variation in morphology and morphometrics of eggs of culex quinquefasciatus mosquitoes from...

Vol. 34, no. 2 Journal of Vector Ecology 191

Variation in morphology and morphometrics of eggs of Culex quinquefasciatus mosquitoes from different ecological regions of India

D.S. Suman1, A.R. Shrivastava1, B.D. Parashar1, S.C. Pant1, O.P. Agrawal2, and Shri Prakash1

1Defence Research and Development Establishment, Gwalior - 474002, India2School of Studies in Zoology, Jiwaji University Gwalior - 474011, India

Received 20 January 2009; Accepted 12 June 2009

ABSTRACT: Variation in egg surface morphology and morphometrics of Culex quinquefasciatus mosquitoes of the Jodhpur, Bikaner, Jamnagar, and Bathinda strains were correlated with geographical distribution in different ecological regions of India. We report the geographic variation in Cx. quinquefasciatus based on 44 attributes of micropylar and conical-shaped regions of eggs, including micropylar apparatus (corolla, disc, and mound), micropylar tubercles, and the exochorionic tubercle, pores, and network in anterior, middle, and posterior regions. No remarkable differences were observed in the surface morphology of eggs of these strains except the absence of small tubercles in the anterior and middle region of the JMN strain. However, a statistical analysis indicated significant morphometric variations in 66% of the attributes of the eggs. The cluster analysis of all egg attributes showed that the JD, BKN, and BTH strains are closer to each other than the JMN strain. The positive correlation (r = 0.95) also indicated an effect of geographical distribution on morphometry of various egg attributes of these strains. The present study suggests that ecological variation may have affected the morphometric attributes of the egg of four strains of Cx. quinquefasciatus from different geographical areas. Journal of Vector Ecology 34 (2): 191-199. 2009.

Keyword Index: Culex quinquefasciatus, egg morphology, morphometrics, geographical variations, ecological regions, cluster analysis.

INTRODUCTION

Culex quinquefasciatus Say, 1823 is a common domestic mosquito species that has been recorded throughout the world (Sirivanakaran 1976). It serves as a primary vector of Bancroftian filariasis in India and tropical and subtropical Oriental, African, and South American countries (WHO 1989, NVBDCP 2008). West Nile and St. Louis encephalitis viruses have been isolated from this species in the U.S.A. (WHO 1989, Reisen et al. 2004). About 21 million cases of symptomatic filariasis and about 430 million individuals potentially at risk of infection have been reported in India (Sabesan 2000). Presently, 1.3 billion people are at risk of this disease and 120 million people are affected in 83 countries (WHO 2006).

Variations in geographical and ecological factors have been reported to induce variations in genetic profile, morphological attributes, life table parameters, and mosquito bionomics (Walter and Hacker 1974, Reisen et al. 1979, Linley et al. 1996, Manguin et al. 1999, Cornel et al. 2003, Yurttas et al. 2005, Yurttas and Alten 2006). Differences in the length of eggs and floats and in the number of float ridges in the eggs of Anopheles stephensi from various parts of India have been described by Rao et al. (1938). Differences in the sizes of Cx. pipiens eggs have been observed (Christophers 1945, Hinton 1968, Chadee and Haeger 1986). Variations in egg morphology and morphometrics in strains of anopheline species have also been reported, including Anopheles quadrimaculatus (Linley

et al. 1993), Anopheles nuneztovari (Linley et al. 1996), and members of the Anopheles gambiae complex (Lounibos et al. 1999) belonging to different geographically isolated regions. Recently, Suman et al. (2008) have identified Cx. quinquefasciatus and Cx. tritaeniorhynchus on the basis of egg morphometrics. However, no report is available on the variations in morphology and morphometrics of eggs of Cx. quinquefasciatus from different geographical areas in India. Eggs of all mosquito species have a chorion comprised of the external exochorion and internal endochorion (Clements 1996). The surface morphology and morphometric analysis of mosquito eggs is based mainly on these exochorionic structures.

In the present study, we investigated the possible effects of geographical distribution in varying ecological regions on variations in egg morphology and morphometrics of Cx. quinquefasciatus found in the Thar Desert region (Jodhpur and Bikaner), a semi-desert region (Bathinda), and the coastal region of the Arabian Sea (Jamnagar) of India.

