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Use of different coloured ovitraps in the surveillance of Aedes mosquitoes in an arid-urban area of western Rajasthan, India.

INTRODUCTION

Dengue, a mosquito-borne flavivirus infection of humans is caused by four serologically distinct viruses, namely dengue virus-1, 2, 3 and 4 and is mainly transmitted by Aedes mosquitoes. Dengue and its severe manifestations--dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the serious public health problems in the tropics (1-2). In India, dengue virus infections have been frequently encountered in epidemic proportions in several states (3-6).

Aedes aegypti, the main dengue vector in many dengue-endemic countries including India, is highly adapted household-container breeder (7). Vision plays a principle role in adult mosquito biology, including location of hosts, food sources, mates, resting sites, and oviposition sites (8). Large number of studies has examined the visual parameters of shape, size, contrast, light intensity, texture, and colour attraction to host seeking mosquitoes, while few studies have explored that which of these parameters are attractive to gravid adult females (9). Most oviposition attraction studies seek to uncover specific odours generated from microbial agents that are responsible for attracting gravid females to a potential oviposition site; however, site selection is also dependent on tactile and visual cues with vision possibly as important as olfactory cues in site selection among some mosquito species (10). For diurnally active artificial-container breeders, such as Aedes aegypti (L.) and Ae. albopictus (Skuse), vision undoubtedly influences oviposition site selection; however, very little is known of the degree to which the visual parameters of colour and contrast influence oviposition site selection of either species. Diurnally active mosquitoes are believed to have better developed colour sensitivity than crepuscular or nocturnally active species (8). Ovitraps are being employed as a sensitive method in detecting the presence of Ae. aegypti (11). They are considered more sensitive than larval surveys where the population density is low and larval surveys are largely unproductive (12-13). Several improvements and modifications have been made in order to enhance the efficacy of ovitraps. Size, colour, material, paddle and oviposition attractant have been reported to be the important factors that influence the efficacy of the ovitrap (14).

In the desert areas due to shortage of water, the people have tendency to store water for a longer period in different colour containers which generally support and enhance the mosquito breeding, and their colour may play an important role in container choice among gravid females. Keeping in view this aspect, the present studies, have been planned and since ovitrap in the recent years, have been considered for the surveillance of Aedes mosquitoes, as an important surveillance tool, different coloured ovitraps have been used to study the colour preference of the gravid females of Ae. aegypti.

MATERIAL & METHODS

Study area

Jodhpur City, located in the western part of Rajasthan was selected for carrying out the experiments. Jodhpur comes under arid zone of the Rajasthan state and represents a part of Great Indian Thar Desert. Extreme of heat in summer and cold in winter are the characteristics of this city. The temperature varies from 49[degrees]C in summer to 1[degrees]C in winter. The rainy days are limited to maximum 15 in a year with an average rainfall of 302 mm. The scarcity of water and food remains throughout the year. So the common people store large amount of available water in the man-made artificial containers for rather prolonged periods, which in turn serve as potential breeding sites for the dengue vectors.

The locality Bamba Mohalla was selected for ovitrap study as the earlier studies also showed that the area has most prominent and potential breeding sites favourable for the dengue vectors (15). Bamba locality is situated in the old city area of Jodhpur with thickly populated human dwellings. Both experimental houses as well as control houses were randomly selected in the same locality. The study was conducted in March and April which was considered as transmission period of dengue in this part of desert (15).

Data collection

In all, 24 houses were randomly selected for the experiment of coloured ovitrap studies; in each experimental household five different coloured ovitraps were placed. In the experimental houses, ovitraps were fixed for seven days. Numbers of eggs laid in each ovitrap in each house were recorded for seven days on daily basis, keeping in consideration of presence of eggs larvae and pupae, the data were started collecting from the following day of installation, which is designated as Day 1 in the text. The experiment was repeated for four times in the same houses of the locality. In each household, the transparent ovitrap was considered as the control ovitrap. The number of eggs laid in each ovitrap was counted and compared with the control ovitrap (transparent) in each household. Since, the variations of colour in ovitraps were same in each house hold, therefore, only 24 houses were selected randomly so that representation of different house types can be included in the locality.

