Entomofaunal diversity of tree hole mosquitoes in Western and Eastern Ghats hill ranges of Tamilnadu, India

Accepted Manuscript Title: ENTOMOFAUNAL DIVERSITY OF TREE HOLE MOSQUITOES IN WESTERN AND EASTERN GHATS HILL RANGES OF TAMILNADU, INDIA Author: Senthamarai P. Selvan A. Jebanesan D. Reetha PII: DOI: Reference:

S0001-706X(16)30121-8 http://dx.doi.org/doi:10.1016/j.actatropica.2016.03.029 ACTROP 3901

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

14-10-2015 9-3-2016 20-3-2016

Please cite this article as: Selvan, Senthamarai P., Jebanesan, A., Reetha, D., ENTOMOFAUNAL DIVERSITY OF TREE HOLE MOSQUITOES IN WESTERN AND EASTERN GHATS HILL RANGES OF TAMILNADU, INDIA.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.03.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ENTOMOFAUNAL DIVERSITY OF TREE HOLE MOSQUITOES IN WESTERN AND EASTERN GHATS HILL RANGES OF TAMILNADU, INDIA

Senthamarai Selvan P*1., Jebanesan A 1., and Reetha, D2

1Division

of Vector Biology, Department of Zoology, Annamalai University, Annamalainagar-608 002.

2

Department of Microbiology, Faculty of Agriculture, Annamalai University, Annamalainagar–608 002.

Graphical abstract:

Highlights:



Mosquitoes are known for transmission of various disease causing organisms to humans and other living organisms.



To understand the distribution patterns of mosquito species may provide basic information about the disease incident.

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Information about mosquito’s species diversity, distribution in tree holes are fundamental to take proper control strategies in hills fringe villages.



Mosquito species distribution and diversity were studied by several workers in India and worldwide.



However, reports on diversity and distribution of tree hole mosquito species in the selected study areas are sparse.



Hence, this research contribution has more significance for basic biological research and developing control strategies for vector borne diseases.



Results of the present study highlights diversity and distribution patterns of tree hole mosquito species in Western and Eastern Ghats hill ranges of Tamilnadu, India.



ABSTRACT:

Most of the studies has been concentrated in urban areas but here we concentrate rural forest fringe villages.

The distribution and abundance of various mosquito vectors is important in the determination of disease prevalence in disease endemic areas. The aim of the present study was to conduct regular entomological surveillance and to determine the relative abundance of tree hole mosquito species in Tamilnadu, India. In addition to this, the impact of weather-conditions on tree hole mosquito population were evaluated between June, 2014 and May, 2015. Six hills ranges viz., Anaimalai hills, Kodaikanal hills, Sitheri hills, Kolli hills, Yercaud hills, and Megamalai were selected, the immatures collected from tree holes by the help of suction tube. Collections were made at dusk and dawn at randomly selected 15 different tree species. The collected samples were stored and morphologically identified to species level in the laboratory. Mosquito diversity was calculated by Simpson’s and Shannon- Weiner diversity indicies with spatial and temporal aspects. Over 2,642 mosquitoes comprising the primary vectors of dengue, chickungunya, malaria, filariasis were identified. Other species collected from the fifteen sites in each hill during the study included Christophersiomyia annularis, Christophersiomyia thomsoni, Downsiomyia albolateralis, Downsiomyia nivea and Toxorhynchites splendens, etc. Study revealed high species diversity and relative density associated with different study sites. Based on the Shannon diversity index high number of species was recorded with Aedes pseudoalbopicta (0.0829) followed by Ae. aegypti (0.0805) and least species was recorded as Anopheles elegans (0.0059). The distribution of the primary vectors of DF along the high occurrence was evident with most study sites representing proportions of this vector population. This showed the high risk level associated with the livestock movement in amplification and circulation of the virus during the outbreaks. The findings of this study, therefore, demonstrated the potential vulnerability of nomadic communities to infection by arboviral diseases transmitted by mosquito vectors.

Key words: Tree hole mosquitoes, diversity, Western and Eastern Ghats, Tamilnadu, India.

1. INTRODUCTION: Dengue, malaria, Japanese encephalitis (JE) and filariasis are the most important mosquito borne prevalent in the Southern states of India especially in hilly regions of tribals. North-eastern regions of India, though contributes to about 3.7%

Indian population, yet shares 8-10% of all malaria cases and 13-15% malaria deaths reported in the country putting severe burden on the economy of the region (NVBDCP, 2014). Predominantly Plasmodium falciparum malaria (80%), highly endemic in the hills, foot hills and forest areas of North, East and Southern India, is chiefly maintained by Anopheles stephensi and An. minimus mosquitoes. Dengue is endemic in Tamilnadu (Vellore and Chennai are highly endemic district) with the annual average morbidity of about 300 cases and 40% case fatality rate (Tyagi et al., 2015; WHO, 2013). This is the first systematic effort to determine the prevalence of the tree hole mosquito vectors with special reference to Aedes, Culex, and Anopheles mosquitoes in Western and Eastern Ghats of Tamilnadu, India. Mosquitoes are well known group of insects, which transmit many dreadful diseases causing serious public health problems to human beings. Amongst the veterinary and medically important arthropods, mosquitoes rank first in the spread of such diseases as yellow fever, dengue fever, Japanese encephalitis, malaria, filariasis as well as other viral and bacterial diseases (Service, 1980; Onyido et al., 2008). In India, mosquito control programme is generally linked with sanitation and solid waste disposal, which is carried out by local bodies like municipalities (or) panchayats. Most of them are unable to undertake effective mosquito control due to various technical and operational reasons, which largely increase the mosquito and mosquito borne disease problems in the country (Paramanik et al., 2012). There are two known tree holes (phytotelmata) types namely, the rot holes and the pan holes. Rot-holes are formed mainly due to damage of the tree woods and the damage penetrates into the heartwood. Pan-holes are formed by the branches or buttresses growing together and are lined with bark completely. Phytotelmates chiefly contain leaf litters and other detritus. These resources act as nutrients and positively influence individuals, populations, and the entire insect community. Many insects use tree holes as breeding sites and some of those are also vectors of diseases (Galindo et al., 1951). Thus, tree holes provide detritus-based resources within the forest canopy that support a wide range of wildlife species (Elliott et al., 1996). We chose tree holes for this study, because the tree holes are fully neglected when compared with other breeding sources. Tree holes are among the most abundant standing water in many tropical and temperate forests, they are the primary breeding sites for many disease vectors especially mosquitoes. Tree holes and bamboo stumps with standing water for long

