, Culicidae, numbering more than 3,200 species, could be ranked as a family of only a small-to-moderate size. Even though yield losses caused by weevils could be estimated in billions of dollars, mosquitoes are able to carry many lethal diseases in their bodies. Apart from being the well-known vectors of life-threatening diseases, in some parts of the world, mosquitoes may also occur in enormous numbers thus causing a signiﬁcant reduction in human life quality and serious economic damage, for instance, in livestock (Norbert et al., 2010). The risk of becoming infected with a vector-borne disease has increased again not only in the tropics but also in Europe and USA, as shown by the outbreak of Chikungunya fever in Italy in 2007 (Angelini et al. 2007) and West Nile virus in the USA. Italy’s outbreak of Chikungunya is the first known disease transmission by the invasive vector, the Asian tiger mosquito (Ae. albopictus [St. albopicta]) in Europe, and triggered action by both experts and public in the EU community. Overall, the greater mobility of people to and from endemic areas, the intensified international trade, as well as the changing climate, will further encourage the spread and establishment of exotic diseases and invasive species in formerly safe areas. The Asian tiger mosquito originates from Southeast Asia, where its developmental stages occur in water-filled tree-holes, coconut shells, bamboo stumps and similar water collections. Over time, this species has adapted to breeding in artificial containers such as water barrels, car tyres or other places where small pools of water may collect. This mosquito has undergone an astonishing expansion of its range within the last few decades. Since 1979, Ae. albopictus has been found in Africa, the Americas and Europe, and more recently also in the Pacific region. It is expected to spread to tropical and subtropical regions, and occasionally to regions with moderate climates. Like Ae. albopictus, the “Asian bush” or “Asian rock pool” mosquito Oc. japonicus japonicus [Hulecoeteomyia japonica] is also an invasive species that has been established outside of its native range (Japan, Korea, China and Russia) for more than a decade. It was first recorded in North America in 1998 and in Europe (France) in 2000 (Schaffner et al. 2003, 2009; Williges et al. 2008). Since Oc. j. japonicus is generally found in more northern climates within its native range (whereas Ae. albopictus was originally a more tropical species), this species is able to establish itself more successfully in moderate climate zones like Central Europe (e.g. Switzerland, Belgium, Germany). Both species are able to colonise a wide range of natural and artificial breeding sites (discarded tyres, flower vases, catch basins, bird baths, tree-holes, rock pools, etc.). Due to the resistance of their eggs to desiccation, cold (diapausing larvae in eggs), and the relative lack of preference concerning their host type (e.g. humans, mammals and birds), these two invasive species have rapidly built up populations in newly colonised geographic regions (Pluskota et al. 2008). The international trade in used tyres and ornamental flowers has facilitated their spread over large distances and between continents.

The essential foundation for successful action against the mosquitoes requires not only an integrated mosquito management (IMM) concept, in which all appropriate methods for control are used, but also knowledge of the biology and ecology of the target organisms. The importance of a vector or nuisance species is determined above all by its physiological characteristics, such as reproduction, migration, host-seeking and biting behaviour. Accurate identification is a basic pre-requisite to a study of the autecology of a species as well as its biocoenotic relationship in the ecosystem. All these efforts should result in an improvement of the quality of life for humans by reducing the mosquito abundance by enhancing control measures based on IVM principles. All approaches should favour effective methods with low toxicological profile and minor environmental impact to contribute to the preservation of wetland biodiversity. Exchange of information and knowledge in the broadest sense should support sound mosquito control programmes worldwide.

Vector borne disease burden has increased considerably worldwide in recent decades, globally cases have increased from few millions to several billion per year (WHO Report, 2010). During the year 2010 - 2011 in India alone the vector-borne disease cases were about 781603 including 1390 deaths which included 745599 malaria cases with 233 deaths, 14047 cases with 93 deaths of dengue, 14820 cases of chickungunya and 7137 cases with 1064 deaths reported due to Japanese encephalitis (National Vectorborne Disease Control Program Report, 2011) (Himmat et l., 2009).

