Morphology Aedes Sp English Journal

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*Corresponding author: Email: [email protected];

Annual Review & Research in Biology3(1): 1-21, 2013

SCIENCEDOMAIN internationalwww.sciencedomain.org

Morphology and Morphometry of Aedes aegyptiLarvae

Ananya Bar1* and J. Andrew1

1Department of Zoology and School of Entomology, St. John's College, Agra, 282002, India.

Authors’ contributions

This work was carried out in collaboration between all authors. All authors read andapproved the final manuscript.

Received 22nd October 2012Accepted 24th December 2012

Published 6th February 2013

ABSTRACT

Aims: To observe the morphology of the larva of A. aegypti in detail and to evaluate theirmorphometry.Place and Duration of Study: Place – St. John’s College, Agra. Duration – June, 2011 toMay, 2012.Methodology: Morphology of larval head, antennae, compound eyes, median brush(palatum), lateral brush; neck; thorax; abdomen, comb spine, siphon, siphon teeth and analpapillae were observed and photographed under an image documentation system andtheir size (length/width) measured by using image J software for morphometric study.Dyar’s rule was also applied to see the increase in width of head, neck, thorax andabdomen of A. aegypti larvae.Results: Morphology and morphometry of various instars of Aedes aegypti (dengue vector)larvae collected from Agra city were studied. Larval key characters of head, antenna,compound eye, palatum, lateral brush, neck, thorax, abdomen, comb spine, siphon, pectenteeth and anal papilla are studied by morphometry. The head capsule grows in size(length/width) to attain globular shape. The width of head, neck, thorax and abdomen weremeasured and the Dyar’s rule was applied to find out the growth rate. The width of thehead doesn’t follow the Dyar’s rule. The factors between I-II, II-III and III-IV larval instarsincrease by a constant factor. The increase in size of antennae, compound eye, medianand lateral brush of larvae in every instar of larval development were observed. In A.aegypti larvae the size of the neck, thorax and abdomen were also studied. The number ofcomb spine varied from 4.4 -10.6 from I-IV instar. The mode in I instar stage is 4 and from

Research Article

Annual Review & Research in Biology, 3(1): 1-21, 2013

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II-IV instar is 10. In the respiratory siphon of A. aegypti larvae the ratio of its length andwidth measure approximately less than the double of its width. The number of pecten teethand their length increase in each instar. Four anal papillae increase in length and widthduring larval development.Conclusion: This study reveals the morphologic features of A. aegypti larvae in Agra forbetter understanding of the key characters.

Keywords: A. aegypti; dengue vector; morphology; morphometry.

1. INTRODUCTION

The dengue vector Aedes aegypti (Linnaeus) belongs to the family Culicidae and the orderDiptera. The female A. aegypti preferably lay eggs in artificial collections of water. Thehatched larvae undergo growth and metamorphosis. In their life cycle, four larval stages andthe pupal stage are aquatic and their adults are aerial. Growth changes in form and sizeoccur during their larval development. The first instar A. aegypti larva is only about 1 mmlength, whereas in the fourth instar stage it reaches a length of approximately 8 mm [1]. Onshedding the IV instar larval cuticle, pupa emerges with most of the adult organs and afterthe pupal moult a complete mosquito appears. Identification of A. aegypti larvae, pupae andadults by their morphologic features immediately after collection is of considerable value inrecognizing vector prevalence. Most of the identification keys of the A. aegypti are basedexclusively on the adult characteristics and on the 4th instar larvae whereas I, II and III instarlarvae are also available in samples which need identification. In larval collections of Aedesaegypti, various other species of larvae (eg. A. albopictus, A. vittatus) also co-exist [2]. Itbecomes very difficult to identify all species of mosquito larvae in the samples becausetaxonomic keys are on all the larval instars are not included [3].

The morphology of A. aegypti larval body parts of head, neck, thorax and abdomen likemouth brush, palatum, preclypeal spines, mentum, compound eye, antenna, comb spines,siphon tube, pecten teeth and anal papilla were described by various researchers [4,5,6]. InIst instar stage A. aegypti larval head is narrow and triangular. In later stage, in the headcapsule, convexity appears. The head becomes large and attain globular shape [7].

Klingenberg and Zimmermann [8] applied Dyar's rule on the head width of waterstriders,Gerris and Aqzlarius (Heteroptera: Gerridae) and found that the data followed the rule insome areas. The growth ratio differed between moults. Mohammadi et al. [9] used Dyar'srule on the size of the sclerotized body parts of different larval instars of cotton bollworm,Helicoverpa armigera and found that the ratio of the size of the sclerotized body parts werein a constant range. Ghafoor [10] applied Dyar's rule on the width of head capsule of larvalinstars of Agrotis ipsilon and found that the head capsule width was 0.28 mm in I instar and3.42 mm instar VI. In this study, Dyar's rule was used on the width of head capsule, neck,thorax and abdomen.

