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Asian Pacific Journal of Tropical Biomedicine journal homepage: www.apjtb.com
Document heading
doi:10.12980/APJTB.4.2014C941
襃 2014
by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.
R evised morphological identification (Diptera: Culicidae) of Sri Lanka 1
1
key to the larval anopheline
1
2*
Nayana Gunathilaka , Thilan Fernando , Menaka Hapugoda , Wimaladharma Abeyewickreme
, Rajitha Wickremasinghe3
Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka
1
Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lanka
2
Department of Public Health, Faculty of Medicine, University of Kelaniya, Sri Lanka
3
PEER REVIEW
ABSTRACT
Peer reviewer Dr. Diawo Diallo, Medical Entomology U nit, I nstitut P asteur de D akar, 36 Av. Pasteur Dakar, B.P. 220, Dakar, Senegal. Tel: 221 7754109076 E-mail: [email protected]
Objective: To revise morphological identification keys to the anophelines in Sri Lanka. Method: Samples were collected from selected entomological sites in different districts in the country. Stage III and IV larvae were identified under a light microscope with an objective (伊 10) using standard larval keys developed for Sri Lankan anophelines. Key larval characters were recorded for each species based on original observations and previous usage in literature. Results: This manuscript describes an illustrated key for the identification of 22 of 23 mosquitoes which are currently recognized as local anopheline species in Sri Lanka, as a guide to workers engaged in malaria surveillance and control in the country. Conclusions: Revised morphological keys to the larval of these species may be helpful in easy and accurate identification at the field level.
Comments This study is very important because
the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. Details on Page S227
KEYWORDS Anopheline, Immature, Mosquitoes, Control, Keys
1. Introduction T he identification keys to the immature stages of Anopheles mosquitoes have long been a necessity for entomologists dealing with malaria vectors. Many of the illustrated keys to the Anopheles of Sri Lanka are of limited value[1,2], as these were published more than 20 years ago and significant advances in our knowledge of the Anopheles mosquitoes have occurred in the intervening years. The number of anopheline species has not changed substantially from Carter’s checklist; there have been many changes in the identity of the species actually listed, as evidenced * C orresponding author: P rof. W imaladharma A beyewickrem, D epartment of Parasitology, Faculty of Medicine, University of Kelaniya, P.O. Box 6, Thalagolla Road, Ragama 11010, Sri Lanka. Tel: +94 714914287 Fax: +94 117452130 E-mail: [email protected] Foundation Project: Supported by Global Fund for Aids, Tuberculosis and Malaria (GFATM) (Round 8) through TEDHA (Grant No. SRL809G11M).
by the checklist of Jayasekera and Chelliah[2]. Changes subsequently to this checklist include the invalidation of one species record and description of Anopheles jeyporiensis (An. jeyporiensis ). This paper presents an updated illustrated key for the identification of larval anophelines occurring in the country. The purpose of the keys presented here is to assist field surveillance teams to identify the larval stages of Anopheles mosquitoes. The keys can be used initially to identify specimens to species group and then to species. Discriminating characteristics are highlighted in drawings wherever possible. Article history: Received 12 Dec 2013 Received in revised form 20 Dec, 2nd revised form 29 Dec, 3rd revised form 3 Jan 2014 Accepted 20 Feb 2014 Available online 28 Mar 2014
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2. Materials and methods 2.1. Collecting samples Samples were collected from 17 entomological sites in Ampara, Batticaloa, Mannar, Trincomalee and Killinochchi
districts as part of the malaria elimination program of Tropical and Environmental Diseases and Health Associates (TEDHA) Pvt. Ltd. Further, wet zone specimens were obtained by the entomological teams attached to the Anti Malaria Campaign. 2.2. Identification of field samples S tage III and IV larvae were placed individually in microscopic slides and identified under a light microscope with an objective (伊10) using standard larval keys developed for S ri L ankan anophelines [2] . K ey larval characters were recorded for each species. Further, larval species identification was reconfirmed through adult identification[3].
2.3. Revising larval identification key for Sri Lankan Anopheles The morphological characters used here are based on original observations and previous use in the literature. The
following were referred to during the construction of this key:
Amarasinghe, 1992[2], Christophers, 1933[4], Colless, 1957[5], Reid, 1968[6], Harrison, 1980[7], Harrison and Scanlon, 1975[8], Linton et al., 2001[9], Junkum et al., 2005[10], Linton et al., 2005[11], Rattanarithikul et al., 2005[12], Sallum et al., 2005[13] and Rattanarithikul et al., 2006[14]. Taxonomic characteristics were cross checked in relation to Sri Lankan specimens by examining the reference materials archived at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. Species nomenclature follows that proposed by Knight and Stone[15], and abbreviations used in the text follow that used by Reinert[16,17]. Morphological terminology and chaetotaxy follow that used by Harbach and Knight[18]. Abdominal segments are numbered by roman numerals. Twenty two Sri Lankan anopheline species have been included into this key. The species considered include: Subgenus Anopheles aitkenii James 1903 (An. aitkenii), Anopheles barbirostris Van der Wulp 1884 (An. barbirostris), Anopheles barbumbrosus Strickland and Choudhury 1927 (An. barbumbrosus), Anopheles gigas var. refutans Alcock 1913 , Anopheles interruptus P uri 1929 ( An. interruptus ) , Anopheles nigerrimus Giles 1900 (An. nigerrimus), Anopheles peditaeniatus (Leicester) 1908 (An. peditaeniatus), Anopheles peytoni Kulasekera (An. peytoni), Harrison and Amerasinghe 1988.