Different locations were also selected as a variation in the study for the installation of ovitraps in each household so as to find most suitable installation site of ovitrap also. After a week, the paddles were removed and Ae. aegypti eggs on the walls of the ovitrap were gently dislodged and the water was filtered using a fine strainer. The total number of eggs on the paddle and on the walls was counted. Each ovitrap was then refilled with grass-infusion and a new paddle was used each time. During the study, some eggs were allowed to hatch and develop into adults, in the laboratory, for their species identification.

Ovitrap preparation

The ovitraps consisted of one litre transparent round plastic containers of 12 cm tall by 9.5 cm wide. The oviposition substrate was a 12 x 2 cm strip of a 9 mm plywood wooden paddle covered by 9 x 2 cm strip of Whatman 42 filter paper fixed by a rubber band at one end and then placed vertically inside the container. The ovitraps were painted with four colours--black, green, orange and red, besides one, i.e. transparent. These traps were used to find out the effect of different colours on the oviposition response of Ae. aegypti females. A grass infusion of Cyndon dactylon (Fig. 1) of 50% concentration was used as oviposition attractant (250 ml/ trap) (16). The grass infusion stock solution was prepared by adding 31.25 g of grass to 7.5 liters of tap water and was kept for seven days (17). The grass without seeds was plucked and cleaned to remove the soil and only its green portion was used for the preparation of infusion after drying in shade for 2-3 days. The time of ovitrap laying was between 0900 and 1200 hrs, the time of lowest oviposition activity of Ae. aegypti (18). All ovitraps were thoroughly rinsed with de-ionized water to remove any organic matter before replacing with fresh infusion and paddle. Later to it fresh 500 ml grass infusion was added. In each house, five different coloured ovitraps were laid together at different locations, i.e. bedrooms, bath rooms, store, lobby, etc. Each coloured ovitrap was given an identification number for households and a separate number for its location specific placement. Mosquito eggs were counted and identified to species-wise based on their shape, luster, size, and colour. To prevent misidentification, 25% of all eggs were reared to adults under laboratory conditions and were identified.

[FIGURE 1 OMITTED]

Data analysis

For comparison with the weekly Ae. aegypti larval and adult surveys, the total number of eggs counted during each week, three indices were calculated as follows:

Prevalence = Total positive ovitraps/Total ovitraps installed

Intensity = Total no. of Ae. aegypti eggs collected/Total positive ovitraps

Mean egg density = Prevalence x Intensity

Various entomological indices were calculated to know correlation among them. Chi-square ([chi square]) was calculated for selection of appropriate coloured ovitrap daywise, location-wise, immature stages-wise and overall percent positivity. All levels of statistical significance were determined minimum at p = 0.01 and p = 0.001 by using a statistical programme with student t-test.

RESULTS

The observations on the percent positivity of different coloured ovitraps revealed that red coloured ovitraps have highest positivity (92.7%), followed by black and orange (91.7% each), green (76.3%) and transparent (45.8%). Day-wise percent positivity of ovitraps revealed that highest percentage of positive ovitraps was found on Day 6 (15.21%), followed by Day 2 (14.58%), Day 7 (12.71%), Day 3 (12.08%), Day 5 (10%), and least on Day 1 (7.9%). It indicates that maximum number of gravid females in the study area were present on Day 6 and minimum on Day 1 (Table 1).

On Day 1 highest number of positive ovitraps were recorded in black coloured (21.9%) while on Days 2 and 3 highest number of positive ovitraps were recorded in red coloured (37.5 and 26%, respectively). On Days 4, 5 and 6 highest number of positive ovitraps were recorded in orange colour (12.5, 21.9 and 31.3%, respectively) and on Day 7 in transparent (21.9%), followed by green colored ovitrap (20.8%) (Table 1). To find the difference in the positivity in each type of ovitraps day-wise the chi-square test was applied with the expectation of equal number of positive ovitraps each day.