The antennae are smooth and cylindrical in shape. It has a single hair [7]. In mosquito larvalhead a pair of large compound eyes are present below the antennae on the lateral side.

A. aegypti larvae are found in different aquatic habitats mainly in small water collections.Various environmental factors like temperature, salinity, pH, dissolved nutrients and gases inthe aquatic habitat influence the growth of mosquito larvae. Extremes of temperature, lack offood [7] and increased salinity [11] result in reduced A. aegypti larval growth and delayed


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development. Variations in the larval, pupal and adult morphology were also described bydifferent authors. Various larval forms like sensu strictu, the type form, formosus (walker)and queenslandensis (Theobald) were reported within A. aegypti [12,13]. In India theidentification of A. aegypti variety is not generally mentioned in most of the larval collections.A. aegypti population density in Agra city is considerably more in the collected mosquitolarval samples [14]. Hence, the morphology of A. aegypti larvae in our collection was studiedin detail. Different regions of larval body parts (head, antenna, median brush, lateral brush,neck, thorax, abdomen, siphon, anal papilla) have been chosen for morphologic study andmeasurement.

2. MATERIALS AND METHODS

2.1 Morphological Study

A. aegypti larvae were collected in 2010 from specific sampling sites of Agra in the vicinity ofSt. John’s College area for morphologic study and brought to laboratory. Sample of A.aegypti larvae were preserved in formalin for morphologic observation under a stereobinocular microscope. Various instars of larvae collected from a collection sites were usedfor morphometric study. 20 A. aegypti larvae were used for the each measurement of thebody part.

The larval head, antennae, median brush (palatum), lateral brush; neck; thorax; abdomen,siphon and anal papillae were observed. Their images were transferred on a computer,photographed under an image documentation system and their size (length/width) measuredby using image j software for morphometric study.

In some species of mosquito larvae, the size of the head capsule increase by a constantfactor at each moult - dyar’s rule [6]. In this study, the Dyar’s rule was applied to see theincrease in width of not only the head but also the neck, thorax and abdomen of A. aegyptilarvae.

Fig. 1. Diagrammatic representation of the dorsal side of larval head showing themeasurement of the angle of orientation of the compound eye in relation to the

margin of occipital foramen and the midline of head

Position of the compound eye in the dorsal side of the larval head of A. aegypti at variouslarval stages were observed and measured. The distance from midline of head capsule to


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eye, angle of orientation from the occipital margin towards the midline of head and the widthof compound eye were measured from the various instars of larvae (Fig. 1).

The number of comb spine (VIIIth segment of abdomen) and pecten teeth (siphon tube)were counted and their size measured.

2.1.1 Statistical analysis

The documented morphometric data were analysed statistically. Kruskal-Wallis one wayanalysis of variance (ANOVA) was employed to determine the significant difference amongthe readings using Sigma plot software along with Mean ± Standard deviation (SD).

3. RESULTS

The egg of A. aegypti is dark, oval and boat shaped. On hatching the larvae appeartransparent. They grow in size and appear dark before moulting. After the first ecdysis theemerged second instar larvae appear transparent. In every instar the darkening of larvalcolour occur before moulting and the emerging larvae appear transparent after the moult.

3.1 Larval Size

A. aegypti larvae increase in size during larval development. The larval length in I, II, III andIV instar stages measured 1.745, 2.935, 4.343, and 7.202 mm, respectively.

3.1.1 Head

In the newly hatched A. aegypti larvae, the head capsule is laterally compressed. In II instarstage it grows in size to attain a convex shape and in later instars the head capsule attains aglobular shape.

3.1.1.1 Dorsal view

Dorsally A. aegypti larval head capsule increase in width from 0.235 mm to 0.987 mm (Table1, Fig. 2) and length from 0.260 mm to 0.868 mm (Table 2, Fig. 3) from I to IV instar stagesignificantly. The occipital margin of the I instar larval head in the occipital foramen appearconcave (curved upwards) whereas from II instar stage onwards it attains a convex shape.On the posterior margin of the larval head is the cervical collar surrounding the occipitalforamen. In the midline of the collar on the anterior side arises a ‘Y’ shaped epicranialsuture. It passes in between the compound eyes and antennae of the head capsule i. e. theyreach medial to compound eyes and lateral to the antennae in I to III instar stages. In the IVinstar stage the ‘V’ shaped arms of the epicranial suture in the vertex region moves mediallyand reaches the inner side of the antennal prominence. These frontal suture lines are thepoints of weakness which split during ecdysis at every moult.