Subgenus Cellia: Anopheles aconitus Donitz 1902 (An. aconitus), Anopheles annularis Vander Wulp 1884, Anopheles culicifacies Giles 1901 (An. culicifacies), Anopheles elegans (James) 1903 (An. elegans), Anopheles jamesii Theobald 1901,
An. jeyporiensis (James) 1902, Anopheles karwari (James) 1902, Anopheles maculatus Theobald 1901, Anopheles pallidus T heobald 1901 , Anopheles pseudojamesi S trickland and Chowdhury 1927, Anopheles ramsayi Covell 1927, Anopheles subpictus Grassi 1899 (An. subpictus), Anopheles tessellatus Theobald 1901 (An. tessellatus), Anopheles vagus Donitz 1902 (An. vagus), Anopheles varuna Iyengar 1924 (Figure 1). 7
Thorax
6
6
9
10 12
5
7
1 32
4
7
6 8 6
5
6
3 1 4 2
5 4 3
2
O
14
6 5
4
2
IV
O 1
8
12 11 10 11
10 11 13 12 9
8
1
7
12
13
7
10 11 12 13 11
10
14
V
10 7 121113 9
14 O 13 1 VI 11 12 7 4 2 9 6 5 10 3 8
9
2
3 4
A
O
1
C
7 10
9
M
13
11 12
T
9
10
11
II
12
8
10 9 12
6-Mx
14 15
5
6
P
7
11
III
O
4
13 12
14
8 2 O 3 41
Head
10 9 9
13
12 II 13 10 9
8 3 21 4 6 5
3
11
14
T
1
5 4 2 1 3
8
13
M 14
5 3 1 4 2 5 6
P
8
10 9
12
11
Tropacic pleural groups
9
VII
14
7 4
O
13 8 6432 9 3 11 1 7 10 14 2 O 5 4 1 VIII 2 6 3-5 1
2
9
S
3
Dorsal Ventral
Figure 1. Larval morphology. Head: A=Antenna, C=Cranium head capsule; Thorax: P=Pro-thorax, M=Mesothorax, T=Meta-thorax, 1-14=setae on designated areas (seta 1M, setae 1-T); Abdomen: I-VII, X=Abdominal segments, S=Spiracular apparatus, 1-14= Setae on designated areas (seta 1-I, seta 5-IV).
3. Results
The revised larval morphological key is shown below. This key presents an illustrated key for the identification of larvae of 22 of 23 currently recognized local anopheline species in Sri Lanka. Morphological features noted in different regions of the body were recorded: head (antennal hairs, inner clypeal hairs, outer clypeal hairs, frontal hairs and sutural hairs), thorax (thoracic palmate hairs, shoulder hairs, pro, meso and meta thoracic hairs), abdomen (abdominal tergal plates, palmate hairs in abdominal segments, lateral hairs in the abdominal segments). The inclusion of An. jeyporiensis is based on the reported collection during the TEDHA Malaria Elimination Program in 2011. This species was included in this key as local workers may encounter the species and may difficulty in its identification. G enerally, members of subgenus Anopheles are characterized by branched seta 1-A and closely situated setae 2-C. However, exceptions such as An. aitkenii sens. str., with widely spaced 2-C[2] and An. interruptus, with
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simple 1-A and closely spaced 2-C[2,6,8] have been reported in Sri Lanka. Figure 2 in the key has been worded to avoid initial misidentification of larvae. 4. Discussion The key to the Sri Lankan anopheline larvae has been
revised according to the currently accepted classification of sections, series and groups in the Anophelinae. Brief notes are provided at each step in the identification process along with illustrative diagrams. It is emphasized that taxonomic keys are only a rapid and convenient guide
to identification based on the examination of a few important characters. I n case of doubt, it is essential to consult published literature with detailed descriptions of species characters. 4.1. Subgenus Anopheles 4.1.1. Myzorhynchus series The Myzorhynchus series can be separated from other Sri Lankan representatives of subgenus Anopheles by the outer clypeal hair ( 3 - C ) being strongly branched. T wo species groups are present in Sri Lanka: the Hyrcanus and
Siphon absent; abdominal seta 1 palmate Antennal hair (1-A) branched
Antennal hair (1-A) simple
Inner clypeal (2-C) hairs close together
Inner clypeal (2-C) hairs well separated
5, 6 & 7 frontal hairs (5-C, 6-C & 7-C) long & branches 1
3
A 7
2 2 4 5
6
1
1
C
A 3
7
Subgenus Anopheles Outer clypeal (3-C) hair
4
A
3
1
2 1
4 5
6
1
C
2
3 4
Subgenus Cellia Abdominal tergal plates very large
strongly branched
Abdominal tergal plates small, transverse
IV-VII 2 3 1
3 3 4
1
C
5
2 2
C
5, 6 & 7 frontal hairs (5-C, 6-C & 7-C) short, simple or with few branches
Outer clypeal (3-C) hair
simple or few branches 1
6
1
2
2
4
4
2
C
13
IV-VII
Inner clypeal (2-C) hair with 9-18 Inner clypeal (2-C) hair with 1-4 short lateral short lateral barbs. Post clypeal (4-C) barbs. Post clypeal (4-C) simple or bifid
Figure 3
with 2-5 branches.