MATERIALS AND METHODS

Mosquito collection and colonizationAdult Cx. quinquefasciatus mosquitoes were collected

from four different geographical regions of India, i.e., JD (Jodhpur, Rajasthan), BKN (Bikaner, Rajasthan), JMN (Jamnagar, Gujarat), and BTH (Bathinda, Punjab), belonging to three ecological regions (Figure 1 and Table 1). These mosquito strains were maintained in the laboratory at 27 1 C, 75 5% relative humidity, and a 12:12 h light:dark

192 Journal of Vector Ecology December 2009

period. Adult mosquitoes were provided ad libitum with a 10% sugar solution dispensed through cotton wicks. Chickens were offered as a blood source to the mosquitoes twice a week. Egg rafts of Cx. quinquefasciatus of JD, BKN, JMN, and BTH strains were obtained from approximately one-month-old field colonies. Data on temperature and annual rainfall were obtained from the meteorological departments of corresponding areas.

Morphometrics, statistical analysis, and terminologyFor SEM examination, eggs (24-36 h) were processed

according to Junkum et al. (2004), mounted on stubs, sputter-coated with gold, and length and width measured under an FEI Quanta 400-EDAX (Netherlands) scanning electron microscope (Suman et al. 2008).The length of the egg was measured between the anterior and posterior ends, not including corollar length. The corolla was attached to the micropylar apparatus that had been removed by fine drawing brushes under the microscope. The tubercles of the eggs were classified according to Suman et al. (2008) as small (0.8 m and 1.6 m diam.). Exochorionic pores were categorized as small (< 0.6 m) and large (> 0.6 m).

For morphometric study, we considered 44 attributes of eggs. These included length, width, and ratio of length to width. Attributes of the micropylar apparatus included the diameters of the corolla, disc, mound, tubercle of small, medium or large size, length and the number of tubercular rows and the conical-shaped regions (diameter and density of small and medium tubercles and small and large exochorionic pores, width of exochorionic bridges, and the area of small and medium tubercular wheel units from the anterior, middle, and posterior regions).

Statistical comparisons among the four strains were performed with a one-way ANOVA for significance of

differences (p


Table 2. Dimensions of entire eggs and some attributes of the micropylar regions of eggs of four strains of Cx. quinquefasciatus.

Attributes JD strain BKN strain JMN strain BTH strain LSDEntire egg

Egg length 590.5026.16a 659.7328.92b 619.4020.64c 611.0026.95ac 23.45

Egg width 194.509.18a 155.379.01b 169.5011.59c 159.0012.20b 9.61

ELWR 3.030.13a 4.250.20b 3.670.23c 3.860.39c 0.23

Micropylar region

Crl (diameters) 44.401.67

a 42.701.48a 40.732.49b 44.501.93a 2.59

Tl (diameters) 2.600.26ac 2.390.43ab 2.880.34c 2.240.15b 0.28

Tm (diameters) 0.990.14

a 1.240.19a 1.130.24a 1.120.18a NS

Ts (diameters) 0.740.07a 0.750.05a 0.700.08a 0.750.05a NSMeans within rows followed by the same letter do not differ significantly. All values are (m) Mean SD. LSD (least significant difference at 95%).Abbreviations: Crl, micropylar corolla; ELWR, ratio between egg length and width; NS, non-significant; Tl, large tubercle; Tm, medium tubercle; Ts, small tubercle; JD, Jodhpur; BKN, Bikaner; JMN, Jamnagar; BTH, Bathinda.

N

Arabian Sea

Figure 1. Locations of collections of Culex quinquefasciatus in India. 1, Bathinda; 2, Bikaner; 3, Jodhpur; 4, Jamnagar.

12

3

4


Attributes JD strain BKN strain JMN strain BTH strain LSDAnterior region

Tm (diameters) 0.880.07 a 0.990.42 a 1.250.20 a 1.150.21a NSTs (diameters) 0.380.14a 0.780.02b - 0.80.00b 0.117Pl (diameters) 0.950.17a 0.910.11a 1.550.21b 1.100.20a 0.24Ps (diameters) 0.270.17a 0.270.15a 0.330.13a 0.260.22a NSTm (density) 8.201.09a 7.800.44a 4.200.83b 3.400.89b 1.142Ts (density) 1.601.14a 1.200.44a - 1.600.54a NSPl (density) 7.601.14a 8.400.54a 4.401.14b 5.602.40b 1.97Ps (density) 10.001.58a 6.001.00b 4.200.83c 10.001.22a 1.66

Middle regionTm (diameters) 0.840.06a 1.150.11b 1.290.23b 1.250.15b 0.209Ts (diameters) 0.410.10a 0.630.09b - 0.740.8b 0.13Pl (diameters) 0.830.05a 0.900.13a 1.180.24b 0.950.10a 0.201Ps (diameters) 0.400.09a 0.220.11a 0.230.07a 0.270.17a NSTm (density) 8.801.48a 7.000.70b 4.401.14c 4.600.89c 1.469Ts (density) 3.401.14a 2.400.54a - 0.800.83b 1.27Pl (density) 6.401.67a 3.601.14b 4.001.00b 1.801.09c 1.68Ps (density) 15.201.48a 15.402.96a 5.401.14b 7.000.70b 2.39