3.1.1.2 Ventral view

On the ventral side of the larval head, the occipital margin is concave in shape and the lowerportion of the Y shaped cervical cleft is visible in the occipital margin close to the cervicalcollar. The width of the occipital foramen also increases (0.175 mm, 0.252 mm, 0.326 mm,0.519 mm) from I to IV instar larval stage.


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Table 1. Width of the head, neck, thorax and abdomen of I-IV instar larvae and thefactors of the Dyar’s rule on A. aegypti

Region Parameter(mm)

Larval stage (instars) FactorsI II III IV Between I-II

instars (a)Between II-IIIinstars (b)

Between III-IV instars (c)

Head Width 0.235a±0.012

0.392b±0.057

0.640c±0.050

0.987d±0.104

0.15* 0.25* 0.35*

Neck Width 0.096a±0.012

0.151b±0.016

0.254c±0.030

0.464d±0.060

0.06 0.10 0.21

Thorax Width 0.248a±0.054

0.491b±0.081

0.749c±0.076

1.401d±0.172

0.24 0.26 0.65

Abdomen Width 0.152a±0.024

0.293b±0.054

0.487c±0.074

0.858d±0.117

0.14 0.20 0.37

a, b, c, d – the different letters shows the significant difference (p< 0.001) of growth among the larvalinstars in same row for specific parameters.

*- to show the same difference (10) in the factor in the growth of larval head width between the instars(i.e. I-II, II-III and III-IV).

Fig. 2. Graphical representation of the width of head, neck, thorax and abdomen ofA. aegypti.


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Table 2. Growth changes in size (length) of A. aegypti during larval growth

Regions Parameters(mm)

Larval stage (instars)I II III IV

Head Length 0.260a ± 0.027 0.380a ± 0.0440.608b ± 0.0450.868c ± 0.098Neck Length 0.064a ± 0.012 0.066a ± 0.0220.087b ± 0.0240.108c ± 0.031Thorax Length 0.222a ± 0.047 0.409b ± 0.0740.639c ± 0.073 1.107d ± 0.138Abdomen Length 1.199a ± 0.225 2.080b ± 0.3633.009c ± 0.251 5.119d ± 0.542Total Length 1.745 ± 0.311 2.935 ± 0.503 4.343 ± 0.393 7.202 ± 0.809a, b, c, d - significant difference (p< 0.001) at various larval instars in same row for specific parameters

Fig. 3. Graphical representation of the length of head, neck, thorax and abdomen of A.aegypti

3.1.1.3 Dyar’s rule

In this study the width of A. aegypti larval head capsule was measured and the Dyar’s rulewas applied to observe the growth rate of A. aegypti larval head. The factors between thevarious instars of larvae were calculated (the width of head capsule of successive instars)and the values among I and II is 0.15, II and III is 0.25, III and IV is 0.35. The calculatedfactors among various instars are not a constant number (different among successiveinstars). Though A. aegypti larval head capsule is not following the Dyar’s rule, the headcapsule of A. aegypti larvae has a same factor (0.10*), found occurring between thecalculated factors (i. e. among 0.15 and 0.25, 0.25 and 0.35). The observation of a commonfactor in between the calculated factors of A. aegypti reflects a definite pattern of the growthof larval head capsule.

In the same way the width of the neck, thorax and abdomen were also measured and theDyar’s rule was applied to find the growth rate. Though the growth of the head, neck, thoraxand abdomen at every instar stage increase significantly, the rate of growth don’t follow aconstant factor (Table 1). Since the factors are different, they don’t follow the Dyar’s rule.

0

1

2

3

4

5

6

I instar II instar III instar IV instar

Length

Instars

Head

Neck

Thorax

Abdomen


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3.1.1.4 Antennae.

In A. aegypti larval head, a pair of antennae is visible which are straight, broad at base andnarrow towards the tip (Fig. 5). The antennae increase in size significantly during larvaldevelopment from 0.078 mm in I instar to 0.258 mm in IV instar in length (Table 3, Fig. 4). Inthe IV instar stage the larval antennae attain a medially curved shape and without spicules.