periods are used more likely to have mosquito population than those containing water for shorter periods (Sota et al., 1994). Mosquito species groups, sub genus and genus have their own preferred habitat based on location and condition of the water body (Hopkens, 1952). Among the disease breeding habitats the phytotelmata are the small volumes of water held in terrestrial plant structures. The mosquito family Culicidae is regarded as ancestrally part-time dwellers, with two of its small families, Sabethini and Toxorhynchitinae are exclusively phytotelm dwellers (Kitching, 2001). Tree holes are influenced by direct precipitation and stemflow, with precipitation being frequent during spring and early summer (Carpenter, 1982; Walker and Merrit, 1988). A variety of macro-organisms use trees as breeding sites, and many species breed exclusively in this habitat, larvae of true flies (Diptera) are generally the most common and diverse inhabitant of this system. Besides abiotic risk factors and the hazards posed by invading mosquitoes, biotic factors linked to indigenous and exotic species can also play a role in possible transmission and outbreaks of mosquito borne diseases. Alterations of these factors can produce significant differences in the distribution of adult mosquito populations which may have important implications for current risk on (re)emergence and epidemiology of vector borne diseases and the implication of control strategies. Tree holes are heterotrophic microbial ecosystem because secondary production of insects living in them is dependent upon microbial assimilation and processing of detritus of external origin (Kitching, 2001). Mosquito population in tree hole ecosystems is considered primarily a function of microbial-mediated decomposition of senescent plant detritus (Fish and Carpenter, 1982, Walker et al., 1988, Kaufman et al., 2001). Microorganisms provide nutrients directly to mosquito larvae in the form of their own biomass and presumably are also instrumental larval development via release of nutrients from the leaf material. The selected study areas are Anaimalai hills, Kodaikanal hills, Yercaud hills, Kolli hills, Sitheri hills and Megamalai in Tamilnadau, India are well known tourist places in Western and Eastern Ghats. The people visit these tourist places from all over the world frequently. So we select these places for tree hole mosquito diversity studies, because study areas are acting as a suitable place for vector borne disease transmission from one place to another place. Undoubtedly the tree holes might be crucial role in propagation of mosquito species in the forest ecosystem. Understanding tree hole breeding mosquitoes distribution and ecological requirements of larval mosquito populations are important for managing potential breeding habitats

and also for predicting vector-borne disease prevalence. In India several studies have examined the relationship between mosquito abundance and physico-chemical parameters of the samples in agricultural land, cattle sheds, cess pits, drainages in plains not concerned in hills areas especially in tree holes. The present study deals with tree hole mosquitoes diversity and physio-chemical parameters in various tree species water samples, which influences the tree hole mosquito survival, growth and development. Thermal and cold fog spraying is advisable for tree hole mosquito control by National Vector Borne Disease Control Programme of India (NVBDCP, 2014). 2. MATERIALS AND METHODS: 2.1. Study Areas: Altogether survey tours were carried out between June, 2014 to May, 2015 viz., Monsoon (Jun-Oct), Winter (Nov-Feb) and Summer (Mar-May) around six hill ranges viz., Anaimalai hills (Coimbatore), Kodaikanal hills (Palani), Yercaud hills (Salem), Kolli hills (Namakkal), Sitheri hills (Dharmapuri) and Megamalai (Theni) (Figure.1). Since the studies were confined to Tamilnadu, it is mainly the Western and Eastern slopes of the Western and Eastern Ghats which have been surveyed. Studies were repeated at least twice in every month to randomly selected trees in each hill ranges. During the tree hole mosquitoes survey, we mostly concentrated forest fringe villages and tourist places. Due to the climatic variability recorded in the different hills areas, it is possible to observe large differences in the average temperatures and precipitations registered between the different categories of the natural tree holes studied. 2.1.1. Anaimalai Hills: Anaimalai hill is one of the parts of a Western Ghats, which is lying between 100 18’ 00.85” N latitude and 770 00’ 00.00” E longitude. One of the most picturesque reserves of the South, the Anaimalai wild life sanctuaries is located in these hills. It’s boundaries encompass both the dry, eastern (rain shadow) slopes and the wet western (windward) slopes of the Western Ghats. It has rich and varied vegetation including extensive forests of teak. The elevation varies from 340 m to 2400 m (1100-

7874 feet) and covers as area from the foot hills of the ghats consisting of dry scrub forests to the rolling grasslands, interspersed with pockets of true shoals in the upper reaches. The annual rainfall varies from 0.5 mm to 157.1 mm and the temperature varies from a high of 31.8 0C in the lower altitudes, in summer to a low of 22.8 0C in the higher elevations during winter. 2.1.2. Kodaikanal hills: The Palani hills are an eastern offshoot of the Western Ghats, radiating from the “Cardamom hills” south of the Palaghat gap. Which is lying between 100 14’ 18.53 N latitude and 770 29’ 20.18” E longitude. This spur is aligned on an east-west axis with a length of 65 kms and a maximum north width of 40 kms. Climate varies over the range, but much of the plateau receives an average of more than 1500 mm of rainfall annually, with no more than four dry months. The temperature of Kodaikanal is quasi-temperature, with summer (April - May) temperatures touching 23 0C max. during the day and 13 0C min. at night. Winter (December – January) temperature between 16 0C max and 14.1 0C min. Rainfall is well distributed throughout the year, with an average precipitation of 0.2-13.5 mm annually. Dindugul district is endemic for malaria and in the last few years chikungunya and some viral fever has been a regular occurrence soon after the rainy season especially in villages of the forest hill (Amala and Anuradha, 2012). Hence the present study is carried out in Kodaikanal, as one of the study area of Western Ghats.

2.1.3. Sitheri hills: The hill has an area about 654.52 sq.km which is located in Eastern Ghats of Tamilnadu, within the geographical limit of 110 53’ 29.09” N latitude and 780 30’ 38.71” E longitude located in Dharmapuri district. It is one of the segments of Eastern Ghats of Tamilnadu, which is situated at an altitude of 1097.3 meters (3600 feet) and the areas comprises vegetation such as the evergreen, semi-evergreen. The minimum and maximum temperature is 20.7 0C in winter and 33.4 0C in summer respectively. The average annual rainfall is 95 mm attained both northeast and southwest monsoons. The area is undulating with an altitude varying from 240 to 1266 m. The total area of Sitheri hills is found to be 400 km2 (Suganthi et al., 2014). Dengue has now

extended to areas including rural areas of the 30 districts in Tamilnadu, dengue cases have been reported from 29 districts between 1998 and 2005 which include Dharmapuri and Krishnagiri districts (Victor et al., 2002). 2.1.4. Kolli Hills: The Kolli hills/Kollimalai is situated in the Namakkal district lying between 110 14’ 38.68” N latitude and 78 0 20’ 10.10” E longitude. The mountains are about 1000 to 1300 m in height and cover an area of approximately 280 km2. These hills are in the southern part of Eastern Ghats, which is a mountain range that runs mostly parallel to the east coast of peninsula India. The minimum and maximum temperature ranged from 16.8 0C – 25.5 0C and the average rainfall is 1.0-16.8 mm recorded annually during study period. 2.1.5. Yercaud Hills: Yercaud hills is located at Eastern Ghats an altitude of 4,970 feet from the sea level in Salem district of northern Tamilnadu with 110 46’ 33.99” N latitude and 780 12’ 35.25” E longitude. There are 3000 trees and 1800 shrubs are grown in Yercaud hills, it spreads in an area about 382-67 Sq. km area which includes the reserve forest, while rest of this region predominantly short and rocky hills of Eastern Ghats. It has moderate humid climate in sub-tropical region. Winters are fairly mild, starting in December and ending in February. During winters the temperature recorded from 170 C to 18.6 0C, and summers from 16 0C to 23.8 0C and the annual rainfall is about 66.8-98.4 mm. 2.1.6. Megamalai: Meghamalai is a cool and misty mountain range situated in the Western Ghats in Theni district, near Kumuly Idukki district, South India. It is situated at an elevation of 1,500 m above the sea level. The latitude and longitude of Meghamalai is 9 0 42’ 54.93” N and 770 24’ 31.57 E respectively. The annual rainfall is varied from 86.5 mm – 98.1 mm and temperature varied from 19.8 0C – 30.6 0C.

