INTRODUCTION
Anopheles stephensi is an important vector of malaria in urban areas of India and is predominantly dis- tributed in other countries also, viz. Myanmar, Thailand, China, Pakistan, Afghanistan, Iran and Iraq. Many stud- ies have been carried out on An. stephensi, but till date there is no proof to specify that it is a sibling species or a species complex (cryptic species). Instead it comprises of three ecological variants, namely ‘type’ form, ‘inter- mediate’ and ‘mysorensis’ which can be characterized by egg morphometry1-3. Gakhar et al4 have also reviewed various published studies on population genetic struc- ture and differentiation of An. stephensi variants using different markers in various parts of world but unable to clearly differentiate the three variants. In India, the ‘type’
form of An. stephensi is an efficient vector of malaria in urban areas, while ‘mysorensis’ has very limited role in malaria transmission, because it is primarily zoophilic in nature1, 4-6. The ‘intermediate’ form is typically recorded
in rural and periurban areas, but its role in malaria trans- mission is not wellknown till now. The existence of eco- logical variants of this vector has also been evidenced by Y-chromosome variation7, spiracular index8, and frequen- cies of inversion polymorphism in urban and rural popula- tions in range of its distribution9-10.
It is well documented that morphological and mor- phometric characters of egg can be exploited to differenti- ate the sibling species of mosquito as well as various forms of a species to better understand their ecology, bionomics and vector potential. This approach has successfully iden- tified the sibling species of An. albimanus Wiedemann and An. dirus Peyton & Harrison as established in earlier studies11-12. Recently, the eggs of An. laneanus Correa &
Cerqueira were compared with those of An. cruzii Dyar
& Knob, and An. bellator Dyar & Knob, likewise, An.
antunesi Gatvao & Amaral eggs were also compared with those of An. lutzii Cruz and other Nyssorhynchus spe- cies, based on description of egg surface morphology13. Furthermore, variations in the eggs not only reflect mor-
Morphometric and morphological appraisal of the eggs of Anopheles stephensi (Diptera: Culicidae) from India
Varun Tyagi
1, Sunil Dhiman
1, Ajay K. Sharma
2, A.R. Srivastava
2, Bipul Rabha
1, D. Sukumaran
2&
Vijay Veer
11Medical Entomology Division, Defence Research Laboratory, Tezpur, Assam; 2Vector Management Division, Defence Research and Development Establishment, Gwalior, Madhya Pradesh, India
ABSTRACT
Background & objectives: Anopheles stephensi is one of the most important urban malaria vectors in India and contribute about 12% of total malaria cases. An. stephensi has three ecological variants; type, intermediate and mysorensis that can be differentiated on the basis of differences in number of ridges on egg float and on the basis of spiracular indices. Because of its anthropophilic nature the ‘type’ form is an efficient malaria vector. In the present study, the egg surface morphometry and morphology of An. stephensi ‘type’ form was studied and detail distinguish- ing characters were recorded for its correct identification.
Methods: Eggs of An. stephensi ‘type’ form were studied by scanning electron microscopy (SEM) after sputter- coating with gold. In total 23 egg characters were analysed morphologically and morphometrically, which included egg attributes, deck attributes, ventral tubercles, micropyle and float attributes.
Results: The dorsal surface of the egg of ‘type’ form was curved while the ventral surface was concave and both anterior and posterior ends were blunt. The average length and width of egg was 473.94 + 11.18 and 154.69 + 2.66 µm respectively. The number of float ribs observed was 20.33 ± 0.33. The maximum length of float was found to be 246.57 + 15.27 µm, whereas maximum width was 87.16 + 3.83 µm.
Interpretation & conclusion: The present study has generated some important data which is specific to An. stephensi
‘type’ form and provided significant morphological and morphometric standards for its correct identification. This information could be useful in differentiation of An. stephensi ‘type’ form from other ecological forms of the same species as well as other species of Anopheles.
Key words Anopheles stephensi; ecological variants; morphology; morphometric; scanning electron microscopy
phological difference, but also indicate ecological prefer- ences. Anopheles eggs are boat shaped and float on the water surface, however the number of ribs depends upon the quality of natural breeding water.
Our previous study14 has elaborated the microscopic differences among the eggs of the sibling species of An.
culicifacies and emphasized that morphological and mor- phometric variations exist in eggs, even in sibling species.