Materials and methods

Basic knowledge about the distribution, abundance, seasonality, and ecology of different mosquito species is essential for a successful control campaign against these insects. For example, knowledge of the population dynamics and migration behaviour of the target organisms are crucial to the design of a control strategy. In parasitological and epidemiological studies, the interaction between the parasite or pathogen, and the vector and host, must be evaluated in order to suppress mosquito-borne diseases successfully. In the initial phases of all mosquito control campaigns, detailed entomological studies are likely to be carried out. In this chapter, the most important methods of mosquito research are presented. A complete review of mosquito sampling techniques and the analysis of collected data are given by Silver (2008).

Table 1. The details of the localities, districts, GPS locations, date of collection and name of the species. Sl.NO. Localities Districts GPS Date of Identities readings Collection 1. Keishamthong Imphal 24047'80.0" 06/04/2018 Culex West 93055'93.3" 2. Ghari Kids’ Imphal 24044'80.0" 12/04/2018 Culex Foundation West 93072'81.3" 3. Khuman Imphal 24081'97.0" 15/04/2018 Culex Lampak East 93094'53.8" 4. Jiribam Bazar Jiribam 24081'97.0" 23/04/2018 Armigeres 93094'53.8" sp. 5. Khangabok Thoubal 24059'64.6" 25/04/2018 Culex 93099'55.7" 6. Ukhrul Bazar Ukhrul 24079'25.4" 24/04/2018 Culex 92001'02.2" 27/04/2018 7. Senapati bazar Senapati 25027'13.7" 30/04/2018 Culex 94002'24.3" 8. Urenjam Imphal 24047'16.6" 13/05/2018 Lutzia West 93052'16.0" 9. Urenjam Imphal 24047'16.6" 13/05/2018 Lutzia West 93052'16.0" 10. Urenjam Imphal 24047'16.6" 13/05/2018 Lutzia West 93052'16.0" 11. Daluan Senapati 24°58'20.4", 24/05/2018 Aedes 93°73'36.7" albopictus 12. Urenjam Imphal 24047'16.6" 31/05/2018 Lutzia West 93052'16.0" 13. Kwakeithel Imphal 24079'34.9" 01/06/2018 Culex sayang West 93091'99.1" Sambalei Sekpin 14. Singjamei Imphal 24°77'55.1", 19/06/2018 Culex West 93°93'67.2" 15. Nambol Bishnupur 24°72'04.5" 22/06/2018 Culex Chingmang 93°83'91.0" 16. Moreh 1 Tengnoupal 24°24'84.1" 24/06/2018 Culex 94°29'26.6" 17. Moreh 2 Tengnoupal 24°24'54.6" 24/06/2018 Neoculex (Premnagar) 94°30'45.0" 18. Moreh 3 Tengnoupal 24°24'54.6" 24/06/2018 Armigeres (Shinoi I) 94°30'45.0" 19. Moreh 4 Tengnoupal 24°25'17.6" 24/06/2018 Neoculex (Shinoi II) 94°30'76.9"

20. RIMS Imphal 24°81'26.6", 28/06/2018 Culex West 93°92'63.4" 21. Langol Imphal 24°83'37.1" 12/07/2018 Armigeres Wangdei west 93°91'22.2" Meitei Leikai 22. Moreh 5 (Sir Tengnoupal 24°24'546.5" 13/07/2017 Lutzia Ishwar) 94°30'45.0" fuscanus 23. Moreh 6 (Sir Tengnoupal 24°24'54.6" 13/07/2017 Culex Ishwar) 94°30'45.0" 24. Leimakhong Imphal 24°94'24.2" 16/07/2018 Culex West 93°83'64.2" 25. Heirok Thoubal 24°34'44.6" 25/07/2018 Armigeres 94°05'19.1" (17teeth) 26. Moreh 7 Tengnoupal 24°25'17.6" 25/07/2018 Anopheles (Ishwar) 94°30'76.9" 27. Moreh 8 Tengnoupal 24°25'17.6" 25/07/2018 Pseudoculex (ishwar) 94°30'76.9"