Figure.1. Map showing the tree hole mosquito sampling areas of Tamilnadu, India (1-Anaimalai hills, 2-Kodaikanal hills, 3-Sitheri hills, 4-Kolli hills, 5-Yercaud hills, 6-megamalai)

2.2. Mosquitoes Collection and Processing: The main aim of this study is to monitor naturally occurring water-filled tree hole mosquitoes and their population (Figure.2). The major age old tree species such as Delonex regia, Delonex elata, Millettia pinnata, Ficus benghalensis, etc., were observed and identified as mosquito productive sites. A random sampling method was carried out across the study areas by selecting all the suitable trees (having tree holes) to accommodate immature forms of mosquitoes. Mosquito collection was carried out in atleast 25 tree holes in each site at dawn (06:00 – 09:30) and dusk (18:00 – 21:30) hours at frequently twice per

month of June 2014 – May 2015. Immature mosquitoes (larvae and pupae) were sampled by sucking 20-40 (500 mL) samples followed by a standardized larval sampling protocol at each tree holes (WHO, 2013; Silver, 2008; Senthamarai Selvan et al., 2015a). The adult mosquitoes were collected from the tree holes with the help of suction tube (having small sieve, a siphon or by a rubber suction bulb to remove the mosquitoes from the tree holes. A rubber tube of about one half inch diameter and 2 feet in length with an eight inch piece of glass tubing inserted made satisfactory siphon) and human bait protection net also used for adult collection which prevented adults from escaping during collection (Jebanesan et al., 2012). 2.2.1. Culture: Field collected immature from each tree holes were maintained in the laboratory as separately at 27±2 0C, 75-85% Rh, under 14L: 10D photoperiod cycles. The larvae were fed with dog biscuits and yeast at 3:1 ratio. The feeding was continued till the larvae were transformed into the pupal stage. The pupae were collected from the culture trays and transferred to plastic containers (12X 12 cm) containing 500 ml of water with the help of a dipper. The plastic jars were kept in 90 X 90 cm size mosquito cage for adult emergence. The cage was made up of wooden frames and covered with polythene sheets on four sides (two laterals, one back and other one upper) and the front part covered with a muslin cloth. In the bottom of the cage, placed cotton pad soaked in 10% sugar solution is provided in a petridish for a period of three days and after they are killed with chloroform and/or ethyl acetate vapours for adult identification (Senthamarai Selvan and Jebanesan, 2014a).

Figure.2. Tree hole mosquito breeding habitats (rots and pans) in the study areas (Western and Eastern Ghats) Tamilnadu, India.

2.2.2. Identification: Species of the family Culicidae differ morphologically from each other by their size, coloration and pattern of scales, setae and bristles. Principle characters distinguishing the sub families Anophelinae and Culicinae can be found in all developmental stages. The collected tree hole mosquitoes were identified into genera and species level according to the morphological keys adopted by Christophers, 1933 and Barraud, 1934. Each labeled petridish containing several collected mosquitoes were photographed in a magnified form and then identified. Again the trinocular stereo zoom microscope with digital system is used for further identification with particular reference to the head, thorax, wings and hind legs. The voucher specimens are present in the Department of Zoology, Annamalai University, Chidambaram. 2.3. Data Analysis: Community analysis was carried out during rainy season when majority of the mosquitoes were at the peak of their growth. In every study sites, 25 trees per species of 15 tree species were randomly selected in all the quadrats of 10 m X 10 m (100 sq. m). The important quantitative analysis such as density, frequency, and abundance of tree hole mosquitoes were determined as per Curtis and Mclntosh (1950). The faunal structure with environmental variables among sampling sites and effect of seasonality on mosquitoes were subjected multivariate analysis of Principal Component Analysis (PCA) by using PAST version 3.06 (Hammer et al., 2001). (a) Relative abundance: Relative abundance is the study of numerical strength of a species in relation to the total number of individuals of all the species and can be calculated as: Relative abundance = (b) Relative frequency:

Number of individuals of the species X 100 Number of individuals of all the species

This term refers to the degree of dispersion of individual species in an area and usually expressed in terms of percentage occurrence. It was studied by sampling the study area at several places at random and recorded the name of the species that occurred in each sampling units. It is calculated by the equation: Relative frequency =

Number of occurrence of the species X 100 Number of occurrence of all the species

Mosquito sampling were analyzed quantitatively to determine the total abundance, percentage abundance of each species identified during the study period, as well as determining Shannon-Wiener diversity index (H) and Simpsons dominance index (C) in the area. (c) Shannon–Weaver (1949) index of diversity: The formula for calculating the Shannon diversity index is H’ = – Σ pi In pi Where, H’ = Shannon index of diversity pi = the proportion of important value of the ith species ( pi = ni / N), ni is the important value index of ith species and N is the important value index of all the species. (d) Simpson (1949) index of Dominance: The equation used to calculate Simpson’s index was D = Σ (pi)2 Where, D = Simpson index of dominance As D increases, diversity decreases and Simpson’s index was therefore usually expressed as 1 – D or 1/ D.

2.4. Water Quality Parameters 2.4.1. Physico-Chemical Characteristics: Water samples were collected monthly twice during June 2014 – May 2015 from all selected mosquito breeding tree holes in all the study areas by small container each 500ml. Before sample collection containers were sterilized by 70% alcohol. The bottles were covered with perforated caps, labeled properly with date and place of collection. The collected water samples carried carefully and transferred to the laboratory. Within 8-10 hrs after collection the breeding water characteristics of pH, dissolved oxygen (mg/L), conductivity (µS/cm), total dissolved solids (mg/L), salinity (%), turbidity (FTU), were recorded using standard procedure (APHA, 2005). Temperature was measured by mercury thermometer at the time of sample collection (Senthamarai Selvan et al., 2015b). 2.4.2. Microbial analysis: Tree hole water samples processed for microbial analysis under sterile conditions and processed 8-10 hrs after sampling. The samples were processed and seeded onto petri dishes containing Potato Dextrose Agar (PDA), Malt Extract Agar (MEA), or Czapeck Yeast Agar (CYA) (DIFCO), to which 0.05g per 1 of chloromphenicol was added. Plates were incubated at 28 0C and examined every three days for 20 days. Cultures were identified by microscopic characteristics (sexual and asexual) using slide culture techniques and specific literature (Humber, 1998; Klich, 2002).

3. RESULTS In this study a total of 2,642 tree hole mosquitoes (immature and adults) (Table-1) were collected during June 2014 May 2015. These represented 30 different species belonging to 9 genera. The genus Aedes was the most dominant represented by 6 species of 880 individuals followed by Culex (6 species) 560 individuals, Ochlerotatus (5 species, 387 individuals), Anopheles (4 species, 281 individuals), Armigeres (137 individuals), Christophersiomyia (108 individuals), Downsiomyia (132 individuals), and Toxorhynchites (118) represented by 2 species each and Heizmania (39 individuals) represented by 1 species. Different tree species habitat types harboured one to 30 species of mosquitoes. Delonex regia harboured maximum number of tree hole mosquito (immatures and adults) individuals (650) followed by Delonex elata (417), and least mosquitoes were collected from Kaya senegalensis (29) (Figure.3). Forest areas were more species rich and 22 species were recovered from them whereas non forest areas yielded 8 mosquito species.