4
rarely.
Figure 4b
Thoracic palmate hair well developed Thoracic palmate hair rudimentary 5 53
4
O
21
II
8 53
Figure 4a
O
2 II 4 1
3
2
3
4
An. gigas
An. aconitus
4
2
An. varuna
Figure 2. Sri Lankan anopheline mosquito - key to the fourth instar larvae. Barbirostris Group
Hycanus Group
Inner shoulder (1-P) hair simple or split at the tip
Inner shoulder (1-P) hair branched from the base
Palmate hairs in the abdominal segment I-II un-pigmented
Palmate hairs in the abdominal segment I-II usually
pigmented (Except An. barbumbrosus)
1-II
1-II 3 2
1 P
Outer clypeal (2-C) hair with 12 – 36 thin
attenuated branches usually loose and spread out.
3
2
An. barbumbrosus Figure 3. Outer clypeal hair strongly branched.
3 2 1 P
II Outer clypeal (2-C) hair with more and thick
branches (19-95), usually stiff and broom like
3
2
An. barbirostris
Inner sutural (8-C) hair with 4 - 9 branches
Outer sutural (9-C) hairs with 3 – 7 branches
An. peditaeniatus
II Inner sutural (8-C) hair with 12- 24 branches. Outer sutural (9-C) hairs with 8-14 branches
An. nigerrimus
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with 7-M resulting in misidentification as An. nigerrimus. In this instance, branching of setae 8-C and 9-C are useful in confirmation. The correct identification of An. nigerrimus is important as it is a suspected vector of malaria in Sri Lanka while An. peditaeniatus is considered a non-vector (Figure 3).
Barbirostris group (Figure 2).
4.1.1.1. Hyrcanus group The Hyrcanus group can be separated from the Barbirostris group by the morphological characters given in the key. This group bears a superficial resemblance to members of the Hyrcanus group, all of which have pale or un-pigmented I–II; the separation of An. peditaeniatus and An. nigerrimus needs care. The definitive identifying feature is the basally branched, sinuous seta 4-M of An. peditaeniatus which is small and often difficult to observe. This may be confused
4.1.1.2. Barbirostris group Within the Barbirostris group of subgenus Anopheles, the orientation of the inner shoulder hair (1-P) can be used as the primary character: 1P seta with 4 or more branches Figure 4b
Figure 4a Inner clypeal (2-C) hair
Inner clypeal (2-C) hair
long stout & simple branched Outer clypeal (3-C) hair Post clypeal hair (4-C) simple or bifid branched from the base
3
Inner clypeal hair simple, outer clypeal (3C) hair branched. 4A & 11C branched 4
A
1 3 2
2
4
C
An. peytoni
hairs simple
C
2
10 9
Figure 5
Meso (M) & Metathoracic (T) plural hairs simple
7
6
5
4
3
with many branches. Root large and dark
21 P
6
An. tessellatus
7 5
4
3
2
6
Post clypeal hair (4-C)
with 2-5 branches
P
An. elegans
8
C
An. jeyporiensis
Palmate hair on abdominal segment I rudimentary
6
Palmate hairs with sharp
pointed filaments Abdominal seta 6 in segment V, VI with 3-6 branches
6
II
An. maculatus
8 3
3
3 2
5 4
1
6
M
Thoracic palmate hair rudimentary; roots of shoulder (M) hairs small & pale
Palmate hairs with blunt filaments Abdominal seta 6 in segment V, VI
with 6-16 branches
1-1 8 6
4
35
2
O
V
7
6
76 3 5
5 4
3
21
Outer (3-C) and post (4-C) clypeal hairs very
short, situated posteriorly between the inner clypeals (2-C) Base of the post clypeal (4-C) hairs wide apart & length of the outer clypeal (3-C) hair is about 1/3 of the length of inner clypeal hair (2-C)
An. karwari
Figure 5. Palmate hairs on abdominal segments.