28. Moreh 1 Tengnoupal 24°24'84.1" 24/06/2018 Culex 94°29'26.6"

A total of eight districts of Manipur covering both the valley and hilly districts were covered in the for present study. The districts were Imphal west, Imphal east, Bishnupur, Jiribam, Senapati, Thoubal, Tengnoupal and Ukhrul. A total of 28 study sites were selected randomly and collected the immature stages as well as the adult mosquitoes from habiatat were collected. The immature larval stages were kept in beaker with enough foods and wait till the pupae and adults emerged. The adults were fed with rat blood and wait for egg laying. The oviposition of Culex was successful and whole life cycle could be understood. Table 2. Summary of the species diversity in respective district in the present study. Sl. Districts Habitat No. of study Species recorded NO. sites 1. Imphal East Valley 1 Culex sp. 2. Imphal West Valley 11 Culex sp., Armigeres, Lutzia, Aedes albopictus, Aedes agyepti

3. Bishnupur Valley 1 Culex sp. 4. Jiribam Valley 1 Armigeres obturbans 5. Senapati Hilly 3 Culex sp., Aedes albopictus 6. Tengnoupal Hilly 7 Culex sp., Armigeres, Lutzia, Anopheles sp 7. Thoubal Valley 2 Armigeres, Culex sp. 8. Ukhrul Hilly 2 Culex sp.

Results Systematic The genera so far studied in the present study were: 1. Aedes: Aedes albopictus, Aedes aegypti 2. Anopheles sp. 3. Armigeres: Armigeres (Armigeres) obturbans (Walk.), 1860 and Armigeres (Armigeres) aureolineatus (Leicesyer), 1908 4. Culex: Culex sp. and Culex pipiense 5. Neoculex 6. Pseudoculex 7. Lutzia: Lutzia tigripes, Lutzia fascanus

AEDES The Asian tiger mosquito is about 2 to 10 mm length with a striking white and black pattern. The variation of the body size in adult mosquitoes depends on the density of the larval population and food supply within the breeding water. Since these circumstances are only seldom optimal, the average body size of adult mosquitoes is considerably smaller than 10 mm. The males are roughly 20% smaller than the females, but they are morphologically very similar. However, as in all mosquito species, the antennae of the males in comparison to the females are noticeably bushier and contain auditory receptors to detect the characteristic whine of the female. The maxillary palps of the males are also longer than their proboscises whereas the females’ maxillary palps are much shorter. In addition, the tarsus of the hind legs of the males is more silvery. Tarsomere IV is roughly three-quarters silver in the males whereas the females’ is only about 60% silver. The other characteristics do not differentiate between sexes. A single silvery-white line of tight scales begins between the eyes and continues down the dorsal side of the thorax. This characteristic marking is the easiest and surest way to identify the Asian tiger mosquito. The proboscis is dark colored, the upper surface of the end segment of the palps is covered in silvery scales, and the labium does not feature a light line on its underside. The compound eyes are distinctly separated from one another. The scutum, the dorsal portion of an insect’s thoracic segment, is black alongside the characteristic white midline. On the side of the thorax, the scutellum, and the abdomen there are numerous spots covered in white- silvery scales. Such white-silvery scales can also be found on the tarsus, particularly on the hind legs that are commonly suspended in the air. The base of tarsomere I through IV has a ring of white scales, creating the appearance of white and black rings. On the fore legs and middle legs, only the first three tarsomeres have the ring of white scales whereas tarsomere V on the hind legs is completely white. The femur of each leg is also black with white scales on the end of the “knee”. The femurs of the middle legs do not feature a silver line on the base of the upper

