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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
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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
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Vol. 34, no. 2 Journal of Vector Ecology 193
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
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194 Journal of Vector Ecology December 2009
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.
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Vol. 34, no. 2 Journal of Vector Ecology 195
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%).
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196 Journal of Vector Ecology December 2009
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
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Vol. 34, no. 2 Journal of Vector Ecology 197
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.
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198 Journal of Vector Ecology December 2009
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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