Table 3. The size (length and width) of various larval organs and the increase innumber of comb spines and pecten teeth in A. aegypti

Organ Parameters(mm)

Larval stage (instars)I II III IV

Antenna Length 0.078a±0.011 0.116b±0.019 0.166c±0.024 0.258d±0.045Median brush Width 0.026a±0.005 0.056a±0.005 0.108b±0.008 0.166b±0.005Lateral brush Length 0.081a±0.012 0.149b±0.013 0.191c±0.008 0.274d±0.013Siphon Length 0.217a±0.044 0.452b±0.079 0.645c±0.048 0.792d±0.122

Width 0.127a±0.188 0.178b±0.038 0.277c±0.040 0.389c±0.065Anal papilla Length 0.148a±0.161 0.222a±0.049 0.389b±0.055 0.649c±0.047

Width 0.021a±0.010 0.042a±0.011 0.076b±0.016 0.135c±0.031CombSpine

Number 4.4a± 0.50 9.05b±0.94 9.8b±0.62 10.6c±0.94Length 0.012a±0.003 0.018b±0.004 0.036c±0.03 0.132d±0.19

Pecten teeth Number 4.68a±0.58 10.79b±1.13 14.11b±1.29 19.42c±3.11Length 0.011a±0.004 0.015a±0.005 0.021b±0.008 0.061c±0.07

a, b, c, d - significant difference (p< 0.001) at various larval instars in same row for specificparameters

Fig. 4. Graphical representation of the length of antenna and mouth brush of A.aegypti larvae

3.1.1.5 Compound eyes

In A.aegypti larvae the compound eyes (Figs. 6 and 7) are situated distinctly on the lateralsides of the head below the antennae. The size of the compound eyes increase in widthfrom 0.014 to 0.053 mm from I to IV instar stage (Table 4). Position of the eye in the headduring larval growth is found shifting anterolaterally. The shift in position of eye during larvaldevelopment was measured by measuring the angle of orientation of the larval eye from the

0

0.05

0.1

0.15

0.2

0.25

0.3


Length

Instars

Antenna

Mouth brush


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midline of the head capsule and it shifts from 74.83º to 61.5º from I to IV instar larval stage(Table 4). The relative distance between larval eye and the midline of the head capsule alsowas measured at various larval stages and was found increasing from 0.08 to 0.349 mmfrom I to IV instar stage.

Fig. 5. Antenna of A. aegypti larvae (I-IV instar) magnification-108X

Table 4. Position (Angle of orientation & distance from midline of head) and the widthof the compound eye in A. aegypti larvae in the head capsule

Parameter Larval stage (Instars)I II III IV

Angle of orientation ofcompound eye (°)

74.83a±1.37 71.25a±3.19 64.55b±2.47 61.5b±1.66

Distance from the ommatidialmargin to the mid line of headcapsule (mm)

0.08a±0.011 0.168b±0.010 0.249c±0.047 0.349c±0.033

Width of the compound eye(mm)

0.014a±0.005 0.0275b±0.004 0.034c±0.005 0.053d±0.005

a, b, c, d - significant difference (p< 0.001) at various larval instars in same row for specificparameters

3.1.1.6 Mouthparts

i) Dorsal view- Median brush (Palatum): In the larval head, the median brush or palatum(Figs. 6, 7 and 8) is found arising from the preclypeal- labral region. The size of the medianbrush increases during larval development. Its width (Table 3) increases from 0.026 mm to0.166 mm from I to IV instar stage. Ventrally the median brush is continuous with thepostpalatal lobe.

ii) Dorsal view- Lateral brush (Mouth brush): In the anterolateral margin of the larval head, apair of lateral brushes (Figs. 6, 7 and 8) are present and they help to capture the foodparticles . They arise from the preclypeal/ labral region. From I to IV instar larval stage theirlength increase significantly from 0.081 mm to 0.274 mm (Table 3 & Fig. 4).

i) Ventral view- Labium: On the larval head, labium is found medially located and is archshaped and a mental sclerite is visible below the arch shaped structure and is toothed. Onthe lateral sides of the labium is the hypostomal area (Fig. 7).


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ii) Ventral view- Mandible and Maxillae: Fenestrae for the articulation of mandibles andmaxillae are found on both sides of the labium. In the articulating area of mandible andmaxilla are a pair of simple spur like submaxillary apodemes (Fig. 7).

Fig. 6. Dorsal view of head of A. aegypti larvae (I-IV instar) magnification-108X (Pal-Palatum, Mo Br- Mouth brush, Ant- Antenna, Ey- Eye, Nk- Neck)

Fig. 7. Ventral view of head of A. aegypti larvae (I-IV instar) magnification-108X [Pal-palatum, MoBr- mouth brush, Ant- antenna, Mdo- Fenestrae for the articulation of

mandibles and maxilae, Ms- mental sclerite, Ey- eye, Hy- hypostomal area, La- labium,Cr Co- cervical collar]

Fig. 8. Lateral brush of A. aegypti larvae (I-IV instar) magnification-108X


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3.2 Neck

In A. aegypti larvae a narrow cylindrical and membranous neck (Fig. 6 and 7) connects thehead and thorax. The larval neck increases in length (Table 2, Fig. 3) from 0.064 mm to0.108 mm and in width (Table 1, Fig. 2) from 0.096 mm to 0.464 mm from I to IV instarrespectively. The upper part of the neck is surrounded by a cervical collar.