Posterior regionTm (diameters) 1.050.86a 0.920.13b 1.140.04a 1.020.09ab 0.125Ts (diameters) 0.540.12a 0.530.10a 0.620.10a 0.620.11a NSPl (diameters) 0.790.35ab 0.730.11a 0.940.09c 0.920.10bc 0.125Ps (diameters) 0.300.07a 0.290.06a 0.250.1 a 0.360.96a NSTm (density) 9.001.00a 8.601.14a 4.200.83b 4.800.83b 1.289Ts (density) 2.200.44a 3.401.34b 3.000.70ab 6.800.44c 1.102Pl (density) 12.402.88a 2.600.89b 3.401.14b 6.201.30c 2.33Ps (density) 17.004.63ab 13.601.67a 19.002.73b 8.601.67c 3.94

Table 3. Diameter and density of tubercles and exochorionic pores in the conical regions of eggs of four strains of Cx. quinquefasciatus.

Means within rows followed by the same letter do not differ significantly. All values are Mean SD. LSD (least significant difference at 95%). Measurement of density in number of tubercles and exochorionic pores per 25m2 and diameter in m.Abbreviations: NS, non-significant; Pl, exochorionic large pores; Ps, exochorionic small pores; Tm, medium tubercles; Ts, small tubercles; JD, Jodhpur; BKN, Bikaner; JMN, Jamnagar; BTH, Bathinda.


respectively. In the micropylar region, small, medium, and large tubercles were present around the micropylar apparatus in the form of tubercular rows. Among these tubercles, only large tubercles showed significant differences among the four strains. Both the number and length of tubercular rows were 25.80 0.83 and 22.20 2.79 in the JD strain, 27.20 1.30 and 18.92 1.56 in the BKN strain, 26.80 1.64 and 19.56 3.17 in the JMN strain, and 26.80 0.45 and 21.10 4.87 in the BTH strain, indicating no significant differences among all four strains.

The conical-shaped region of the egg includes the anterior, middle, and posterior regions. It is broader at the anterior region and becomes narrower gradually towards the middle region and slightly pointed at the posterior region. As in the micropylar region, the conical-shaped regions are also ornamented with tubercles, but there are small and medium tubercles present in this region without the large tubercles. Tubercles present in the anterior and middle regions of the eggs are rounded and become sub-fusiform in the posterior region (Figures 2d, 2e, and 2f). Small tubercles have been observed in the anterior, middle, and posterior regions of the eggs of the JD, BKN, and BTH strains, except for the anterior and middle regions of the JMN strain eggs. The medium tubercles are present in all these regions of eggs for all four strains. Dimensions and density of these tubercles of eggs of the four strains indicated significant to non-significant variations for various attributes (Table 3).

The exochorionic membrane is perforated with small and large, rounded or polygonal, pores. These exochorionic pores are situated around the tubercles in all four strains of Cx. quinquefasciatus (Figures 2d, 2e, and 2f). The size of small and large exochorionic pores in various regions of eggs ranged between 0.22 to 0.40 m and 0.73 to 1.55 m, respectively, indicating significant differences in the large exochorionic pores only (Table 3). The density of both small and large exochorionic pores in different regions of the eggs of all four strains showed non-significant to significant differences.

Exochorionic bridges connect the tubercles. The width of these bridges in eggs of the JD, BKN, JMN, and BTH strains was 0.270.17 m, 0.450.30 m, 0.610.49 m, and 0.470.44 m in the anterior regions, 0.250.15 m, 0.360.37 m, 1.020.81 m, and 0.700.58 m in the middle regions, and 0.240.11 m, 0.200.07 m, 0.220.11 m, and 0.270.17 m in the posterior regions, with no significant differences among all the four strains.

The area of both medium and small tubercular wheel units of the anterior, middle, and posterior regions of eggs of all the four strains differed significantly among these strains (Figures 2d, 2e, 2f, and Table 4).

Cluster and regression analysis of various egg morphometric parameters

The cluster analysis of all 44 attributes of eggs of the four strains of Cx. quinquefasciatus showed two clusters, one comprised of the JD, BKN, and BTH strains, and the other of the JMN strain alone. The distance coefficient between both of the clusters is 90.89. The BTH and BKN (59.82) strains are closer to each other and neighboring the JD strain (67.53) and farthest to the JMN strain (90.89) (Figure 3).