side, whereas, the femurs on the hind legs have short white lines on base of the upper side. The tibias are black on the base and have no white scales. The terga on segments II through VI of the abdomen are dark and have an almost triangular silvery-white marking on the base that is not aligned with the silvery bands of scales on the ventral side of the abdomen. The triangular marking and the silvery band are only aligned on abdominal segment VII. The transparent wings have white spots on the base of the Costas. With older mosquito specimens, the scales could be partially worn off making the previously mentioned characteristics not stand out as much. The Aedes albopictus mosquito has a rapid bite that allows it to escape most attempts by people to swat it. By contrast the male member of the species primarily feeds on nectar. The female mosquito also bites other mammals and birds besides humans. They are always on the search for a host and are both persistent and cautious when it comes to their blood meal and host location. Their blood meal is often broken off short without enough blood ingested for the development of their eggs. This is why Asian tiger mosquitoes bite multiple hosts during their development cycle of the egg, making them particularly efficient at transmitting diseases. The mannerism of biting diverse host species enables the Asian tiger mosquito to be a potential bridge vector for certain pathogens, for example, the West Nile Virus and Dengue Virus. The female lays her eggs near water; not directly into it as other mosquitoes do, but typically near a stagnant pool. However, any open container containing water will suffice for larvae development, even with less than an ounce of water in. It can also breed in running water, so stagnant pools of water are not its only breeding sites. It has a short flight range (less than 200 m), so breeding sites are likely to be close to where this mosquito is found.

ANOPHELES

CULEX

Fig. 3. The larval structures of Culex pipiens A) Siphon and paddle if the IV instar, B) the enlarge view of siphon with 2 lateral setae and other setae in double row, C) Pecten teeth of 4 spines arising proximally, D) All scales of the comb evently fringed atsides and apex, E) the single setae 1-X. Bar represents 0.5 mm.

Fig. 4. Adult structures of Culex pipiens A) the wing showing subcostal intersects costa at or beyond level of furcation of R2+3, B) lateral view of adult female with foreungus simple , C) male genitalia showing broad dorsal arms truncate at apex and narrow and shaply curved ventral arms and D) two males lateral view from Leimkhong. Bar represents 0.5 mm.

LUTZIA

Fig. 5 . The morphometric of Lutzia tigripes (A, B, C, D, E, F, G, H and I) and Lutzia fuscana (J, K and L) from Manipur.

According to Lane (1953), for the Old World, Bram (1967), Tanaka (1979), Hopkins (1953) the keys for identification of the species of the Lutzia (Lt) 1. Siphon index 3.0 or greater, setae shorter than width of siphon at point of attachment (subgenus Lutzia) …………………….2 - Siphon index 2.0 or less, setae much longer than width of siphon at point of attachment (0ld World species) ……………. 3

2 (1). Pecten confined to basal 0.4 of siphon, usually 1 1 or more ……… allostigma - Pecten extending to 0.7 length of siphon usually 10 or fewer………bigoti

3 (1). Pecten closely spaced, confined to middle 0.3 of siphon; with 18-20 comb scales (sub-genus Insulahitzia) shinonagai - Pecten more widely spaced, extending to near apex of siphon, with more than 30 comb scales (subgenus Metalutzia) ………. 4