5 4
6
9
8
10
1
2 3 1
Jamesii Group
54 76
2 V 5 4 1
C
Figure 6
developed;
P
O
8
Palmate hairs on abdominal segment no I well
Mesothoracic Pleural hairs not both simple; one branched
7 6
9
An. pallidus
An. annularis
a: Thoracic palmate hair well developed; b: Abdominal tergal plates small, transverse.
21
10
8 9 10
Figure 4. Thoracic palmate hair and abdominal tergal plates.
5 4
simple or split at the tip
Palmate hair on abdominal segment Palmate hair on abdominal segment I rudimentary without obvious leaflet I, with well developed leaflets
Inner shoulder (1-P) hair well developed,
1
Inner sutural (8-C) hair
branched from the base
C
Palmate hair on abdominal segment I & II rudimentary
branches. Root small and pale
hairs strongly branched
Inner sutural (8-C) hair
32
3
An. interruptus
Inner shoulder (1-P) hair weak with 2- 5
Inner (2-C) & outer (3-C) clypeal
2
3 2 4
II
C
inconspicuous fraying
Inner (2-C) & outer (3-C) clypeal
1
1
An. aitkenii
Inner (2-C) & outer (3-C) clypeal hairs with
C
423
P
2 1
M
5 3
Thoracic palmate hair moderately developed; roots of shoulder (M) hairs strong and dark P
1-1
M
7 65 4 O 3 6 7
An. culicifacies
Outer (3-C) and post (4-C) clypeal hairs longer, situated posteriorly below and outside the inner clypeals (2-C) Base of the post clypeal (4-C) hairs close together & length of the outer clypeal (3-C) hair is about 1/2 of the length of inner clypeal hair (2-C)
An. vagus 3 2
3
3
2 2 3 4 C 4
An. subpictus
On abdominal segment II always well developed; hairs on segment I sometimes rudimentary; Meso and Metathoracic Pleural hairs not all simple; one or more branched.
P M
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is characteristic of this group. This character should be checked carefully during identification. The pigmentation of seta I-II is an useful secondary character in separating barbirostris from barbumbrosus (pale or colorless seta). In addition, seta orientation of the outer clyepeal hair (3-C) can be used to separate these two species as the optional character in order to confirm the identification. It can be described as follows; 3-C of An. barbumbrosus has 1236 thin attenuated branches usually loose and separated out, whereas 3-C of An. barbirostris has more branches which are thick (19-95), usually stiff and broom like (Figure 3). The larval morphology of An. reidi has still not been reported. 4.1.2. Lophoscelomyia series An. interruptus is the only species in this series reported in the country. I t can be distinguished by the simple inner clypeal hair (2-C), and branched outer clypeal hair (3-C), 4-A and 11-C. In the previous key for Sri Lankan anopheline larvae, the identification was confirmed through the presence of reduced setae 5 , 6 and 7 - C , some or all of which were short and a few-branched or simple; seta 4-C simple or branched only in distal half[2]. These characters are rather difficult in identification and controversial. Therefore, the new character which has been included in this key is useful to minimize misidentification of this species (Figure 4). 4.1.3. Anopheles series There are three species in the country, namely: Anopheles gigas, An. aitkenii and Anopheles peytoni (An. peytoni). An. peytoni is an endemic species in Sri Lanka and had been confused with An. insulaeflorum; the larvae of An. peytoni has bases of setae 2-C nearly as wide apart as 2-C and 3-C on one side which is different from the larval morphology of An. insulaeflorum. In this key the following characters have been included to confirm identification of An. peytoni: 2-C long and simple, 3-C simple or bifid. These features facilitate the separation of An. aitkenii which has branched 2-C and 4-C (Figure 4). 4.2. Subgenus Cellia 4.2.1. Myzomyia series Separating Anopheles varuna and An. aconitus in the Myzomyia series of subgenus Cellia is sometimes difficult when the barbed nature of setae 2-C, 3-C and branching of 4-C in An. aconitus are observed by drift or the larval mouth brushes. I t is more difficult to distinguish An. jeyporiensis as it separates only by having a small tergal plate and 4-5 branches in 4-C (Figure 2). An. culicifacies, the main vector of malaria in S ri Lanka can be separated very easily among the members in Myzomyia series of subgenus Cellia by the presence of small tergal plates, unbranched 2 - C and 3 - C and moderately developed thoracic palmate hairs (Figure 5).