Number of Mosquitous

Figure.3. Tree hole mosquito collection in different tree species in Western and Eastern Ghats, Tamilnadu, 550 500 450 400 350 300 250 200 150 100 50 0

Delonex regia Pinusrox burgii Cupressus sempervirens Salix alba Millettia pinnata Memecylon angustifolium Azadirachta indica Ficus religiosa Ficus benghalensis Mangifera indica Aegle marmelos Kaya senegalensis Ailanthus excels Delonex elata Polyalthia longifolia

Monsoon (Jun-Oct)

Winter (Nov-Feb)

Summer (March-May)

Seasons June 2014 - May 2015

India Mosquitoes were collected from different altitudes, ranging between 340 meters to 4970 m above mean sea level. Maximum species were found from altitudes between 1000-3500 m. Besides larval collections, and adult mosquitoes were also collected during dusk hours, (18:00-21:30) and during day time. A total of 2,642 tree hole mosquitoes were collected representing 30 species under 9 genera. Among the species of mosquitoes encountered, one hundred and ninty are potential vectors of JE reported from elsewhere in India and 358 mosquitoes are known potential dengue and chickungunya vectors. In order to study the organization of mosquito community in different tree species habitats, and composition in various tree hole aquatic habitats in the study area was analyzed using index of Relative abundance, Relative frequency, Simpson’s index and Shanon-Weaver's diversity index. Of the 15 tree species mosquito habitats examined, Delonex rigia and Delonex elata had the highest mosquito species richness followed by Tamarindus indica. Figure.4.Distribution and larval abundance of mosquito species in sampling sites

The mosquitoes collected in the selected study areas are highest in Anaimalai having 20.6% (618), followed by Yercaud, Salem district having 15.26% (458) and least values are recorded in Sitheri hills 10.5% (315). Mosquito species collected

belonged to 30 species of 9 genera: Aedes: 6 spp., n=880 (Anaimalai-196, Kodaikanal-111, Sitheri hills-87, Kolli hills-157, Yercaud-153, Megamalai-176), Culex: 6 spp., n=560 (Anaimalai-122, Kodaikanal-86, Sitheri hills-87, Kolli hills-117, Yercaud-78, Megamalai-70), Oclerotatus: 5 spp., n=387 (Anaimalai-94, Kodaikanal-67, Sitheri hills-15, Kolli hills-59, Yercaud-99, Megamalai53), Heizmannia: 1 spp., n=39 (Anaimalai-8, Kodaikanal-25, Sitheri hills-2, Kolli hills-1, Yercaud-2, Megamalai-1), Toxorhynchites: 2 spp., n=118 (Anaimalai-40, Kodaikanal-44, Sitheri hills-6, Kolli hills-4, Yercaud-23, Megamalai-1) and Anopheles: 4 spp., n=281 (Anaimalai-48, Kodaikanal-23, Sitheri hills-85, Kolli hills-43, Yercaud-21, Megamalai-48) (Figure.5). Figure.5. Variations of tree hole mosquitoes in Western and Eastern Ghats hills of Tamilnadu, India during June 2014 – May 2015

Mosquitoes collected

250 200

196 176 157

150 100 50

122 94

153 117

111

4841 43 40 26 8

86 67 43 44 25 232631

8785

99

87

78 43

105 18156 2

59

710 11 4 1

48 21 28

6

61 23 2

70

53

5 8 11 1 1

0 Anaimalai

Kodaikanal

Sitheri hills

Kolli hills

Yercaud hills

Study Areas Aedes

Anopheles

Armigeres

Christophersiomyia

Culex

Downsiomyia

Ochlerotatus

Toxorhynchites

Heizmannia

Megamalai

The diversity can be measured using Shannon-Weaver Diversity Index together with Simson’s Dominance Index. Based on the survey, the highest diversity index was recorded for Ae. pseudoalbopicta (0.0829) followed by Cx. quinquefasciatus (0.0822) and least values were recorded in An. elegans (0.0259). Computations for dominance index for tree hole mosquitoes sampled at six different hill stations of Western and Eastern Ghats of Tamilnadu, the highest values were recorded in Ae. psedoalbopicta (0.0053) followed by Cx. quinquefasciatus (0.0051) and least in An. elegans (0.0001). Ecological statistics demonstrated the differences in diversity between species and hills stations of tree holes (Table 1 & Figure 4). The results of any field study will be more valuable if conducted in an area typical regions that may potentially be treated by any future control programme. Finding a field site which is representative of the hills stations as a whole, which is highly varied in terms of climate, land use and population density is therefore clearly impossible. The sites have a human and wild animals population abundance typical of others. The highest population of mosquitoes and species abundance observed in the rainy season of 2014, when the maximum and minimum humidity and temperature was 0.5%/157.1%; 31.8 0C/22.8 0C, respectively, and rainfall was 21.7-78.3 mm (Table 3). While minimum abundance of mosquito species was observed in the summer 2015, when the maximum and minimum humidity was 91%/59%; temperature was 33.4 0C/20.7 0C. Thus the maximum and minimum temperature 31.8 0C/22.8 0C, humidity 78.3%/21.7%, and rainfall 0.5-157.1 mm is an ideal condition for the proliferation of mosquito species. Environmental parameters around these levels can be used as early warning for the outbreaks of mosquito population which is directly related to mosquito vector borne diseases. These finding indicated that in winter the diversity is highest because of stagnant water bodies in tree holes. The ecological stability of a field sites is also important, as the less stable or the environmental conditions are more variables at present, it affecting the usefulness and reliability of surveillance results. A high value of tree hole mosquitoes was obtained in the rainy seasons (November-January), with a lower one in the cold seasons (February-March), and the lowest value was in the hot season (April-June) at all sampling sites. In the rainy season, the Shannon index was highest at Anaimalai sampling sites followed by Yercaud and Kodaikanal hills (Figure.6).

Mosquitoes collected

Figure.6. Monthly collections of tree hole mosquitoes from study areas (Anaimalai, Kodaikanal, Sitheri, Kolli hills, Yercaud and Megamalai hill ranges) of Western and Eastern Ghats, Tamilnadu, India during June 2014 – May 2015 160 140 120 100 80 60 40 20 0

Anaimalai hills Kodaikanal hills Sitheri hills Kolli hills Yercaud hills Megamalai hills

Months

Mosquitoes are cold-blooded insects whose development and behavior is strongly linked to prevailing climatic conditions such as temperature, rainfall, extreme flooding or drought, relative humidity and wind (Paaijmans et al., 2010). The global atmospheric temperatures rise began 250-300 years ago have greater impacts on mosquito borne disease that are still continuing (Reiter, 2001). The out breaks of malaria, yellow fever, and dengue reveals that climate has been the principal determinant for mosquito vectors to extend their geographic ranges and enhance their disease transmission rates. Other important considerations were socio-demographic influences such as human migration and transportation; drug resistance and nutrition; as well as environmental influences such as deforestation, agricultural expansion water projects and urbanization.