3.3 Thorax

Fig. 9. Thorax of A. aegypti larvae (I-IV instar) magnification-108X [ Pro- prothorax,meso- mesothorax, Meta- metathorax]

In A. aegypti larvae the thorax (Fig. 9) is globular and it has pro, meso and meta thoracicsegments. In meso and meta thoracic region a pair of thorn like large processes are visibledorsally. Lateral hairs arise from the lateral sides of the pro, meso and meta thoracicsegments. The increase in size of the larval thorax during larval development i.e. the lengthand the width of thorax (Tables 1 and 2, Fig. 2 and Fig. 3) show that the thoracic width ismore than its antereo-posterior length. The thoracic size increase significantly (length andwidth from 0.222 mm to 1.107 mm and 0.248 mm to 1.401 mm respectively) during larvalgrowth from I to IV instar stage.

3.4 Abdomen

A. aegypti larval abdomen is 8 segmented, long, cylindrical and dorsoventrally flat (Fig. 10).It appears more transparent after ecdysis. Tufts of lateral hairs arise from the abdominalsegments and the number of hairs in each segment varies. The larval abdomen increase insize significantly (from I to IV instar stage i. e. length and width of abdomen increase from1.199 mm to 5.119 mm and 0.152 mm to 0.858 mm respectively- Tables 1 and 2, Fig. 2 andFig. 3).

The VIII th abdominal segment of A. aegypti larvae has a row of comb spines (Fig. 11).Comb spines increase in number during larval development (Table 3, Fig. 15). Their numberincrease from 4.4 to 10.6 from I to IV instar. They are arranged in a row of which the medianone is close to the siphon and the lateral ones arranged backwards laterally in a semi lunarfashion. Each comb spine is pointed and curved at the tip having apical and subepicaldenticles. The average size of the comb spine increase in length from 0.012 mm to 0.132mm during larval development from I to IV instars stage (Table 3, Fig. 16). The repeated


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occurrence of number of comb spines in various samples within a particular instar (mode)was estimated statistically. In I instar the mode is 4 and II-IV it is 10.

Fig. 10. Abdomen of A. aegypti larvae (I-IV instar) magnification-22X

3.5 Siphon

A. aegypti larval abdomen has a respiratory siphon (Figs. 11,12,13 and 14). At the tip of thesiphon is the respiratory opening - the spiracle, surrounded by perispiracular lobes.

In the newly hatched larva the siphon is soft and in later instars it becomes darker andhardened. Inside the siphon tube tracheal trunk and strands of muscle fibres are visible. Thesize of the respiratory siphon increase during larval growth from I to IV instar stage (length -0.217 mm to 0.792 mm and width- 0.127 mm to 0.389 mm). The ratio of the length and widthof the respiratory siphon shows that the length of the siphon is measuring close to thedouble of its width (Table 3, Fig. 17 and Fig. 18).


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Fig. 11. Comb spine of A. aegypti larvae (I-IV instar) magnification-196X.

Fig. 12. Respiratory siphon of A. aegypti larvae (I-IV instar) magnification-108X.

Fig. 13. Anal papilla of A. aegypti larvae (I-IV instar) magnification-108X.


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The respiratory siphon bears a row of pecten teeth on both sides laterally (Figs. 11,12,13and 14) and have basal denticles. They are arranged in a row orienting towards the spiracle.The spines closer to the spiracle are longer and the distal ones are smaller. The number ofpecten teeth increase in successive instars significantly from I to IV instars from 4.68 to19.42 (Table 3, Fig. 15). The denticles of the pecten teeth are variable in size in every larvalstage. The average size of the pecten teeth increase in length from 0.011mm to 0.061 mmfrom I to IV instar (Table 3, Fig. 16).

Fig. 14. Respiratory siphon of A. aegypti larvae (I-IV instar) magnification-196X.


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Fig. 15. Number of comb spine and pecten teeth of A. aegypti larvae.

Fig. 16. Length of comb and pecten teeth of A. aegypti larva

0

5

10

15

20

25


Number

Instar

Comb

Pecten

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14


Length

Instar

Comb spine

pecten teeth


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Fig. 17. Graphical representation of the length of siphon and papilla of A. aegypti

3.6 Anal segment

At the terminal portion of A. aegypti larval abdomen is the anal segment. It has ventral brushwith 5 pairs of setae of variable size and four anal papillae which are of equal size. Analpapillae are transparent and boat shaped. Anal segment has incomplete saddle.

They grow in size in each instar during larval development. The size of papillae increase inlength from 0.148 mm to 0.649 mm and width from 0.021 mm to 0.135 mm from I to IV instarstage (Table 3, Fig. 17 and Fig. 18). Significant growth of the anal papilla was observed in IIIand IV instar stage.