The present study is the continuation of our work of docu- menting microscopic morphological and morphometric characters of closely related ecological variants and sib- ling species of malaria vectors. The eggs of An. stephensi
‘type’ form were used to identify and record detail mor- phological and morphometric characters using SEM for better appraisal and correct identification.
MATERIAL & METHODS Mosquito culture
Anopheles stephensi ‘type’ form mosquitoes were maintained in insectary of the Defence Research Lab- oratory, Tezpur (Assam) at 27 + 1°C temperature, 75 + 5% relative humidity and 12:12 h light : dark period. The mosquitoes were provided 10% sugar solution ‘ad libi- tum’ through cotton wicks. In this insectary, different spe- cies of mosquitoes are reared regularly for experimental purpose. The present study was approved by the Institu- tional Ethical Committee (No. 1127/bc/07/CPCSEA) for animal care.
Scanning electron microscopy (SEM) studies on eggs Oviposited eggs of An. stephensi ‘type’ form were placed in 2.5% glutaraldehyde and subsequently washed with phosphate buffer (PB) (pH 7.4) (10 min, with two changes), and post-fixed for 1 h in 1% osmium tetra-oxide (OsO4) at room temperature. The eggs were dehydrated in graded ethanol series (30, 50, 70 and 80% for 10 min each; and 95% for 15 min); followed by absolute ethanol for 10 min with two changes14. The eggs were then dried and mounted on stubs and sputter-coated with gold and examined under SEM (FEI–Quanta 400, Netherlands) in the Electron Microscopy Division, Defence Research and Development Establishment, Gwalior, India. The standard terminology15 was followed for the description of eggs.
Morphometric and morphological analysis of egg attributes
In total, 23 attributes were considered for morpho- metric and morphological analysis of eggs. At least 10 replicates of eggs were randomly selected for morpho-
metric and morphological analysis using SEM. The eggs were randomly taken from different oviposition events, different females and generations from the same labora- tory culture. The measurement of different attributes was done manually and data were presented as mean along with the standard error of mean (Mean ± SEM).
RESULTS
Scanning electron microscopic descriptions of the An.
stephensi ‘type’ form are depicted in Fig. 1(a–g), whereas the morphometric values have been shown in Table 1. The general as well as morphological characters observed in the present study have been described below:
(a) Overall appearance: In general, the eggs of An. ste- phensi ‘type’ form were black in colour and appeared slightly boat shaped in ventral, dorsal and lateral views. Anterior and posterior ends were blunt, but sometimes pointed. Ventral surface was concave and dorsal surface was curved (Fig. 1a).
(b) Size: The mean length of An. stephensi ‘type’ form was 473.94 + 11.18 µm and the width (at the broad- est point) was 154.69 + 2.66 µm. The length-to-width ratio was 3.06 + 0.12.
(c) Ventral surface: Deck was continuous and the width of anterior part of deck was almost similar to the pos- terior part (Fig. 1b). The maximum length of deck was measured as 479.76 + 22.97 µm, while the maximum width of anterior deck was 74.10 + 1.03 µm. The max- imum width of middle deck was 73.94 +4.66 µm and the area of deck region was calculated as 35370.33 + 2037.37 µm2.
(d) Lateral surface: Floats were present on both sides of the egg surface which is filled with air that helps in floating. They were short and closer to ventral than dorsal surface (Fig.1c). The number of float ribs mea- sured was 20.33 + 0.33. The maximum length of float was 246.57 + 15.27 µm, while maximum width of float was calculated as 87.16 + 3.83 µm. The length- to-width ratio of float was 2.83 + 0.16. The float length as percentage of egg length was calculated as 52.02 + 3.42%. The ratios between float length and oth- er parameters like egg width and number of ribs were 1.59 + 0.08 and 12.14 + 0.88, respectively. Simi- larly, the ratios between float width and egg width, and number of ribs were measured as 0.56 + 0.01 and 4.28 + 0.16, respectively.
(e) Anterior end, micropyle: As shown in Fig. 1, the an- terior and posterior ends were blunt, The micropylar apparatus was situated at the apex of dorsal side of the anterior pole. Outline of micropylar collar was
Fig. 1: Scanning electron micrograph of Anopheles stephensi ‘type’ form egg: (a) Lateral aspect; (b) Ventral aspect showing deck area; (c) Lateral aspect showing floats and ribs; (d) Micropylar disc showing ‘six’ sectors; (e) Micropylar disc showing ‘seven’ sectors; (f) Anterior; and (g) Posterior rosette tubercles.