Regression analysis (Figure 4) between the cluster analysis distance coefficients of egg attributes of Cx. quinquefasciatus and geographic distance (kilometers) for the JD, BKN, JMN, and BTH strains showed a strong positive correlation (r = 0.95).

DISCUSSION

Mosquitoes from different geographically isolated locations and living under different ecological conditions normally become adapted to those conditions after a varying period of time, often leading to the development of geographically isolated strains of mosquitoes that may differ in various biological traits, such as morphology (Linley et al. 1996, Belen et al. 2004, Yurttas et al. 2005), genetics (Manguin et al. 1999, Cornel et al. 2003), and

Attributes JD strain BKN strain JMN strain BTH strain LSDAnterior region

MTWU 9.991.54a 20.026.21b 24.136.57b 19.464.68b 6.907STWU 5.851.02a 9.411.74b - 10.262.07b 2.298

Middle regionMTWU 8.791.24a 14.401.17ac 23.168.47b 17.594.06bc 6.407STWU 5.191.02a 7.311.45a - 11.012.12b 2.205

Posterior regionMTWU 10.091.22a 10.882.03a 12.842.36a 28.287.46b 5.48STWU 5.120.53 a 5.531.71 6.652.45 19.865.05b 3.954

Table 4. Area of medium tubercle wheel unit (MTWU) and small tubercle wheel unit (STWU) in the conical regions of eggs of four strains of Culex quinquefasciatus.

Means within rows followed by the same letter do not differ significantly. Area in m2 (Mean SD). LSD (least significant difference at 95%).


Figure 2. Representative scanning electron micrographs of eggs of the JD strain of Culex quinquefasciatus; a: entire egg (400X); b: micropylar end with corolla (2000X); c: micropylar end without corolla (2000X); and d: anterior region; e: middle region; and f: posterior region (20000X). Abbreviations: A, anterior region; Cr, conical-shaped region; Eb, exochorionic bridges; MC, micropylar corolla, Md, micropylar disc; MP, micropylar pore; Mr, micropylar region; Mmd, micropylar mound; MTW, medium tubercle wheel unit, P, posterior region; Pl, large exochorionic pore; Ps, small exochorionic pore; Rt, rounded tubercles; Sft, sub-fusiform tubercles; STW, small tubercle wheel unit; Tl, large tubercle; Tm, medium tubercle; Tr, tubercular row; Ts, small tubercle.

MC

Tr

MP

b Mr Cr

a

A P

Md

Mmd

Tr c

Tl

Ts

Tm

Tm

STW

MTW

Ts

Pl Ps

Eb

Rt d

MTW

STW

Ts Tl

Eb Ps

Pl

e Rt

Sft


life-table parameters (Walter and Hacker 1974, Reisen et al. 1979, Yurttas and Alten 2006). The morphological variations in structural features, such as length and banding pattern of appendages, have been previously reported in geographically isolated strains of Anopheles darlingi (Manguin et al. 1999). Variation in egg morphology and morphometrics have also been reported in geographically isolated strains of different species of mosquitoes including the Anopheles quadrimaculatus Complex (Linley et al. 1993), An. nuneztovari (Linley et al. 1996), An. gambiae, An. arabiensis, An. bwanbae, and An. merus (Lounibos et al. 1999).

In the present study, variation in morphology was only observed for the small tubercles of the Jodhpur, Bikaner, Jamnagar, and Bathinda geographic strains of Cx. quinquefasciatus. However, there were statistically significant differences in the morphometrics of eggs as a whole, and different structural features were observed among JD, BKN, JMN, and BTH strains. Differences in length and width of the eggs among the four strains were

significant; the JD and BKN strains showed the minimum and maximum mean length of eggs, respectively, whereas the mean width of eggs is minimum in BKN strain and maximum in JD strain. Variations in egg length and width ratios also showed significant differences among the four strains of Cx. quinquefasciatus belonging to the different ecological regions of India. Although these dimensions are nearer to a description of Cx. quinquefasciatus, they are greater than those of Cx. tritaeniorhynchus (Suman et al. 2008). Similarly, significant differences in egg length have been recorded in some strains of the An. quadrimaculatus complex from five different locations of Florida, whereas variations were not significant in width and length-width ratios of the egg (Linley et al. 1993). In another study, significant differences in the width and egg length-width ratio of eggs were reported in eight populations of An. nuneztovari collected from western Venezuela, Suriname, and Brazil, but the differences were not significant for egg length (Linley et al. 1996). Lounibos et al. (1999) also reported significant variations in various egg dimensions

Figure 3. Cluster analysis of egg attributes of JD (Jodhpur), BKN (Bikaner), JMN (Jamnagar), and BTH (Bathinda) strains of Cx. quinquefasciatus.