It was found that on Day 1 transparent and green coloured ovitraps were significantly less positive ([chi square] = 10.9; p [greater than or equal to] 0.001 each) and black coloured ovitraps were significantly highly positive ([chi square] = 9.30; p >0.01). On Day 2 red and black coloured ovitraps were found more positive which was significantly high in comparison to other ovitraps ([chi square] = 57.6 and 26.7; p [greater than or equal to] 0.001). On Day 3 red coloured ovitraps were highly positive ([chi square] = 18.2; p [greater than or equal to] 0.001), whereas transparent ovitraps were found to be significantly less positive ([chi square] = 9.0; p [greater than or equal to] 0.01). On Day 4 the variability in the positivity of ovitraps did not differ much. On Day 5 orange coloured ovitraps were found significantly less positive ([chi square] = 9.30; p [greater than or equal to] 0.01), whereas on Day 6 orange ([chi square] = 33.3; p [greater than or equal to] 0.001) and green ([chi square] = 18.2; p [greater than or equal to] 0.001) coloured ovitraps exhibited significantly high positivity (Table 1). On Day 7 the transparent ovitraps found highly positive ([chi square] = 9.3; p [greater than or equal to] 0.01). The day-wise variability observed in positiveness of ovitraps was also found statistically significant ([chi square] = 25.13; p [greater than or equal to] 0.001 and at df = 6).

Each experiment was repeated four times and the collected data were pooled for number of eggs laid in different coloured ovitraps. The results showed that black coloured ovitraps fetched highest percentage of eggs (37.78%) which was found significantly higher ([chi square] = 1664; p [greater than or equal to] 0.001) than green, orange, red and transparent coloured ovitraps, whereas least egg percentage was found in the transparent ovitraps (4.33%--Table 2).

The location-wise positivity of the ovitraps revealed that highest positivity was recorded among the ovitraps installed inside bathrooms (92%), followed by bedrooms (85%), lobby (66.7%), and stores (48.3%--Table 3). The highest positivity of all coloured ovitraps, except trans parent one, was found in bathrooms, followed by bedrooms. The black, green and red coloured ovitraps were found least positive when installed inside stores (41.7, 33.3 and 66.7%, respectively), whereas orange coloured ovitraps inside lobby (66.7%) (Table 3). The transparent ovitraps were not found positive inside lobby and stores. The overall percent positivity was found highest in case of red coloured ovitraps (92.7%), followed by black and orange (91.7% each), green (76%) and least in transparent ovitraps (45.8%) and the difference between transparent and orange coloured ovitraps was found to be statistically significant ([chi square] = 7.2; p [greater than or equal to] 0.01) and between transparent and black or red colored ovitraps was also found to be more statistically significant ([chi square] = 15.8; p [less than or equal to] 0.001).

The prevalence was calculated as 0.9 for orange coloured ovitraps, whereas the intensity, mean egg density and relative percentage of eggs results exhibited higher values in case of black ovitraps (Table 4), which indicated that the orange coloured ovitraps having grass infusion, as attractant, can be used for the detection of the presence of the vector species, but for quantification or correlation purposes with adults, the black coloured ovitraps may be preferred.

DISCUSSION

Epidemiologically, gravid females are the most important component of the mosquito population and are targeted in mosquito control programmes in active surveillance of disease for the early detection of epidemic events. Field mosquitoes are also collected using the ovitrap technique, the cheapest and easiest method for collecting Aedes mosquitoes (19). Fay and Perry (20) were first to use ovitraps for Ae. aegypti (Linn.) surveillance, and Fay and Eliason demonstrated that the ovitrap was in some aspects superior to larval surveys (21). Ovitraps were also shown to be useful sampling devices in determining Ae. aegypti distribution and seasonal population fluctuation (22). Ovitraps are relatively easy to construct and are sensitive in detecting the presence of gravid females, even at low population densities, making them ideal for surveillance and control of vector species (23).

The studies have demonstrated the advantages in time and labour savings and increased sensitivity in using artificial oviposition devices as compared to various adult traps for surveillance of Ae. aegypti (22-24). The addition of insecticides or insect growth regulators to the traps renders them lethal ovitraps (25). Behavioural choices of female mosquitoes are clearly affected by colour, either as a substrate or as light. Snow surmised that Ae. aegypti were most sensitive to green-orange light (470-610 nm) and thus avoided those colours while seeking more cryptic oviposition sites (26). Earlier studies have also exhibited that black containers are the most attractive coloured targets for male (27) and female (28) mosquitoes.