The breeding habitat is crucial for mosquito survival and its dynamics, and vital location where metabolic processes takes place like oviposition, larval and pupal development, adult emergence and probably resting, swarming and mating place. Physico-chemical factors of tree holes are influenced by atmospheric inputs, and atmospheric decomposition is a pervasive form of pollution that can disrupt community assemblages inhabiting temporary ponds and increase metal concentrations in tree bark lichens. Physico-chemical factors of tree holes in the collected mosquito breeding tree hole water indicated that the average pH value are 7.54 ± 0.2 in Kodaikanal, Palani district, and lowest are recorded (5.89 ± 0.5) in Kolli hills of Namakkal district. Whereas the conductivity (μS/cm) ranged from 6.02 ± 1.1 in Kodaikanal hills to 162.9 ± 22.3 in Anaimalai hills. The lowest phosphate (mg/L) values recorded in Kodaikanal hills (0.25 ± 1.1) and the highest in Yercaud (2.15 ± 0.25), whereas the highest total dissolved solids (mg/L) values are recorded in Yercaud hills (561.07 ± 6.65) and the lowest in Kolli hills (124 ± 3.2). The turbidity of breeding water ranged from 22.7 ± 3.08 to 253 ± 27 in over all study sites and the dissolved oxygen (mg/L) ranged from 3.64 ± 0.08 in Yercaud to 6.78 ± 0.06 in Sitheri hills (Table 4). Available data on physico-chemical characteristic of water in mosquito larval breeding tree hole habitats in Western and Eastern Ghats, Tamilnadu revealed that temperature, pH, alkalinity, hardness, phosphate, turbidity, and dissolved oxygen, varied significantly among the various tree hole habitats in different tree species. These are evidence of organic pollution in forested tree species water in the areas. Quantitative tests of tree hole mosquitoes distribution in each tree holes of the study sites, clearly showed that Aedes (33.30) mosquitoes had the highest relative abundance followed by Culex (21.19) genera and least values were recorded in Christophersiomyia (4.08) (Figure.7). Comparisons among all of the study sites, Aedes and Culex mosquitoes are significantly occur followed by Ochlerotatus and Anopheles mosquitoes during the study period. The correlation between the physicochemical characteristics and the larval density of tree hole breeding mosquitoes indicated that pH (6.8 ± 0.94) and the dissolved oxygen (6.21 ± 0.13) showed positive correlation with the larval relative density.

Relative abundance (%)

Figure.7. Relative abundance of the collected tree hole mosquitoes genera from the study areas during June 2014 – May 2015 35 30 25 20 15 10 5 0

33.3

21.19 14.64 10.63 5.18

4.08

4.99

4.46

1.47

Mosquitoes Genera

During the study period of tree hole mosquito survey (larvae, pupae, and adults), the Aedes (45.59 %) genera had a highest relative frequency in Megamalai hills followed by Kolli hills and Yercaud hills viz., 38.38 %, 33.40 % respectively. The least values of relative frequency were recorded in Heizmannia and Toxorhynchites species in all the study areas (Table 5). From our study results clearly revealed the mosquitoes species frequently lay their eggs much more in Delonex rigia tree species when compared with others.

To study the spatial pattern, six hills were selected in Western and Eastern Ghats hill ranges of Tamilnadu. Anaimalai hill areas was selected for studying temporal pattern, physical characteristics of temperature, pH, Conductivity, TDS and TSS were choosed for spatial variations. The Principal Component Analysis (PCA) to measure the relationship between distribution of mosquito species and some environmental variables. Cumulative variance was 92.55 for PC1 and 6.18% for PC2. PCA loadings of environmental variables on the mosquito species distribution in sampling sites exhibited that conductivity and total dissolved solids were important factors in PC1 and PC2 axes (Figure.8). Figure.8. PCA ordination showing the relationship between environmental variables and mosquito species distribution in sampling sites of Southern Western Ghats

Seasonal pattern analysis in Western and Eastern Ghats hills of Tamilnadu, resulted in the identification of mosquitoes representing 9 genera, 30 species and 2,642 individuals distributed in three seasons. Of these 55% of individuals account to monsoon (July-Oct). The Anaimalai hill ranges had the highest abundance of mosquitoes among 618 individuals. The effect of seasonality on

mosquito distribution was analyzed by Correspondance Analysis (CA). Figure.9 shows changes in mosquito species among seasons. Although the first axis of CA explained 43.36 % of mosquito variability and all analyzed mosquito species influenced by seasonality revealed by CA results. Figure.9. Corresponding analysis representing the distribution of mosquito species and seasonality in first and second axes in Anaimalai hills of Southern Western Ghats.

The microbial analysis revealed that the fungi Aspergillus sydowii, Fusarium merismoides,

Penicillium citrimum,

Metarhizium anisopliae, Beauveria bassiana, Alatosopora acuminate were isolated and identified from tree hole water samples

collected from the study areas. Under laboratory conditions, the isolates of B. bassiana killed An. stephensi mosquitoes, although the rate differed significantly among the isolates. M. anisopliae and B. bassiana were pathogenic to mosquitoes and they influenced abundance and occurrence of mosquitoes in tree hole water. 4. DISCUSSION Both quantitative and qualitative characters of the tree hole breeding habitats have contributed to understanding the similarity of habitat requirements of different species. Study reports the abundance of mosquitoes varies with the season and availability habitats, with other factors such as water temperature, water turbidity, nutrients, predation, parasitism, pathogens, competition, and plants can either attract or repel mosquito oviposition, similar to Imbahale et al., (2011) findings. Devi and Jauhari (2007) has studied the frequency of association of immature mosquitoes collected from the Himalayan range of India and recorded the highest association of Ae. psedoalbopicta with Cx. quinquefasciatus which is similar to the present observation. Considering the results of the present study in comparison to earlier findings, it has been found that positive associations between mosquito species from particular tree holes habitat. Maximum immature associations, as recorded in the tree hole habitats based on the physic chemical parameters of the water. Gimnig et al., (2002) have also reported that the heavy rainfall may flush out the predators and pathogens that may have previously colonized the same habitats and this could increase the mosquito larvae survival rate. Furthermore, rainfall decreases the water temperature as the raindrop temperature is less than that of surface water, and this decrease, which is larger in smaller water pools may affect the larval development and survival as well. Monsoon season brings precipitation and mosquito density is highly associated with the levels of rainfall, moderate rains may prove beneficial to mosquito breeding and abundance, but may destroy breeding sites and flush out the mosquito larvae when it is excessive. Similarly, our result concludes that some of the mosquito entomopathogenic fungus viz., Beauveria bassiana, Metarhizium anisopilae, etc., are passed out from tree holes during heavy rainy seasons, this will cause the mosquito species richness in tree holes. Sathe (2011) was stated, from November to March, Aedes and Culex mosquitoes were more prevalent leading to dengue, chikungunya, JE and filarial in the region. The

combination of favorable environment temperatures, rainfall and high relative humidity were responsible for mosquito larvae harvestation at onset of rainfall and biggest in July – August and gradual fall in October. Humidity can greatly influence transmission of vector borne disease, particularly for mosquitoes. Rainfall increases the near-surface humidity associated with rainfall enhances the mosquito flight activity and host-seeking behavior (Shaman and Fay, 2007; Senthamarai Selvan and Jebanesan, 2014b). Mosquitoes can desiccate easily and survival decreases under dry conditions and saturation deficit (similar to relative humidity) is one of the most critical determinants of climate/disease (Bradshaw and Holzapfel, 2006). Rainfall provides breeding sites for mosquitoes to lay their eggs, and ensures a suitable relative humidity of at least 50 to 60% to prolong mosquito survival. The peaks in relative humidity coincided with higher amounts of rainfall and led to increases in larvae in the following month. The environmental variables are influencing the distribution of adult and larval mosquito populations (Smith et al., 2004). From this study, we reported that during the summer, mosquitoes were recorded lowest due to rise in atmospheric and water temperatures while the mosquito habitats were dried out. During summer, the mosquito productivity was distributed and the offspring production was reduced due to high surface-water temperature this correlated with the findings of Kolivras (2006) and Senthamarai Selvan and Jebanesan, (2014a). Godwing et al., (2005) also accounted climatic parameters like temperature, relative humidity and rainfall are responsible for tree hole mosquitoes occurrence. Nutrients including sulfates and nitrates, can also adversely affect mosquito populations in tree holes, in part by changing bacterial population dynamics. Kitching et al., (1971) reported the dehydration and high temperatures may have indirectly reduced species richness in canopy of tree holes by influencing nutrient availability. Nagpal and Sharma (1985) studied the tree hole breeding and resting mosquitoes in Orissa. They reported to An. barbirostris, An. culicifacies, An. fluviatilis, An. jeyporiensis, An. nigerrimus, Cx. tritaeniorhynchus, Cx. vishnui and Ae. aegypti are breeding and resting in tree holes. The species they reported refers Butticker (1958), studied the resting and breeding of malaria vectors, An. culicifacies in tree holes in Anuradhapuram, Sri Lanka and corelated with the epidemiology of malaria. The abundance of the vector species varies in different months and seasons of the