4.2.2. Pyretophorus series An. subpictus and An. vagus are the only two recorded species in this series in Sri Lanka. These two species can be easily separated from An. culicifacies by the moderately developed thoracic palmate hair and strong and dark roots of shoulder hairs. An. vagus has very short 3-C and 4-C, 4-C is situated between 2-C with the base of 4-C being wide apart, and the length of 3-C being about 1/3 of the length of 2-C; An. subpictus has longer 3-C and 4-C, and 4-C seta is situated below and outside 2-C; the base of the 4-C seta and length of the 3-C is about ½ of the length of 2-C (Figure 5). 4.2.3. Neocellia series Six species of Sri Lankan Neocellia can be divided into those with bushy setae 3-C (Anopheles jamesii, Anopheles annularis, Anopheles pallidus) and those with simple or barbed 3 - C ( Anopheles maculatus, Anopheles kawari, Anopheles pseudojamesi). A further character that separates the two groups is the length of the filaments in abdominal palmate setae IV –VII, these being equal to or greater than ½ the length of the blades in the former and 1/3rd or less in the latter (Figure 6). Inner (2-C) & outer (3-C) clypeal hairs
has short barbs
3
2
An. pseudojamesi
Inner clypeal (2-C) hair with short barbs
& outer clypeal (3-C) hair with brush like branches
3
2
An. jamesii
Figure 6. Palmate hair on abdominal segment I rudimentary without obvious leaflet.
4.2.4. Neomyzomyia series Two species, An. tessellatus and An. elegans (the latter belonging to the leucosphyrus group) have been reported in Sri Lanka. An. elegans is an endemic species in the country. Identification of An. tessellatus is important in malaria vector surveillance programmes in the Sri Lanka as it has been incriminated as a secondary vector of malaria transmission in the country. Identification of this species can be confirmed by the presence of 2-5 branches in the inner shoulder hair (1-P) with small and pale roots. Individuals with opposite morphological features can be regarded as An. elegans (Figure 4). Sri Lankan anopheline larvae can be differentiated easily by observing basic characters. However, this may become a difficult task when processing whole larvae in routine malaria entomological surveys.
Nayana Gunathilaka et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S222-S227
Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements would like to thank Dr. Panduka Wijeyarathne, the of the Malaria Elimination Program of Tropical and Environmental Diseases and Health Associates (TEDHA) Pvt. Ltd for his encouraging support and financial assistance from the Global Fund for Aids, Tuberculosis and Malaria (GFATM) (Round 8) through TEDHA (Grant No. SRL809G11M). I
P rogram D irector
Comments Background Morphological taxonomic keys are indispensable tools in malaria vectors surveillance and control. They help field operators to easily and accurately identify anopheline species in the field. I n S ri L anka, the published keys are outdated and need to be revised as knowledge on anophelines have advanced. Research frontiers This paper is carried out to revise a larval morphological identification key of the anopheline species of Sri Lanka. Related reports This key has reported one more species than the guide to the identification of anopheline mosquitoes of Sri Lanka published by Amarasinghe in 1992. Innovations and breakthroughs This paper contains the first illustrated key that allows the identification of the immature of 22 anopheline species currently known in Sri Lanka. Applications This key will assist field workers involved in malaria surveillance and control in Sri Lanka to firmly identify the anopheline species currently known in this island. Peer review This study is very important because the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. References [1] C arter HF . C eylon mosquitoes list species and names of
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mosquitoes recorded from Ceylon. Ceylon J Sci Sect B Zool 1950;
24: 85-115.
[2] Amarasinghe FP. A guide to the identification of anopheline mosquitoes (Diptera; Culicidae) of Sri Lanka. II. Larvae. Ceylon J Sci 1992; 22: 1-13. [3] Amarasinghe FP. A guide to the identification of anopheline mosquitoes (Diptera; Culicidae) of Sri Lanka. I. Adult females. Ceylon J Sci 1990; 21: 1-16. [4] Christophers SR. The fauna of British India, including Ceylon and Burma. London: Taylor & Francis; 1933, p. 371. [5] Colless DH. Further notes on the systematic of the Anopheles leucosphyrus group (Diptera: Culicidae). Proc R Entomol Soc London 1975; 26: 131-139. [6] Reid JA. Anopheles mosquitoes of Malaya and Borneo. England: Staples Printers Limited; 1968, p. 518. [7] Harrison BA. Medical entomology studies–XIII. The Myzomyia series of Anopheles (Cellia). In Thiland, with emphasis on intrainterspecific variations (Diptera: Culicidae). Contrib Am Entomol Inst 1980; 17(4): 1-195. [8] Harrison BA, Scanlon JE. The subgenus Anopheles in Thailand (Diptera: Culicidae). Contrib Am Entomol Inst 1975; 12(1): 1-307. [9] Linton YM, Harbach RE, Chang MS, Anthony TG, Matusop A. M orphological and molecular identity of Anopheles ( Cellia ) sundaicus (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia. Syst Entomol 2001; 26: 357-366. [10] Junkum A, Komalamisra N, Jitpakdi A, Jariyapan N, Min GS, Park MH, et al. Evidence to support two conspecific cytological races of Anopheles aconitus in Thailand. J Vector Ecol 2005; 30: 213-224. [11] Linton YM, Dusfour I, Howard TM, Ruiz LF, Duc Manh N, Ho Dinh T, et al. Anopheles (Cellia) epiroticus (Diptera: Culicidae), a new malaria vector species in the Southeast Asian Sundaicus Complex. Bull Entomol Res 2005; 95: 329-339. [12] Rattanarithikul R, Harrison BA, Panthusiri P, Coleman RE. Illustrated keys to the mosquitoes of Thailand I. Background; geographic distributions; list of genera, subgenera, and species; and a key to genera. Southeast Asian J Trop Med Public Health 2005; 36(Suppl 1): 1-80. [13] Sallum MA, Peyton EL, Wilkerson RC. Six new species of the Anopheles leucosphyrus group, reinterpretation of Anopheles elegans and vector implications. Med Vet Entomol 2005; 19: 158199. [14] Rattanarithikul R, Harrison BA, Harbach RE, Panthusiri P, Coleman RE. Illustrated keys to the mosquitoes of Thailand IV Anopheles. Southeast Asian J of Trop Med & Pub Health. 