Similarly, as observed during present studies, Yap (29) also found that ovitraps which are black in colour are more attractive to mosquitoes. Yap (19) in another study in Malaysia found that gravid Ae. albopictus in rural habitats oviposited more in red and black ovitraps than in blue, yellow, green, white, and plain (unpainted) ovitraps. Later, in the laboratory studies, gravid females of Ae. albopictus laid significantly more eggs in black, red, and blue ovitraps than in green, yellow, white, or clear (unpainted glass) ovitraps (30), which correlated well with Snow's (26) findings for colour preferences of gravid Ae. aegypti. Yap et al (30) also reported preference for dark coloured glass jars, especially black, blue, and red ones over light coloured jars by Ae. albopictus mosquitoes during their study.

Hoel et al (31) tested five choices of colours against Ae. albopictus in Florida and based on the mean eggs collected, the choices of colours were black > blue > checkered > orange > striped and white. Like our studies, they also found preference of black colour more than the other competing colours and also observed the positive response of gravid Ae. albopictus to black and orange targets and concluded that orange is perceived as an attractive stimulus. As concluded by Hoel et al (31) that traditionally used black ovitraps do produce superior results, in our study too, the black coloured ovitraps proved to be fastest attracting colour for ovipositing females of Ae. aegypti. Burkett and Butler (32) demonstrated that orange lighted targets attract host seeking Ae. albopictus, and Muir et al (28) also reported that Ae. aegypti can detect orange light based on the electoretinographic examination and also revealed spectral sensitivity ranging from UV to orange coloured light.

The study reveals that the colour of the ovitraps plays an important role in attracting the ovipositing females of Ae. aegypti species and due attention can be given while considering the colour of the ovitraps, to be used for different objectives of the investigations. The ovitraps of black colour were found to be appropriate. The grass infusion of Cyandon dactylon due to its easy availability can be a very useful for attracting gravid female and this combination can be a good vector surveillance tool in case where gravid females are not traceable during the household surveillance and would be helpful in detection of gravid females more appropriately even in the area of low density.

ACKNOWLEDGEMENTS

The authors are thankful to the Director, Desert Medicine Research Centre (ICMR), Jodhpur for facilitating and permitting the study. They are also thankful to the staff of Vector Biology Laboratory who supported in the field during the conduct of this study.

REFERENCES

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(32.) Burkett DA, Butler JF. Laboratory evaluation of coloured light as an attractant for female Aedes aegypti, Aedes albopictus, Anopheles quadrimaculatus, and Culex nigripalpus. Fla Entomol 2005; 88: 383-9.

Rina Kumawat [1], Karam V. Singh [2], S.K. Bansal [2] & Himmat Singh [3]

[1] Epidemiologist, Office of CMHO, Jodhpur; [2] Desert Medicine Research Centre (ICMR), Jodhpur; [3] National Institute of Malaria Research, New Delhi, India

Correspondence to: Dr Karam V. Singh, Scientist 'F', Vector Biology Laboratory, Desert Medicine Research Centre (ICMR), New Pali Road, Jodhpur-342 005, India.

E-mail: karamvs@gmail.com

Received: 8 March 2014 Accepted in revised form: 25 July 2014
Table 1. Day-wise percent positivity of individual ovitraps recorded
during the study

Colour type       Day-wise percent positive ovitraps

                      Day 1              Day 2

Transparent       0 *                2.08
Black            21.88 ([dagger])   29.17 ([dagger])
Green             0 *                1.04
Orange            3.13               3.13
Red              14.58              37.5 ([dagger])
Total ovitraps    7.92              14.58
  positive/day
  (n = 480)

Colour type       Day-wise percent positive ovitraps

                      Day 3         Day 4    Day 5

Transparent       1.04 *             3.13    7.29
Black            19.79               9.38    4.17
Green             8.33               7.29   12.5
Orange            5.21              12.5    21.88 *
Red              26.04 ([dagger])    3.13    4.17
Total ovitraps   12.08               7.08   10
  positive/day
  (n = 480)