year. The influence of the climatic and the environmental conditions on the density pattern of Ae. aegypti and Cx. quinquefasciatus have been studied by different workers. However, under natural conditions in the field, the interpretation of response of the mosquito species to the changes in climate alone is difficult because of other variables in the environment (Senthamarai Selvan et al., 2015a). Some species of trees are more attractive than others to arboviral mosquitoes are supposed to depend in some way upon the particular physicochemical characteristics which the water in the cavities derives from the surrounding wood. Very few studies have addressed the physico-chemical factors that are potentially limiting to tree hole mosquitoes population (Wilton, 1968; Paradise and Chapman, 1969). Mosquitoes inhabiting tree holes show increased mortality, decreased growth rates and smaller size when stem flow containing high concentrations of hydrogen ions (Carpender, 1982; Walker et al., 1991). Tree holes with larger volumes of water have higher densities of mosquitoes and lower concentrations of nitrate, and these larvae may be actively taking up the limited amount of nitrate in the water column. Paradise and Dunson, 1997 also reported that water and nitrate are critical to the dynamics of tree hole food webs, and that nitrate may be a limiting resource to some tree hole mosquitoes. Nutrient limitations is quite possibly a major influence on growth and survival of tree hole mosquitoes. Energy in the form of leaf litter may also be critical to some tree hole food web structure. Stem flow brings nutrients for microbes into tree holes, this would stimulate microbial respiratory metabolism, growth, and also detritus decomposition on leaf and surfaces and in the water column, thereby providing mosquito food. The heavy rain flushes toxic metabolites (ammonium and hydrogen sulfide) out of tree holes which lesser the possible mosquito mortality. Herms and Gray (1944) stated, the eggs are laid on the side of the hole just above the water surface and are deposited singly (or) separately in irregular groups onto the side wall of the tree hole as the female mosquito walks around the periphery just above the water surface. The eggs hatch when covered by rising water in the hole, provided the oxygen content of the water is sufficiently low. Reduction of dissolved oxygen in the water is said to be an important stimulus in the hatching of the eggs of mosquitoes.

Microbes in tree-hole aquatic habitats are heterotrophic microbial ecosystem. Most of the studies consider the microbes and detritus as single ecological unit and concentrate more on insect populations. Around 45 fungal species including aquatic, aero-aquatic and dematiaceous hyphomycetes were recorded from tree-hole aquatic habitats of Hungary (Gonczol and Revay, 2003) and reported that they influenced the mosquito population. It was concluded that the extreme habitat in terms of nutrient concentration is excluding photosynthetic forms from tree-hole aquatic habitat. Tree hole mosquitoes production is, therefore, thought to be tightly dependent on the microbial community dynamics, and conversely, microbial groups have been shown to respond to larval grazing pressures (Eisenberg et al., 2000; Kaufman et al., 2001). Physico-chemical factors that influence oviposition, survival and the spatio-temporal distribution of important disease vector species include salts, dissolved organic and inorganic matter, the degree of eutrophication, turbidity, presence of suspended mud, presence or absence of plants, temperature, light and shade, and hydrogen ion concentration. This study has documented the presence of 30 Culicine and Anopheline mosquito species and their habitat ecology and represents the first systematic update to the inventory and distribution of mosquitoes in Western (Anaimalai, Kodaikanal, Megamalai hills) and Eastern Ghats (Kolli hills, Sitheri, Yercaud hills) over five decades. This brings the mosquito species composition, distribution, density, seasonality, types of larval habitats and their association with water quality parameters in the study areas, a part of the Southern India. The study has also documented that certain habitat factors and physico-chemical parameters can be used to predict the occurrence of mosquito larvae. Several species of mosquitoes belonging to different genera have been reported from different parts of Tamilnadu (Jebanesan et al., 2012; Barraud, 1934; Suganthi et al., 2014; Miswar Ali et al., 2014). However, these studies were mainly confined to plains and variety of breeding places, where mosquito borne-disease were prevalent but, with regard to tree holes and hilly areas the references are rare. 5. CONCLUSION Knowledge of the taxonomic and functional biodiversity of both endemic and invading vector mosquito species as well as the factors driving change is missing in India. Acquiring this knowledge is an essential step towards understanding current

risk and preparing for future threats. Forest areas are more species rich than non-forest areas. Both the forest and non-forest areas are risk zones by contracting dengue, malaria and Japanese encephalitis. Mosquito fauna shows similarities with respect to geographical zones. Anaimalai hill area has similar mosquito fauna from rest of the study area (based on diversity index analysis). Six species of potential JE and dengue vectors are present in the study area. However, further studies are required to prove their virus transmission capability. Further we conclude complex interactions among environmental factors like minimum and maximum temperature, rainfall and flood level are involved in modulating the seasonal abundance of mosquitoes. In the present study an attempt has been made to understand diversity, distribution and tree hole breeding habitats of mosquitoes in Western and Eastern Ghats of Tamilnadu, India. Forest cover, high rainfall, high relative humidity and moderate temperature along with tree holes water made suitable conditions for mosquito breeding and proliferation. The study has revealed the presence of many vector mosquito species of various communicable diseases such as dengue fever, chickungunya, JE, malaria and filariasis in hill areas (Tyagi et al., 2015). Although sanitation and solid waste management are two of the many methods mentioned the guidelines or vector control advocated by National Vector Borne Disease Control Programme (NVBDCP, 2014), the main reliance continues to be space spraying (thermal and cold fog), larvivorous fishes, Bt and Temephos spraying in urban and rural areas in India. Introducing entomopathogenic fungus to tree holes is essential for the control of tree hole mosquitoes and subsequently the mosquito borne-diseases in forest hill areas. 6. CONFLICT OF INTEREST STATEMENT We declare that we have no conflict of interest. 7. ACKNOWLEDGEMENTS This study was supported financially by University Grants Commission under UGC– Major Research Project Scheme, UGC Ref. Letter No: 42-558/2013 (SR) dated 22.03.2013. The authors also acknowledge, the study participants, local people of study

sites for their constant support during sample collection and the Professor and Head, Department of Zoology, Annamalai University for the facilities provided.