(a) Lateral aspect (e) Seven sectors
Antierior rosette tubercles
Posterior rosette tubercles Deck area
Float and ribs
Six sector (b)
(c)
(d)
(f)
(g)
irregular in shape and well developed. Inner edge was uniformly and deeply excavated, peaks between excavations tapering to form radial ridges extending about halfway across micropylar disc, dividing disc into sectors (Figs. 1 d–e). Anopheles stephensi ‘type’
form has bigger micropyle with 6–7 sectors and num- ber of sectors in micropylar disc was calculated as
6.33 + 0.33. The area of micropylar disc was 267.50 + 12.16 µm2. The ratio between area and number of sec- tors in micropylar disc was found to be 42.56 + 3.55.
Lobed ventral tubercles were usually oval (Fig. 1 f–g).
The number of lobed ventral tubercles at anterior end of deck was 6.66 + 0.33, while lobes in each anterior ventral tubercle were found to be 7.64 + 0.50 (Fig. 1f).
Table 1. Morphometrics of egg attributes of Anopheles stephensi
‘type’ form
Attributes Total No. An. stephensi (Mean ± SE) Egg attributes
EGGL 10 473.94 + 11.18 µm
EGGW 10 154.69 + 2.66 µm
ELWR 10 3.06 + 0.12
Deck attributes
DECL 10 479.76 + 22.97 µm
DECW 10 74.10 + 1.03 µm
DEMW 10 73.94 + 4.66 µm
DECA 10 35370.33 + 2037.37 µm2
Micropyle
MICA 10 267.50 + 12.16 µm2
MICDR 10 42.56 + 3.55
MIDN 10 6.33 + 0.33
Ventral tubercles
ATUN 10 6.66 + 0.33
PTUN 10 4.66 + 0.33
ATLN 10 7.64 + 0.50
PTLN 10 8.18 + 0.51
Float attributes
FLOL 10 246.57 + 15.27 µm
FLOW 10 87.16 + 3.83 µm
FLWR 10 2.83 + 0.16
FRIN 10 20.33 + 0.33
FLELP 10 52.02 + 3.42
FLEWR 10 1.59 + 0.08
FWRR 10 4.28 + 0.16
FLPR 10 12.14 + 0.88
FWEWR 10 0.56 + 0.01
EGGL—Length of egg; EGGW—Width of egg; ELWR—Length/
width ratio of egg; DECL—Maximum length of deck; DECW—
Maximum width of anterior deck; DEMW—Maximum width of middle deck; DECA—Area of deck; MICA—Area of micropylar disc; MICDR—Area/Sector number ratio of micropylar disc;
MIDN—Number of sector of micropylar disc; ATUN—Number of antero-ventral tubercles; PTUN—Number of postero-ventral tubercles;
ATLN—Number of lobes of antero-ventral tubercles; PTLN—Number of lobes of postero-ventral tubercles; FLOL—Maximum length of float; FLOW—Maximum width of float; FLWR—Length/width ratio of egg; FRIN—Number of float ribs; FLELP—Float length as percentage of egg length; FLEWR—Float length/egg width ratio;
FWRR—Float width/number of ribs ratio; FLPR—Float length/
number of ribs; and FWEWR—Float width/egg width ratio.
which can be exploited for correct identification of vari- ants and sibling species. Studies conducted on the egg surface morphology and morphometry using electron microscopy (SEM) have been proved useful in separat- ing closely related mosquito species and their sibling species14.
The floats present on lateral surface on eggs serve as an important morphological marker and can be used in differentiating the eggs of different Anopheles species. In An. atropos, floats are fairly small (about half the length of the egg), whereas in An. darlingi floats measures more than half and in An. punctimacula floats extends 70% of the length of the egg16-17. In the present study, the float was short and closer to ventral surface than the dorsal surface.
Microscopic observations of float have revealed differ- ences among the eggs of species A, B, C and D of An.
dirus complex11.