2006; 37(Suppl 2): 1-128. [15] Knight KL, Stone A. A catalog of the mosquitoes of the world (Diptera: Culicidae). 2nd ed. Maryland: Entomology Society of America; 1977, p. 1-611. [16] Reinert JF. Mosquito generic and subgeneric abbreviations (Diptera: Culicidae). Mosquito Systematics 1975; 7: 105-110. [17] R einert JF . R evised list of abrreviations for genera and subgenera of Culicidae (Diptera) and notes on generic and subgeneric changes. J Am Mosq Control Assoc 2001; 17: 51-55. [18] Harbach RE, Knight KL. Taxonomists’ glossary of mosquito anatomy. Marlton: Plexus Publishing; 1980, p. 415-420.
Asian Pac J Trop Biomed 2014; 4(Suppl 1): S222-S227
Asian Pacific Journal of Tropical Biomedicine journal homepage: www.apjtb.com
Document heading
doi:10.12980/APJTB.4.2014C941
襃 2014
by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.
R evised morphological identification (Diptera: Culicidae) of Sri Lanka 1
1
key to the larval anopheline
1
2*
Nayana Gunathilaka , Thilan Fernando , Menaka Hapugoda , Wimaladharma Abeyewickreme
, Rajitha Wickremasinghe3
Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka
1
Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lanka
2
Department of Public Health, Faculty of Medicine, University of Kelaniya, Sri Lanka
3
PEER REVIEW
ABSTRACT
Peer reviewer Dr. Diawo Diallo, Medical Entomology U nit, I nstitut P asteur de D akar, 36 Av. Pasteur Dakar, B.P. 220, Dakar, Senegal. Tel: 221 7754109076 E-mail: [email protected]
Objective: To revise morphological identification keys to the anophelines in Sri Lanka. Method: Samples were collected from selected entomological sites in different districts in the country. Stage III and IV larvae were identified under a light microscope with an objective (伊 10) using standard larval keys developed for Sri Lankan anophelines. Key larval characters were recorded for each species based on original observations and previous usage in literature. Results: This manuscript describes an illustrated key for the identification of 22 of 23 mosquitoes which are currently recognized as local anopheline species in Sri Lanka, as a guide to workers engaged in malaria surveillance and control in the country. Conclusions: Revised morphological keys to the larval of these species may be helpful in easy and accurate identification at the field level.
Comments This study is very important because
the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. Details on Page S227
KEYWORDS Anopheline, Immature, Mosquitoes, Control, Keys
1. Introduction T he identification keys to the immature stages of Anopheles mosquitoes have long been a necessity for entomologists dealing with malaria vectors. Many of the illustrated keys to the Anopheles of Sri Lanka are of limited value[1,2], as these were published more than 20 years ago and significant advances in our knowledge of the Anopheles mosquitoes have occurred in the intervening years. The number of anopheline species has not changed substantially from Carter’s checklist; there have been many changes in the identity of the species actually listed, as evidenced * C orresponding author: P rof. W imaladharma A beyewickrem, D epartment of Parasitology, Faculty of Medicine, University of Kelaniya, P.O. Box 6, Thalagolla Road, Ragama 11010, Sri Lanka. Tel: +94 714914287 Fax: +94 117452130 E-mail: [email protected] Foundation Project: Supported by Global Fund for Aids, Tuberculosis and Malaria (GFATM) (Round 8) through TEDHA (Grant No. SRL809G11M).
by the checklist of Jayasekera and Chelliah[2]. Changes subsequently to this checklist include the invalidation of one species record and description of Anopheles jeyporiensis (An. jeyporiensis ). This paper presents an updated illustrated key for the identification of larval anophelines occurring in the country. The purpose of the keys presented here is to assist field surveillance teams to identify the larval stages of Anopheles mosquitoes. The keys can be used initially to identify specimens to species group and then to species. Discriminating characteristics are highlighted in drawings wherever possible. Article history: Received 12 Dec 2013 Received in revised form 20 Dec, 2nd revised form 29 Dec, 3rd revised form 3 Jan 2014 Accepted 20 Feb 2014 Available online 28 Mar 2014
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2. Materials and methods 2.1. Collecting samples Samples were collected from 17 entomological sites in Ampara, Batticaloa, Mannar, Trincomalee and Killinochchi
districts as part of the malaria elimination program of Tropical and Environmental Diseases and Health Associates (TEDHA) Pvt. Ltd. Further, wet zone specimens were obtained by the entomological teams attached to the Anti Malaria Campaign. 2.2. Identification of field samples S tage III and IV larvae were placed individually in microscopic slides and identified under a light microscope with an objective (伊10) using standard larval keys developed for S ri L ankan anophelines [2] . K ey larval characters were recorded for each species. Further, larval species identification was reconfirmed through adult identification[3].