Fig. 18. Graphical representation of the width of siphon and anal papilla of A. aegypti.

00.10.20.30.40.50.60.70.80.9


Length

Instars

Siphon

Papilla


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4. DISCUSSION

On hatching, the newly emerged Aedes aegypti larvae appear transparent. As they grow,they appear dark before moulting. In every instar the larvae become transparent immediatelyafter moulting and before the next moult the larval cuticle gets darkened. The larval sizeincreases as they grow and moult. The length of A. aegypti larvae in I, II, III and IV instar are1.745, 2.935, 4.343 and 7.202 mm respectively. Clements [6] observed the length of A.aegypti larvae in I, II, III and IV instar 1.6, 2.7, 4.6 and 7 mm respectively in agreement withthe present study.

The newly hatched Ist instar A. aegypti larval head is narrow and triangular in shape.Gradually the swelling begins in the head capsule, convexity appears and the head becomesenlarged and globular. The length of A. aegypti larval head capsule of I, II, III and IV instarsare 0.25, 0.41, 0.65 and 0.82 mm and width are 0.27, 0.46, 0.74, 0.98 mm respectively [7].In this study, the length of A. aegypti larval head capsule of I, II, III and IV instars measured0.260, 0.380, 0.608, 0.868 mm and the width 0.235, 0.392, 0.640, 0.987 mm respectively.The growth of head width is much than the length of the larval head. The morphologicobservation and the measurement of the length and width of the head capsule of A. aegyptilarvae reveal that the I instar larval head is not globular whereas in the II, III and IV instars,they become globular.

Snodgrass [5] observed that upper surface of A. aegypti larval head is round than lower andthe upper surface of head is differentiated into large, shieldlike central area, narrow lateralareas bearing the antennae, the eyes and a slender transverse anterior sclerite at the basesof the brushes.

The antennae of A. aegypti are much reduced in size compared with many Culicine larvae.The antennae are small, cylindrical and smooth and their length measured 0.10, 0.16, 0.24,0.34 in mm from I-IV instars respectively [7]. Huang [13] described that antenna of A. aegyptiis without spicule whereas other species possess spicules. In this study the length of theantennae of I, II, III and IV instar larvae measured, was in co-incidence with the previousworkers. In early instars of A. aegypti larvae (I –III instar), the antennae are straight, broad atbase, narrow towards tip. In the IV instar stage the larval antennae attain a medially curvedshape.

In A. aegypti larvae compound eyes have no lenses, whereas the ordinary head cuticle iscontinuous over them [5]. In the I instar larval stage, optic placode appears at the posteriormargin of the prospective compound eye area and it expands anteriorly across theprospective eye area throughout the larval stage [6,15]. To study the expansion of opticplacode anteriorly across the prospective eye area during larval development, the angle oforientation of the compound eyes in the head capsule in relation to the midline of the headcapsule was measured. So the shift in the position of the compound eye in the lateral marginof the head capsule was observed. Hence, the anterolateral movement of compound eyeduring larval development is evident in co-incidence with the previous worker. Moreover, inthe present study, it was observed that in the dorsal side of the head capsule, the distancefrom the ommatidial margin to the midline of head capsule also increased considerablyduring the larval development. So the head capsule in the occipital region also expandsduring larval development. Moreover, the ommatidial size also increased considerably duringlarval development.


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In A. aegypti larval head, a pair of lateral brushes and a small median brush (palatum)appear anteriorly from preclypeus and the three brushes are supported by labrumSnodgrass [5]. The labral area has a clypeal portion which terminates anteriorly topreclypeus [7]. The median brush appears between the two lateral brushes dorsally frompreclypeus/ labral region. They help in feeding.

Schaper and Hernandez-Chavarria [1] described that Lateral brushes of the 1st and 2ndinstars appeared as uniformly shaped filaments. In III and IV instars, the arrangement ofsetae is more complex. It is difficult to determine from where it originates because the tips ofthe median brush filaments appear to be intermingled with filaments from other mouthstructures and the median brush in A. aegypti appears to be wider in later instars (III and IVinstars) than in the early instars (I and II instar). In this study it is found also that in I and IIinstar it is easy to determine the lateral and median brushes than III and IV instars inaccordance with the previous workers.

The length of lateral brush of A. aegypti I, II, III and IV instars increases along with theincrease in the length of setae. The width of the median brush in each instar (I-IV) wasmeasured and found increasing.

The large central area of the head capsule of A. aegypti is bounded by lateral cephaliccleavage lines [5]. Christophers [7] also described that, dorsally there are two diverging lineswhich appears ‘Y’ shaped, are the lines of weakness. During ecdysis larva emerges throughthis line. In this study also the ecdyseal lines were observed and they are clearly seen in IIIand IV instars of A. aegypti than in I and II instars.