Figure 4. Regression analysis between the cluster analysis distance coefficient of egg attributes and geographic distance among all four strains of Cx. quinquefasciatus.


of An. gambiae complex collected from nine countries in Africa.

Surface structure morphology of the egg micropylar region did not show any obvious variation in the JD, BKN, JMN, and BTH strains, though the morphometrics of different attributes showed variations among these four strains (Table 2). The diameter of the corolla and large tubercles did differ significantly among the four strains, but non-significant differences were observed in the rest of the micropylar attributes. The dimensions of the micropylar region in the present study were found to be similar to those described by Suman et al. (2008) for Cx. quinquefasciatus. Variation in the morphometrics of micropylar attributes and air floats in anophelines was also reported for strains from different geographical areas (Linley et al. 1993, Linley et al. 1996, Lounibos et al. 1999).

The conical-shaped regions of Cx. quinquefasciatus eggs possess tubercles and exochorionic pores. Morphologically, no significant differences have been observed in these attributes among all four strains except for the absence of small tubercles in the anterior and middle regions of the JMN strain. However, the diameter of tubercles and their density in the conical-shaped region of the eggs showed significant differences in most of the parameters among the JD, BKN, JMN, and BTH strains (Table 3). Linley et al. (1993) reported variation in the morphometrics of deck tubercular dimensions from the eggs of five geographically isolated strains of An. quadrimaculatus. Similarly, dimensions of deck tubercles in the eggs of various populations of An. nuneztovari, An. gambiae, An. arabiensis, An. bwambae, An. merus, and An. melas of different geographically isolated regions have been reported to vary (Linley et al. 1996, Lounibos et al. 1999). Suman et al. (2008) suggested that these exochorionic tubercles in eggs of Cx. quinquefasciatus and Cx. tritaeniorhynchus play very important roles in egg-raft formation and protection. Furthermore, the size of large exochorionic pores in the anterior and middle regions of JMN strain eggs differed significantly from the JD, BKN, and BTH strains, but the size of small exochorionic pores did not differ significantly in any region of the eggs among all four strains (Table 3). The density of large exochorionic and small exochorionic pores was significantly different among these strains. Functionally, the exochorionic pores of the conical-shaped region are involved in fitting together the tubercles of adjacent eggs within the egg raft and allow for a denser packing. The widths of the exochorionic bridges of all the four strains also exhibited variations, although these were not significant. The area of tubercular wheel units in the anterior, middle, and posterior regions of eggs differed significantly among the JD, BKN, JMN, and BTH strains. Suman et al. (2008) have also used these parameters for differentiation of Cx. quinquefasciatus and Cx. tritaeniorhynchus.

Jodhpur, Bikaner, Jamnagar, and Bathinda are located in different geographical and ecological areas. The JMN strain is exposed to a more humid environment with a narrower temperature range and comparatively higher rains than the other three regions due to its location near the

coastal region of the Arabian Sea. In contrast, JD and BKN strains are found in the Thar Desert and the BTH strain is located in a semi-desert zone, with all three strains subject to lower humidity with less rainfall and extreme minimum and maximum temperature conditions. Geographically, Jamnagar is not distinctly related to Jodhpur, Bikaner, and Bathinda, which are comparatively closer to each other. The cluster analysis of egg attributes of all the four strains of Cx. quinquefasciatus also showed that the JMN strain existed separately in an isolated group, whereas the JD, BKN, and BTH strains comprised a separate group with some variations, with the BKN strain being closer to the BTH strain in comparison with the JD strain. Moreover, a strong positive correlation (r=0.95) between the cluster distance of egg attributes and geographic distance indicated that egg morphometry varied according to the geographical distribution. This may be a reflection of the cumulative effect of various conditions that prevail in corresponding geographical areas and ecological regions. We have also recorded variations in life table and genetic profiles of these strains (unpublished data). The present study suggests that geographical distribution in varying ecological regions affects the morphometrics of egg attributes in four strains of Cx. quinquefasciatus belonging to the desert (Jodhpur and Bikaner), Arabian Sea coastal (Jamnagar), and semi-arid regions (Bathinda) of India.

Acknowledgments

The authors express their gratitude to Dr. R. Vijayaraghavan, Director, Defence Research and Development Establishment, Gwalior, for his encouragement and interest in the work.

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