4 (3). Siphon with 10 or more pecten spines . …………tigripes - Siphon with 9 or fewer pecten spines ...... …. fuscana, halifaxii, vorax The larvae were about 0.9 cm or 9 mm twice the size of the Culex species showed the 2.0 (1mm/0.5mm) siphon index, setae much longer than width of siphon, pectin teeth more widely spaced, extending to near apex of siphon, with 32 scale combs and individual scales. the head of larva has two reddish pointed bands and one opposite to the middle (. According to http://www.wrbu.org/mqID/mq_gnra/lutzia.htm the saddle complete, extending dorsally much further than ventrally, siphon short posteroventral hair tufts and pecten along its entire length, pronotal lobes and scutellum with narrow scales, fore- and mid- femora and tibiae with anterior patches of pale scales. The adult female had quite similar features as that of the Culex. The peculiar feature was the wing venation- Cross vein rm at nearly same level as base of vein M3+4 as compare to Culex species. Again according to Ahmad et al., (2011) the key for the Lutzia tigripes was: Siphon as long as or shorter than segment X; pecten extending to apex of siphon; lateral palatal brushes thin and strongly developed for grasping. From above, the identity of the larvae and adult in the present studies was Lutzia tigripes. Report of this predacious mosquito from Manipur is unexpected as this species is exclusively for the Afro-tropical region. Four species of the subspecies of Lutzia (Lt.) are reported but not the Lt. tigripes from India. Is this the remnant of the Pangea or any introgression of the local species will be very valuable question to answer for academic as well as the bio-geographic point of view in future. Further studies on the larvae and adult mosquitoes of the subgenera Metalutzia from more study sites and through molecular as well as cytogenetic studies will be more concrete evidenced of the occurrence of the predacious mosquito from Manipur. In future controlling of the virus borne mosquito could be attempted studies through thorough bionomic studies of the species.

Biology of mosquito (Life cycle studies)

In vitro rearing of Culex pipiens in laboratory The life history of Culex pipiens is carried out at the laboratory of Entomology, P.G. Department of Zoology, D.M. College of Science, Dhanamanjuri University under the temperature 28 ± 20C and 70± 3% relative humidity. Male and female adult mosquitoes are present in about equal numbers after emergence. Generally the male mosquitoes reside near the breeding place and have a shorter lifespan than the female. The female mosquitoes travel some distance to find a blood meal. Only the female mosquitoes feed blood in order to obtain protein to produce fertile eggs. The female mate only once, as the sperm packet introduced by a male during mating act is sufficient for the female to fertilize all batches of eggs female produced. For egg production, female require protein via a blood meal. In the laboratory the female Culex mosquitoes get the blood meal from reared rats in laboratory. Egg: The female Culex mosquitoes oviposit their eggs on the water bodies. They are almost transparent when first laid, but gradually darken to brown or black as they mature. Eggs measure ¼ inch to 1/8 inch in diameter and elongate –oval in shape with the anterior end rounded ad the posterior somewhat pointed. The eggs are laid in clusters above the breeding water level. The egg rafts float on top of the water surface. They hatch after about two days on the water. Larva: The larval stages have an aquatic habitat in which to complete its development to pupal stage. The adult females select a suitable larval habitat when they deposit their eggs. The larvae grow through four instars. Between each stage the moult their rigit outer skin so they can increase in size. The discarded skin is known as exuvium or exuviae. This cast skin is eaten by predaceous mosquito larvae such as Lutzia which have cannibalistic habit. The different instars level is determined by the size of the head capsule not by the body length. Most larvae feed on microscopic organisms in the water and bottom detritus by filtering water through their mouth brushes. The larvae breathe air from spiracles at the tail end of the body through a structure known as a siphon. They hang below the water surface with only the tip of the siphon exposed to the air. They can also remain motionless on the bottom for sometime but need to return to the surface for air to prevent suffocation. The time taken for development through the different larval stages is dependent on a number of environmental factors such as temperature, food and the extent of larval crowding within the habitat. Identification of larvae is most easily accomplished with mature larvae with the help of microscope. The different developmental stages are summarized as below: First instar larva: Just hatching larvae are too small and transparent and whitish in colour. The measure about 2 to 3 mm in length and 55-60 mg in weight. The head capsule is also transparent. The duration of it is about 3-4 days.