Generally the head capsule of some mosquitoes of Anopheles and Culex increase in size bya constant factor at each moult–Dyar’s rule [6]. In A. aegypti it is not there. Abdel-Malek andGoulding [16] also observed that A. aegypti is not obeying Dyar’s rule. In our observationalso we found that A. aegypti is not obeying Dyar’s rule. Whereas in this study though thecalculated factors according to the Dyar’s rule are not obeying the rule (0.15, 0.25, 0.35) inco-incidence with the previous worker. The gaps in between the factors vary by a constantfactor i. e. 0.10 (Table 1).

In this study the growth of neck, thorax and abdomen of A. aegypti were also checkedwhether the growth rate obeys the Dyar’s rule. It was found that the factors between I-II, II-IIIand III-IV of neck, thorax and abdomen does not obey the Dyar’s rule.

On the ventral side of larval head, fenestrae for the articulation of mandibles and maxillaeare present. Its edges give attachment to both mandible and maxilla. On the ventral surfaceof gena on each side, a spur like process, submaxillary apoodemes appears. The bases ofmaxilla extend to occipital foramen forming hypostomal area. Between hypostomal area anarch shaped labium is present. A mental sclerite is visible below the arch [7]. In this studyalso it is found that in between the hypostomal area the labium is located. This area isbounded by triangular mental sclerites. Fenestrae for the articulation of mandibles andmaxillae and submaxillary apoodemes are also present.

In A. aegypti the larval neck is membranous and cylindrical [5]. Not much information isavailable on this parameter and this study gives an account of length and width of it. In thisstudy the length of the neck of A. aegypti were measured in I to IV larval instars as 0.064,0.066, 0.087 and 0.108 mm and the width measured 0.096, 0.151, 0.254 and 0.464 mmrespectively.


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The thorax of A. aegypti larvae is globular in shape. Snodgrass [5] described that it has asimple oval form and on emergence the thoracic segments- pro, meso and meta are distinctand fused. Christophers [7] described that the thorax is roughly globular and it consists ofthe three fused thoracic segments. The segments are indicated by their hair series and theyhave long lateral hairs. Christophers [7] measured the length of thorax as 0.30, 0.50, 0.92,0.83 mm and the width as 0.41, 0.71, 1.23, 1.67 mm in I, II III and IV instar respectively. Inthis study, the length of A. aegypti larval thorax in I-IV instar larval stages measured 0.222,0.409, 0.639, 1.107 mm and the width measured 0.248, 0.491, 0.749, 1.401 in mmrespectively in I, II, III and IV instar larvae in contrast with the previous worker. Snodgrass [5]described that in each thoracic segment of Anopheles larvae has a pair of minute, tapering,transparent lobes arising from a common base which is called notched organ. Barraud [4]described that on meso and meta thoracic segments of A. aegypti has thorn like largeprocesses. Its end has a single point. In this study also meso and meta thoracic segment oneach side thorn like large processes appear in agreement with the previous worker.

In mosquito larvae, the shape of the abdomen is cylindrical and 8 segmented. In A. aegyptithe size of the segments decreases gradually towards the 8th segment. Snodgrass [5]described that the larval abdomen appears to have 9 segments with the respiratoryapparatus on 8th segment and a 9th terminal segment, whereas Christophers [7] describedthat the abdomen, apart from the small terminal anal segment consists of eight roughly equalsegments increasing in size to the third and then decreasing to the 8th. Christophers [7]described that the size of the abdomen of I to IV instar larvae of A. aegypti measured 1.4,2.24, 3.52, 4.10 mm and the width 0.26, 0.47, 0.76, 0.99 mm whereas in this study also thelength and the width of A. aegypti larval abdomen measured in I, II, III and IVth instars in co-incidence with the previous worker.

Moreover each of the abdominal segment carries a number of hairs including decreasingseries of long lateral hairs. The lateral hairs arise from the side of each segment and theirnumber varies in each instar.

In A. aegypti larvae, on the 8th segment of abdomen, comb spines are present in a singlerow in semilunar fashion. Barraud [4] described that in A. aegypti the comb teeth number is8-12. It has strong basal lateral denticles. The teeth of A. aegypti do not arise from chitinisedplates whereas in other species they arise from chitinised plates and are in a single row. InA. aegypti I instar stage, the number of comb spines are described by Schaper andHernandez-Chavarria [1] as 5, Christophers [7] as 5, LaCasse and Yamaguti [17] as 4. In IVinstar the numbers are described by Christophers [7] as 8-10, LaCasse and Yamaguti [17]as about 18. Other instars numbers are described by Schaper and Hernandez-Chavarria [1]as 6-10, Huang [13] as 6-12, Ribeiro and Ramos [18] as 6-12. In this study the number ofcomb spines in I, II, III and IV instars are 4.4±0.50, 9.05±0.94, 9.8±0.62 and 10.6±0.94respectively and the length measured 0.012±0.003, 0.018±0.004, 0.036±0.028 and0.133±0.187 mm in I, II, III and IV respectively.