Colour type           Day-wise percent        Individual    Total %
                      positive ovitraps       % positive   positive
                                               (n = 96)    (n = 480)

                      Day 6          Day 7

Transparent      10.42              21.88 *     45.84         9.17
Black             4.17               3.13       91.69        18.33
Green            26.04 ([dagger])   20.83       76.03        15.21
Orange           31.25 ([dagger])   14.58       91.68        18.33
Red               4.17               3.13       92.72        18.54
Total ovitraps   15.21              12.71                    79.58
  positive/day
  (n = 480)

* Represents test of significance at p > 0.01; ([dagger]) Represents
test of significance at p > 0.001 ([chi square] calculated on
original values).

Table 2. Day-wise egg laying percentage of individual coloured
ovitraps recorded during the study

Colour type   Total no.        Day-wise egg laying (%)
              of eggs

                          Day 1   Day 2   Day 3   Day 4

Transparent    456 *       0       5.26    6.58   20.61
Black         3976 *      33.48   37.63   21.1     5.28
Green         1094         0       3.02   30.16   21.02
Orange        1562        12.74    6.91   10.37   18.05
Red           3435 *      18.43   53.28   24.48   1.08

Colour type    Day-wise egg laying (%)Percentage
                                       of eggs

              Day 5   Day 6   Day 7

Transparent   30.04   19.52   17.98    4.33
Black          1.26    0.98    0.28   37.78
Green         18.65   20.02    7.13   10.4
Orange        22.54   24.46    4.93   14.84
Red            1.31    1.19    0.23   32.64

* Chi-square test significant at p > 0.001.

Table 3. Location-wise positivity of different coloured ovitraps
observed in the study area

Colour type       Different locations of traps
                           installed

                          Bathrooms

                 No.        No.          %
              installed   positive   Positivity

Transparent       20         12          60
Black             20         20         100
Green             20         20         100
Orange            20         20         100
Red               20         20         100
Total            100         82          92

Colour type       Different locations of traps
                           installed

                          Bedrooms

                 No.        No.          %
              installed   positive   positivity

Transparent       52         32         61.5
Black             52         51         98.1
Green             52         41         78.9
Orange            52         48         92.3
Red               52         49         94.2
Total            260        221          85

Colour type       Different locations of traps
                           installed

                           Lobby

                 No.        No.          %
              installed   positive   positivity

Transparent      12           0        0
Black            12          12      100
Green            12           8       66.7
Orange           12           8       66.7
Red              12          12      100
Total            60          40       66.7

Colour type       Different locations of traps
                           installed

                           Store

                 No.        No.          %
              installed   positive   Positivity

Transparent      12           0        0
Black            12           5       41.7
Green            12           4       33.3
Orange           12          12      100
Red              12           8       66.7
Total            60          29       48.3

Colour type       Different locations of traps
                           installed

                           Total

                 No.        No.          %
              installed   positive   Positivity

Transparent       96         44      45.8
Black             96         88      91.7
Green             96         73      76
Orange            96         88      91.7
Red               96         89      92.7
Total            480        382      79.6

Table 4. Observations on trap positivity and egg collection with
respect to different colours

Coloured        Total      Total     Prevalence   Total eggs
ovitrap       ovitraps    positive                in positive
              installed   ovitraps                  ovitrap

Transparent      96          48         0.50          456
Black            96          84         0.88         3976
Green            96          68         0.71         1094
Orange           96          88         0.92         1562
Red              96          84         0.88         3435

Coloured      Intensity   Mean egg    Relative
ovitrap                   density    percentage
                                     of eggs in
                                     different
                                      ovitraps

Transparent      9.5        4.75         4.3
Black           47.3        41.6        37.8
Green           16.1        11.4        10.4
Orange          17.8        16.4        14.8
Red             40.9         36         32.6
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Author:Kumawat, Rina; Singh, Karam V.; Bansal, S.K.; Singh, Himmat
Publication:Journal of Vector Borne Diseases
Article Type:Report
Geographic Code:9INDI
Date:Dec 1, 2014
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