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33. Senthamarai Selvan. P., and Jebanesan, A., 2014a. Survey and Epidemiology of tree hole breeding mosquitoes in Annamalai University campus, Tamilnadu, India. Int. J. Curr. Res. 6(5), 6462-6465. 34. Senthamarai Selvan. P., and Jebanesan, A., 2014b. Distribution study of tree hole mosquitoes (Diptera: Culicidae) during pre- and post-monsoon seasons in Kolli hills of Eastern Ghats, Tamilnadu, India. Scientific Transactions in Environment and Technovation, Vol. 8(1), 40-43. 35. Senthamarai Selvan, P., Jebanesan, A., Divya, G., and Ramesh, V., 2015a. Diversity of mosquitoes and larval breeding preference based on physico-chemical parameters in Western Ghats, Tamilnadu, India. Asian. Pac. J. Trop. Dis. 5(1), S59-S66. 36. Senthamarai Selvan, P., Jebanesan, A., and Makesh Kumar, C., 2015b. Diversity and distribution of tree hole mosquitoes in Puducherry Union Territory, India. J. Coast. Life Med. 3(7), 531-533. 37. Service, M.W., 1980. Guide to Medical Entomology. Macmillion Press Limited, London. 38. Shaman, J., Day, J.F., 2007. Reproductive phase locking of mosquitoes populations in response to rainfall frequency. Plos One. 2, e331. 39. Shannon, C.E., and Weaver, W., 1949. The Mathematical theory of communication. The University of Illinois Press, Urbana. III. 144p. 40. Silver, J.B., 2008. Sampling the larval production. Mosquito Ecology: Field sampling Methods. 3rd Ed. The Netherlands. Springer. Dordrecht. 137-338. 41. Simpson, E.H., 1949. Species diversity and its measurement. Nature. 163, -651-688. 42. Smith, D.L., Dushoff, J., Mckenzie, F.E., 2004. The risk of a mosquito-borne infection in a heterogeneous environment. PLoS Biol. 2 (11), e368. 43. Sota, T., Mogi, M., and Haymiza, E., 1994. Habitat stability and the larval mosquito community in tree holes and other containers on a temperate Island. Res. Popul. Ecol. 36, 93-104.

44. Suganthi, P., Govindaraju, M., Thenmozhi, V., Tyagi, B.K., 2014. Survey of mosquito vector abundance in and around tribal residential areas. J. Entomo and Zoo. Stud. 2(6), 233-239. 45. Tyagi, B.K., Munirathinam, A., Venkatesh, A., 2015. A catalog of Indian Mosquitoes. Int. J. Mos. Res. 2(2), 50-97. 46. Victor, T.J., Malathi, M., Gurusamy, D., Desai, A., Ravi, V., Narayanasamy, G, 2002. Dengue fever out breaks in two villages of Dharmapuri district in Tamilnadu. Indian J. Med. Res. 116, 133-9. 47. Walker, E.D., and Merritt, R.W., 1988. The significance of leaf detritus to mosquito (Diptera: Culicidae). J. Med. Entomol. 28: 581589. 48. WHO, 2013. World Malaria Report, World Health Organization, Geneva, 2013. 1-284. Table.1. Tree hole Mosquitoes of Western and Eastern Ghats of Tamilnadu, India during June 2014 – May 2015 S. No

Name of the Species

Anaimalai hills

Kodaikanal hills

Yercaud hills

Kolli hills

Megamalai hills

Sitheri hills

I

A

I

A

I

A

I

A

I

A

I

A

Total

1

Ae. aegypti

31

19

19

3

26

6

22

9

11

6

19

13

184

2

Ae. albopictus

31

11

13

2

8

9

16

11

26

13

16

18

174

3

Ae. pseudoalbopicta

26

13

21

10

41

12

19

8

30

11

0

2

193

4

Ae. subalbata

28

6

16

2

18

6

24

12

9

2

6

1

130

5

Ae. krombeini

6

3

0

8

6

0

15

4

34

17

3

0

96

6

Ae. stokesi

18

4

11

6

13

8

9

8

12

5

7

2

103

7

An. elegans

11

1

9

0

5

2

0

0

6

2

0

1

37

8

An. stephensi

3

8

0

2

4

1

16

7

21

6

19

9

96

9

An. aitekenii

7

7

6

3

3

1

7

4

12

5

16

8

79

10

An. mirans

9

2

2

1

2

3

6

3

7

2

21

11

69

11

Armigeres joloensis

6

6

1

0

16

0

3

0

2

1

6

2

43

12

Armigeres inchoatus

28

1

17

8

22

10

4

0

0

2

2

0

94

13

Christophersiomyia annularis

5

2

6

4

11

6

5

3

1

0

2

0

45

14

Christophersiomyia thomsoni

19

0

15

6

9

2

2

0

4

3

0

3

63

15

Cx. quinquefasciatus

37

7

9

4

1

3

33

13

16

19

30

18

190

16

Cx. mimulus

16

8

13

0

2

0

8

3

2

0

3

1

56

17

Cx. pseudovishnui

2

0

7

1

8

2

12

7

0

1

6

2

48

18

Cx. flagilis

11

0

2

0

16

4

3

0

3

0

0

3

42

19

Cx. flavicomis

13

0

11

8

12

10

6

8

4

6

13

4

95

20

Cx. uniformis

21

7

17

14

9

11

24

0

8

11

7

0

129

21

Downsiomyia albolateralis

28

2

21

10

3

2

0

1

8

2

11

3

91

22

Downsiomyia nivea

12

1

6

6

1

0

6

4

0

1

2

2

41

23

Ochlerotatus greeni

9

3

7

2

4

1

2

0

13

7

4

2

54

24

Ochlerotatus albocinctus

2

0

1

0

8

1

11

9

16

3

0

1

52

25

Ochlerotatus gubernatoris

10

6

3

1

12

2

18

3

0

2

0

0

57

26

Ochlerotatus khazani

31

7

19

6

16

12

9

0

8

0

3

1

112

27

Ochlerotatus pseudotaeniatus

18

8

17

11

29

14

6

1

4

0

4

0

112

28

Toxorhynchites splendens

21

10

18

14

6

2

1

0

0

1

5

0

78

29

Toxorhynchites viridibasis

9

0

12

0

10

5

2

1

0

0

0

1

40

30

Heizmannia grenii

7

1

21

4

2

0

1

0

1

0

2

0

39

TOTAL

618

456

458

409

386

315

2,642

I = Immature. A = Adult

Table.2. Tree hole mosquitoes diversity in Western and Eastern Ghats of Tamilnadu, India (June 2014 – May 2015)

S. No

Name of the Species

fi

fi log fi

fi log2 fi

Pi

Ni(ni1)/n(N1)

Pi log Pi

Pi In Pi

Pi (In Pi)2

ShannonWeiner Index H=(N log N-∑ fi log fi/N) (or) – (Pi log Pi)