2.3. Revising larval identification key for Sri Lankan Anopheles The morphological characters used here are based on original observations and previous use in the literature. The
following were referred to during the construction of this key:
Amarasinghe, 1992[2], Christophers, 1933[4], Colless, 1957[5], Reid, 1968[6], Harrison, 1980[7], Harrison and Scanlon, 1975[8], Linton et al., 2001[9], Junkum et al., 2005[10], Linton et al., 2005[11], Rattanarithikul et al., 2005[12], Sallum et al., 2005[13] and Rattanarithikul et al., 2006[14]. Taxonomic characteristics were cross checked in relation to Sri Lankan specimens by examining the reference materials archived at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. Species nomenclature follows that proposed by Knight and Stone[15], and abbreviations used in the text follow that used by Reinert[16,17]. Morphological terminology and chaetotaxy follow that used by Harbach and Knight[18]. Abdominal segments are numbered by roman numerals. Twenty two Sri Lankan anopheline species have been included into this key. The species considered include: Subgenus Anopheles aitkenii James 1903 (An. aitkenii), Anopheles barbirostris Van der Wulp 1884 (An. barbirostris), Anopheles barbumbrosus Strickland and Choudhury 1927 (An. barbumbrosus), Anopheles gigas var. refutans Alcock 1913 , Anopheles interruptus P uri 1929 ( An. interruptus ) , Anopheles nigerrimus Giles 1900 (An. nigerrimus), Anopheles peditaeniatus (Leicester) 1908 (An. peditaeniatus), Anopheles peytoni Kulasekera (An. peytoni), Harrison and Amerasinghe 1988.
Subgenus Cellia: Anopheles aconitus Donitz 1902 (An. aconitus), Anopheles annularis Vander Wulp 1884, Anopheles culicifacies Giles 1901 (An. culicifacies), Anopheles elegans (James) 1903 (An. elegans), Anopheles jamesii Theobald 1901,
An. jeyporiensis (James) 1902, Anopheles karwari (James) 1902, Anopheles maculatus Theobald 1901, Anopheles pallidus T heobald 1901 , Anopheles pseudojamesi S trickland and Chowdhury 1927, Anopheles ramsayi Covell 1927, Anopheles subpictus Grassi 1899 (An. subpictus), Anopheles tessellatus Theobald 1901 (An. tessellatus), Anopheles vagus Donitz 1902 (An. vagus), Anopheles varuna Iyengar 1924 (Figure 1). 7
Thorax
6
6
9
10 12
5
7
1 32
4
7
6 8 6
5
6
3 1 4 2
5 4 3
2
O
14
6 5
4
2
IV
O 1
8
12 11 10 11
10 11 13 12 9
8
1
7
12
13
7
10 11 12 13 11
10
14
V
10 7 121113 9
14 O 13 1 VI 11 12 7 4 2 9 6 5 10 3 8
9
2
3 4
A
O
1
C
7 10
9
M
13
11 12
T
9
10
11
II
12
8
10 9 12
6-Mx
14 15
5
6
P
7
11
III
O
4
13 12
14
8 2 O 3 41
Head
10 9 9
13
12 II 13 10 9
8 3 21 4 6 5
3
11
14
T
1
5 4 2 1 3
8
13
M 14
5 3 1 4 2 5 6
P
8
10 9
12
11
Tropacic pleural groups
9
VII
14
7 4
O
13 8 6432 9 3 11 1 7 10 14 2 O 5 4 1 VIII 2 6 3-5 1
2
9
S
3
Dorsal Ventral
Figure 1. Larval morphology. Head: A=Antenna, C=Cranium head capsule; Thorax: P=Pro-thorax, M=Mesothorax, T=Meta-thorax, 1-14=setae on designated areas (seta 1M, setae 1-T); Abdomen: I-VII, X=Abdominal segments, S=Spiracular apparatus, 1-14= Setae on designated areas (seta 1-I, seta 5-IV).