Ribeiro and Ramos [18] also found that the spines are curved and pointed, thorn like, combscales with well developed basal denticles each side of central tooth. Wood and Dalingwater[19] described that the spine number maintain modality. In the I instar of A. aegypti, combspine number showed a single mode at 4 spines, in II instar mode at 8 and a lesser one at10, in IV instar the mode is at 8-10 spines. In II-IV instar bimodality arise as the spinenumber become double from I instar during development. Christophers [7] described thatspine number in the I instar is half that of II-IV instars. In I instar it is about 5 and increases to


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8 or 10 in II instar and further increases in later instars. In this study, in I instar mode is at 4spine and II-IV at 10 spine. The spine number is double in II-IV instars from I instar inagreement with the previous workers.

In A. aegypti larvae a tubular respiratory siphon is present at the posterior part of 8th

abdominal segment. In the newly hatched larvae the siphon is soft and later it becomesdarker and hardened. The larval siphon has a pair of large open spiracles. Christophers [7]described that at the apex of siphon are the terminal spiracle surrounded by certainstructures perispiracular lobes. Christophers [7] described that the length and width ofsiphon of I to IV instars are 0.28, 0.44, 0.67, 0.75 mm and 0.14, 0.26, 0.39, 0.41 mmrespectively. In this study the length and width of siphon of I, II, III and IV instar larvae are0.217±0.044, 0.452±0.079, 0.645±0.048, 0.792±0.122 mm and 0.127±0.188, 0.178±0.038,0.277±0.040, 0.389±0.065 mm respectively. Macgregor [20] described that the length of thesiphon is less than the double of its width. Barraud [4] also described siphon is more thantwice the length of the diameter at base. In this study, the ratio of the length and width of thesiphon in I instar larva is in the limit of the value in agreement with Macgregor [20] whereasin II to IV instar larvae the value is within the limits of SD.

In A. aegypti larvae a row of pecten teeth are present laterally on both sides of therespiratory siphon. Barraud [4] described that pecten teeth are present in one rank. They arenot widely spaced whereas in other species they are spaced. Their numbers are 12-20 withbasal lateral denticles. Christophers [7] observed the two slightly diverging lines of spinesconstituting the pecten and its number. The number of pecten spines in I to IV instars areabout 6, 7-10, 12-15, 12-20. In this study the number of pecten teeth of I, II, III and IV instarsare 4.68, 10.79, 14.11, 19.42 respectively on each side is nearly co-incidence in case of II, IIIand IV instar, with the previous worker and in contrast in case of I instar with the previousworker. Huang [13] also described that the number of pecten teeth in A. aegypti (IV instar)as 8-20. Moreover the length of pecten teeth of A. aegypti in I to IV instars measured0.011±0.004, 0.015±0.005, 0.021±0.008 and 0.061±0.07 mm respectively.

A. aegypti larvae have 4 long elongated anal papillae on anal segment. They are of samesize. Christophers [7] found that ventral to the siphon is the small anal segment with theopening of anus terminally around which 4 transparent anal papillae 2 dorsal and 2 ventralare found and the segment bears 5 pairs of setae. The length of anal papilla of I, II, III and IVare 0.21, 0.33, 0.60, 0.84 respectively. In this study the length of anal papilla of I, II, III andIV are in contrast with the previous worker.

Barraud [4] described that the anal segment is enclosed by chitinisation. Christophers [7]described that the anal segment has saddle-shaped plate. It increases in size in each instarand restricted to its distal half. In this study it is observed that saddle plate on anal segmentis incomplete. It is not present on whole segment in agreement with the previous worker [7].

5. CONCLUSION

In this study the morphology of A. aegypti in Agra was observed in detail and the importantlarval characters of the head, neck, thorax and abdomen were studied by morphometry. Themorphometric evaluations of the various regions of larva revealed that the head region andthe last abdominal segment follow some specific growth pattern than other regions of thelarva. The morphologic features of A. aegypti larvae in Agra will help in better understandingof the larval key characters of this vector mosquito.


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ACKNOWLEDGEMENTS

Authors are grateful to the University Grants Commission, New Delhi for providing the grantsthrough the Major Project F. No. 34-481/2008 (SR).

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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_________________________________________________________________________© 2013 Bar and Andrew; This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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