Second instar larva: After moulting the first instar becomes second instar larva. It measures about 4 mm-5 mm in length and 80-100mg in weight. The head capsule becomes darker. The duration is about 2 days. Third instar larva: After two days it moults into third instar. It measures about 6mm- 7 mm in length in100- 120 mg in weight. The head capsule becomes darker. It also takes about two days. Fourth instar larva: It measures about 8mm- 9mm in length and 130- 160 mg in weight. The size increases and head capsule become dark or black in colour. This is the last larval stage and it takes about two days also. Pupa: The fourth instar larvae moult into a non feeding but highly mobile stage known as pupa. The pupa breathes through a pair of tube like organs known as trumpet situated at the head end of the comma shaped body. The duration of the pupal stage is also dependent on temperature but is 2-3 days. The pupa swims to the water surface and stretches itself out to full length and pupal skin splits along the back and tender adult mosquito emerges above the water surface. After emergence, the adult mosquitoes rest on the water surface for a short time to allow its wings and body to dry. Male mosquitoes develop faster than females and are the first to emerge. Adult: After emergence from the pupal case, the adult mosquitoes rest on the water surface for a short time allowing its wings and body to dry before flying off in search of mate. In a single generation, the males always develop more quickly than the females and the males are first to emerge from the last larval instar. Male mosquitoes do not travel far from the breeding site and feed on plant juices, sugars from flowers and fruit nectars. The adult female also seeks out a sugar meal of nectar to replenish expended energy reserves and then mates with a male near the breeding site at dusk. Female mosquitoes mate only once because the sperm packet introduced by a male during mating is sufficient for the female to fertilize all batches of eggs laid subsequently. In the laboratory the preferred source of blood meal is rat blood. The female searches for speculated refuge where she can rest undisturbed digest the blood meal and develop a batch of eggs again. She will then fly off in search of additional blood meals to repeat this process. Mosquitoes have sense organs which allow them to choose between physical and chemical features of an aquatic site. The identification of adult mosquitoes is very complex. All the stages in the life cycle of Culex are dependent upon environmental factors for their survival and development.

Table 3: Duration of different developmental stages of Culex pipiens in laboratory. INSTAR DATE TEMP RH (%) LENGTH WEIGHT DURATION (oC) (mm) (mg) (days) Egg 8/4/2018 28±2 70±3 1/4́΄-1/8΄́ - 3-4 First instar 11/4/2018 29±2 72±3 2-3 55-60 2 Second 12/4/2018 27±2 70±3 4-5 80-100 3-4 instar Third 15/4/2018 28±2 73±3 6-7 100-120 3 instar Fourth 17/4/2018 28±2 73±3 8-9 130-160 3 instar Pupa 19/4/2018 28±2 70±3 160 3 Adult 21/4/2018 28±2 70±3 17-19

Life Cycle of Culex pipiens

Bar representsManagement 0.5 mm

Management

Biological control

Biological control is most suitable in this context as few attempt for introduction of biological agents like larvivorus fishes i.e. gambusia and guppy have been successful (Chandra et al., 2008) bacteria (Dua et al.,1993) and predatory mosquitoes of subgenera Mucidus (Mattingly, 1961), Culex fuscanus (Ikeshoji, 1966), Toxorhynchites spp. to control Aedes aegypti and Culex quinquefasciatus larvae (Gerberg and Visser, 1978). Utilizing biological organisms to control mosquito larvae is not only eco-friendly, but constitutes a means by which more effective and sustainable control can be achieved. This would be Instars Odonata Hemiptera Diptera of (Sympatrum sp.) (Dilponicus sp.) Lutzia sp insects Replications Replications Replications 1 2 3 3 1 2 3 4 1 2 3 4 1st 2nd 3rd 4th preferable to relying solely upon synthetic insecticides which are not being developed fast enough to combat resistance. As is always the case, the elimination of aquatic larval stages is a proactive measure whereas control of potentially infective adult mosquitoes is a reactive response necessitated by inadequate management. In this context, predators should be seriously considered for they have the advantage that they can adapt to various water bodies that are enormously scattered around and within human settlements. Once established and effectively auto-reproducing, predators can achieve sustainable mosquito control to a degree that no chemical can hope to aspire (Norbert et al., 2010).