Anopheles atropos and An. darling eggs have been well described using scanning electron micrographs. The eggs of An. atropos are conventionally structured and have comparatively large deck than An. darling16. In a study, it was reported that the eggs of two closely related species namely, An. fluminensis and An. shannoni differ consider- ably18. The An. fluminensis eggs are long with narrow deck region overlain by a frill modified into prominent ridges nearly continuous to both ends of the egg, whereas the An. shannoni eggs are unique and possess 22–27 finger- like filaments projecting with regular spacing from each of the floats18.
The number of lobed tubercles in the deck has been reported to vary noticeably in different anophelines11,16. Some variations have been noted in the number of branch- es of the lobed tubercles, which are fewer in An. bellator than in An. cruzii, but this character was not found con- sistent between both the species19. In this study, it was observed that lobed ventral tubercles of anterior and pos- terior end were usually oval and similar in structure.
Studies have revealed that in An. atropos, An. darlingi and An. vestitipennis, the micropylar collar is hexa or sep- tagonal in outline (with smooth surface), and have deeply and uniformly excavated inner edge16, 20. In the present study also, it was found that micropylar collar was hexa or septagonal with smooth surfaces and deeply excavated inner edges. Similar structure was observed by Malhotra et al21 in An. stephensi ‘type’ form mosquitoes. Anoph- eline eggs also vary considerably in number of ribs pres- ent on the egg surface. Earlier studies6, 22 have recorded 18 (± 1.6) and 18–19 ribs respectively, in the ‘type’ form, while the present study noted 16–19 ribs in most of the eggs (> 80%). The results are in conformity with those reported previously1 who grouped An. stephensi into (f) Posterior end: The posterior lobed tubercles were
similar in structure to anterior lobed tubercles (Fig.
1g). Lobed ventral tubercles at posterior end of deck were 4.66 + 0.33, while lobes of each posterior ventral tubercle were found to be 8.18 + 0.51.
DISCUSSION
Differentiable morphological characters are present in the egg stages of many important mosquito vectors
three categories and emphasized that all the categories have different rib number. It was suggested that the ‘type’
form of An. stephensi has 16–19 ribs, the ‘intermediate’
form has 13–16 ribs, whereas ‘mysorensis’ form was re- ported to have 10–14 ribs on egg surface. Earlier stud- ies1-2, 21 have described some details on the egg structure of An. stephensi ‘type’ form, however these descriptions did not elaborate the morphological and morphometric characters sufficient to exploit for differentiating it from other variants. The details provided in the present study completely matches the ‘type’ form description reported in earlier studies and additionally elaborated many other characters which could be used to confirm the identifica- tion of ‘type’ form9, 21. Earlier reports have established that the differences in the proportion of various morphological types were statistically significant, although proportions of egg types were variable among individuals within the same population12. The study had limitation that it could not rear var. ‘mysorensis’ and ‘intermediate’ variants of An. stephensi, and therefore could not characterize egg attributes for comparison among the three variants.
CONCLUSION
The present study is an attempt to elaborately describe the microscopic details as well as differentiable morpho- logical and morphometric characters of the ‘type’ form of an important urban malaria vector which is prevalent in majority of Southeast Asian countries. The study findings showed well precised morphometric and morphological egg surface attributes, that could be helpful in elucidating the classification of An. stephensi particularly between the ‘type’ form, var. ‘mysorensis’ and ‘intermediate’ of this species and also in differentiating them from other morphologically similar anophelines. Both traditional as well as molecular studies are required in corroboration for more information about variants of An. stephensi4. Further, studies are needed to rear var. ‘mysorensis’ and
‘intermediate’ forms; and to elaborate the morphometric and morphological characters of the eggs, so that compar- ison of characters could be done to find out differentiable characters.
ACKNOWLEDGEMENTS
The authors are thankful to the Director, Defence Re- search Laboratory (DRDO), Tezpur (Assam), India for taking interest and providing all necessary facility to con- duct this research work. Sincere thanks to the scientists and staff of the Medical Entomology Division of the De-
fence Research Laboratory, Tezpur for cooperation while carrying out the work. The assistance and SEM facility provided by the Director, Defence Research and Develop- ment Establishment (DRDO), Gwalior, India is gratefully acknowledged.
Conflict of interest
The authors declare that they have no competing interests.
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Correspondence to: Dr Sunil Dhiman, Medical Entomology Division, Defence Research Laboratory, Tezpur–784 001, Assam, India.
E-mail: [email protected]
Received: 7 June 2016 Accepted in revised form: 7 March 2017