Simpson’s Dominance Index C=∑ (ni/N)2

1

Ae. aegypti

184

416.7264

943.7912

0.0696

0.0048

-0.0805

-0.1854

0.4943

0.0805

0.0048

2

Ae. albopictus

174

389.8555

873.4452

0.0658

0.0043

-0.0777

-0.1790

0.4872

0.0777

0.0043

3

Ae. pseudoalbopicta

193

441.1125

1008.1355

0.0730

0.0053

-0.0829

-0.1910

0.5000

0.0829

0.0053

4

Ae. subalbata

130

274.8126

580.905

0.0492

0.0024

-0.0643

-0.1481

0.4463

0.0643

0.0024

5

Ae. krombeini

96

190.2980

377.1936

0.0363

0.0013

-0.0522

-0.1203

0.3991

0.0522

0.0013

6

Ae. stokesi

103

207.3222

417.2839

0.0389

0.0015

-0.0548

-0.1262

0.4100

0.0548

0.0015

7

An. elegans

37

58.0234

90.9904

0.0140

0.0001

-0.0259

-0.0597

0.4183

0.0259

0.0001

8

An. stephensi

96

190.2980

377.1936

0.0363

0.0013

-0.0522

-0.1203

0.3991

0.0522

0.0013

9

An. aitekenii

79

149.9125

284.4632

0.0299

0.0008

-0.0455

-0.1049

0.3683

0.0455

0.0008

10

An. mirans

69

126.8805

233.2959

0.0261

0.0006

-0.0413

-0.0951

0.3469

0.0413

0.0006

11

Armigeres joloensis

43

70.2391

114.7197

0.0162

0.0002

-0.0290

-0.0667

0.2753

0.0290

0.0002

12

Armigeres inchoatus

94

185.4740

365.9514

0.0355

0.0012

-0.0514

-0.1185

0.3956

0.0514

0.0012

13

Christophersiomyia annularis

45

74.3945

122.985

0.0170

0.0002

-0.0300

-0.0692

0.2822

0.0300

0.0002

14

Christophersiomyia thomsoni

63

113.3584

203.9562

0.0238

0.0005

-0.0386

-0.0889

0.3325

0.0386

0.0005

15

Cx. quinquefasciatus

190

432.9631

986.556

0.0719

0.0051

-0.0822

-0.1892

0.4982

0.0822

0.0051

16

Cx. mimulus

56

97.8985

171.1248

0.0211

0.0004

-0.0353

-0.0814

0.3141

0.0353

0.0004

17

Cx. pseudovishnui

48

80.6995

135.6672

0.0181

0.0003

-0.0315

-0.0726

0.2913

0.0315

0.0003

18

Cx. flagilis

42

68.1764

110.6574

0.0158

0.0002

-0.0284

-0.0655

0.2718

0.0284

0.0002

19

Cx. flavicomis

95

187.8837

371.567

0.0359

0.0012

-0.0518

-0.1194

0.3973

0.0518

0.0012

20

Cx. uniformis

129

272.2660

574.5918

0.0488

0.0023

-0.0640

-0.1473

0.4450

0.0640

0.0023

21

Downsiomyia albolateralis

91

178.2727

349.2216

0.0344

0.0011

-0.0503

-0.1159

0.3906

0.0503

0.0011

22

Downsiomyia nivea

41

66.1241

106.6328

0.0155

0.0002

-0.0280

-0.0280

0.2691

0.0280

0.0002

23

Ochlerotatus greeni

54

93.5492

162.0432

0.0204

0.0004

-0.0344

-0.0794

0.3090

0.0344

0.0004

24

Ochlerotatus albocinctus

52

89.2321

153.1192

0.0196

0.0003

-0.0334

-0.0770

0.3030

0.0334

0.0003

25

Ochlerotatus gubernatoris

57

100.0848

175.7196

0.0215

0.0004

-0.0358

-0.0825

0.3169

0.0358

0.0004

26

Ochlerotatus khazani

112

229.5124

470.3104

0.0423

0.0017

-0.0581

-0.1337

0.4231

0.0581

0.0017

27

Ochlerotatus pseudotaeniatus

112

229.5124

470.3104

0.0423

0.0017

-0.0581

-0.1337

0.4231

0.0581

0.0017

28

Toxorhynchites splendens

78

147.5833

279.2088

0.0295

0.0008

-0.0451

-0.1039

0.3662

0.0451

0.0008

29

Toxorhynchites viridibasis

40

64.0823

102.656

0.0151

0.0002

-0.0274

-0.0633

0.2654

0.0274

0.0002

30

Heizmannia grenii

39

62.0515

98.7168

0.0147

0.0002

-0.0269

-0.0620

0.2617

0.0269

0.0002

2642

5288.5996

10712.4128

0.9093

0.0374

-1.417

3.1242

11.1069

1.417

0.0374

∑

fi=Abundance of species, N=total number of individuals, Pi=Proportion of individuals found in the species, In=the natural (Naperian) logarithms (loge), (ni/N)2 = (Pi)2

Table.3. Average meteorological data in study areas during June 2014 – May 2015 Measures Max. Temp. (0C) Min. Temp. (0C)

Anaimalai Kodaikanal Sitheri hills Kolli hills Yercaud hills Megamalai 31.8

22.3

33.4

25.5

22.8 14.1 20.7 16.8 Rainfall (mm) 0.5-157.1 0.2-13.5 88.1-96.0 1.0-16.8 Rh max. (%) 78.3 71.6 31.8 92.3 Rh min. (%) 21.7 94.4 25.9 23.7 Min-minimum; Max-maximum; Temp-temperature; Rh-relative humidity

23.8

30.6

17.0 1.0-129.0 98.4 66.8

18.8 1.5-48.0 98.1 86.5

Table.4. Physico-chemical parameters of tree hole mosquito breeding habitats in study areas of Tamilnadu during June 2014 – May 2015 Name of the Station Temperature

pH

(0C)

Conductivity

Total Alkalinity

Total Hardness

(mg/L)

(mg/L)

Turbidity

(µS/cm)

(NTU)

Magnesium

Chloride

Nitrate

TDS

(mg/L)

(mg/L)

(mg/L)

(mg/L)

Total Suspended Solids (mg/L)

Phosphate

DO

(mg/L)

(mg/L)

Anaimalai hills

28

6.8±0.94

162.9±22.3

22.7±3.08

60.36±1.46

138.55±2.28

7.01±0.3

8.36±0.13

14.0±0.2

226.15±10.27

380±19.1

0.68±0.05

6.21±0.13

Kodaikanal hills

18

7.54±0.2

6.02±1.1

80.3±0.1

41±0.3

20.2±8.1

2.2±0.3

41±1.2

22±3

132±2.4

305±6.1

0.25±1.1

4.9±0.8

Sitheri hills

37

7.5±0.66

148.6±16.3

36.2±6.7

49.17±2.58

144.72±3.11

3.6±1.7

9.57±0.72

31±2.6

258.04±7.65

178±7.3

0.58±0.38

678±0.06

Kolli hills

27

5.89±0.5

241±92

253±27

58.02±11

67.3±21.1

6±0.3

35±10

13±0.5

124±3.2

460±28

10±2.1

5.49±0.7

Yercaud

25

7.1±0.14

263±41

187±19

52.36±4.5

100±5.07

5.1±1.7

26.96±1.36

16.2±2.1

561.07±6.65

368±21

2.15±0.25

3.64±0.08

Megamalai

16

7.2±0.52

98.1±11.03

46.81±6.18

73.75±3.25

127.38±2.72

7.1±3.2

10.45±0.28

17.6±2.8

298.26±9.57

323±12

1.48±0.64

6.18±0.04

Table.5. Relative frequency (%) of the tree hole mosquitoes species in the study areas during the study period June 2014 – May 2015. Name of the AnaimalaiKodaikanalSitheri Kolli Yercaud Megamalai Genera hills hills hills hills hills Aedes 31.71 24.34 27.61 38.38 33.40 45.59 Anopheles 7.76 5.04 26.98 10.51 4.58 15.80 Armigeres 6.63 5.70 3.17 1.71 10.48 1.29 Christophersiomyia 4.20 6.79 1.58 2.44 6.11 2.07 Culex 19.74 18.85 27.61 28.60 17.03 18.13 Downsiomyia 6.95 9.42 5.71 2.68 1.31 2.84 Ochlerotatus 15.21 14.69 4.76 14.42 21.61 13.73 Toxorhynchites 6.47 9.64 1.90 0.97 5.02 0.25 Heizmannia 1.29 5.48 0.63 0.24 0.43 0.25