Introduction of predatory mosquito species is one of the targeted approaches for control of immature forms of mosquitoes. Lutzia (Metalutzia) fuscana is one of such species of mosquito whose larvae is reported to feed upon vector species larvae i.e Anopheles, Aedes and Culex species in several parts of India (Geetha, et al. 1982; Panicker et al.,1982). Its predatory habit was found to be excellent yet non-targeted in the mesic environmental conditions due to availability of several outdoor breeding habitats. Stable breeding of Lutzia (Metalutzia) fuscana has been first time reported from this arid zone (Singh et al., 2013). The genus of predatory mosquito Lutzia fuscana has been elevated from Culex to Lutzia (earlier Lutzia was subgenus) earlier Culex (Lutzia) fuscanus (Wiedmann, 1820) is now Lutzia (Metalutzia) fuscana (Wiedmann) (Tanaka, 2003).

Statistical results Layout of the design of experiment

Table 3. Layout of the design of experiment. 24 hour observation on consumption pattern of three different insects viz., Odonata, Hemiptera and Dipteran on different stages (instars) of mosquito larvae. Analysis of variance two-way classification with 4 observations per cell: The null hypothesis to be tested are:

H01: the different types of insects consume larvae equally. H02: there is no significant difference in the consumption of larvae in respect of different instars H03: there are no significant influences of the different types of insects on different instars of larvae.

For testing the above three hypotheses we use analysis of variance two-way classification with four observations per cell and its ANOVA is given as below:

ANOVA of two-way classification Interaction SS= Between Groups SS – (SS Instar + SS Insects) = 1972506.67- (1164912.00+426834.67) = 1972506.67- 1591746.67 = 380760.00 0r

2 2 2 2 Interaction SS = (360 /4 + 288 /4 + 0 /4 + ……………+ 1896 /4) – CF – SS Instar + SS Insect) Table 4. Two-way ANOVA Source of SS df MS F P- value variation Insects 426834.67 2 213417.34 67.08* <0.05 Instars 1164912.00 3 388304.00 122.06** <0.01

Interaction 380760.00 6 63460.00 19.95** <0.01 Within groups 114528.00 36 3181.33 Total 2087034.67 – (4X3X4) -1 = 48-1 =47 *- Significant at 5% level of significance ** - Significant at 1% level of significance

Conclusion: To find out the insect consuming larvae significantly more than others Turkey’s HSD for 3 means and 2 means are calculated and is found to be 61.19 and 48.79. Comparing the differences between the means, all means are found to be significantly difficult. The insect consuming larvae significantly than others is Odonata. In the same way, to find out the instar of the larvae at which insects consume them in significant amount Turkey’s HSD is calculated for 4, 3 and 2 means and they are 79.77, 70.66 and 56.34 respectively. Comparing the differences in the means with these values, it is found that insects consume larvae at 4th instar significantly more than the larvae at other stages. The three larvae for managing or even controlling the mosquito larvae, Lutzia might be the most efficient/effective candidate releasing in wild to control for it is inhabitable to the any habitats whether polluted or non-polluted but the Odonata and Hemiptera prefer somewhat non polluted aquatic habitats. The main focus of the study is select a bioagent that has habitat boundary or constrain and nature of aquatic habitat. Hence the efficient candidate for controlling the mosquitoes of a particular habitat will be to utilize natures’ best weapon against the mosquitoes that Lutzia tigripes.

FOLDSCOPY DO FAR

Milidew and spiderlings studies from onion root tips (see microcosmos uploaded by Dr. M. Bhubaneshwari Devi)

Chironomus studies (Head region, pupal anal spur and salivary gland)

Mitosis studies from onion root tips (see microcosmos uploaded by Dr. M. Bhubaneshwari Devi)

Pollen grains and mosquito amle genital features of Armigeres

Mosquito studies: a Armigeres and Culex (see microcosmos uploaded by Dr. M. Bhubaneshwari Devi)

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