3. Results
The revised larval morphological key is shown below. This key presents an illustrated key for the identification of larvae of 22 of 23 currently recognized local anopheline species in Sri Lanka. Morphological features noted in different regions of the body were recorded: head (antennal hairs, inner clypeal hairs, outer clypeal hairs, frontal hairs and sutural hairs), thorax (thoracic palmate hairs, shoulder hairs, pro, meso and meta thoracic hairs), abdomen (abdominal tergal plates, palmate hairs in abdominal segments, lateral hairs in the abdominal segments). The inclusion of An. jeyporiensis is based on the reported collection during the TEDHA Malaria Elimination Program in 2011. This species was included in this key as local workers may encounter the species and may difficulty in its identification. G enerally, members of subgenus Anopheles are characterized by branched seta 1-A and closely situated setae 2-C. However, exceptions such as An. aitkenii sens. str., with widely spaced 2-C[2] and An. interruptus, with
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simple 1-A and closely spaced 2-C[2,6,8] have been reported in Sri Lanka. Figure 2 in the key has been worded to avoid initial misidentification of larvae. 4. Discussion The key to the Sri Lankan anopheline larvae has been
revised according to the currently accepted classification of sections, series and groups in the Anophelinae. Brief notes are provided at each step in the identification process along with illustrative diagrams. It is emphasized that taxonomic keys are only a rapid and convenient guide
to identification based on the examination of a few important characters. I n case of doubt, it is essential to consult published literature with detailed descriptions of species characters. 4.1. Subgenus Anopheles 4.1.1. Myzorhynchus series The Myzorhynchus series can be separated from other Sri Lankan representatives of subgenus Anopheles by the outer clypeal hair ( 3 - C ) being strongly branched. T wo species groups are present in Sri Lanka: the Hyrcanus and
Siphon absent; abdominal seta 1 palmate Antennal hair (1-A) branched
Antennal hair (1-A) simple
Inner clypeal (2-C) hairs close together
Inner clypeal (2-C) hairs well separated
5, 6 & 7 frontal hairs (5-C, 6-C & 7-C) long & branches 1
3
A 7
2 2 4 5
6
1
1
C
A 3
7
Subgenus Anopheles Outer clypeal (3-C) hair
4
A
3
1
2 1
4 5
6
1
C
2
3 4
Subgenus Cellia Abdominal tergal plates very large
strongly branched
Abdominal tergal plates small, transverse
IV-VII 2 3 1
3 3 4
1
C
5
2 2
C
5, 6 & 7 frontal hairs (5-C, 6-C & 7-C) short, simple or with few branches
Outer clypeal (3-C) hair
simple or few branches 1
6
1
2
2
4
4
2
C
13
IV-VII
Inner clypeal (2-C) hair with 9-18 Inner clypeal (2-C) hair with 1-4 short lateral short lateral barbs. Post clypeal (4-C) barbs. Post clypeal (4-C) simple or bifid
Figure 3
with 2-5 branches.
4
rarely.
Figure 4b
Thoracic palmate hair well developed Thoracic palmate hair rudimentary 5 53
4
O
21
II
8 53
Figure 4a
O
2 II 4 1
3
2
3
4
An. gigas
An. aconitus
4
2
An. varuna
Figure 2. Sri Lankan anopheline mosquito - key to the fourth instar larvae. Barbirostris Group
Hycanus Group
Inner shoulder (1-P) hair simple or split at the tip
Inner shoulder (1-P) hair branched from the base
Palmate hairs in the abdominal segment I-II un-pigmented
Palmate hairs in the abdominal segment I-II usually
pigmented (Except An. barbumbrosus)
1-II
1-II 3 2
1 P
Outer clypeal (2-C) hair with 12 – 36 thin
attenuated branches usually loose and spread out.
3
2
An. barbumbrosus Figure 3. Outer clypeal hair strongly branched.
3 2 1 P
II Outer clypeal (2-C) hair with more and thick
branches (19-95), usually stiff and broom like
3
2
An. barbirostris
Inner sutural (8-C) hair with 4 - 9 branches
Outer sutural (9-C) hairs with 3 – 7 branches
An. peditaeniatus
II Inner sutural (8-C) hair with 12- 24 branches. Outer sutural (9-C) hairs with 8-14 branches
An. nigerrimus
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with 7-M resulting in misidentification as An. nigerrimus. In this instance, branching of setae 8-C and 9-C are useful in confirmation. The correct identification of An. nigerrimus is important as it is a suspected vector of malaria in Sri Lanka while An. peditaeniatus is considered a non-vector (Figure 3).
Barbirostris group (Figure 2).
4.1.1.1. Hyrcanus group The Hyrcanus group can be separated from the Barbirostris group by the morphological characters given in the key. This group bears a superficial resemblance to members of the Hyrcanus group, all of which have pale or un-pigmented I–II; the separation of An. peditaeniatus and An. nigerrimus needs care. The definitive identifying feature is the basally branched, sinuous seta 4-M of An. peditaeniatus which is small and often difficult to observe. This may be confused
4.1.1.2. Barbirostris group Within the Barbirostris group of subgenus Anopheles, the orientation of the inner shoulder hair (1-P) can be used as the primary character: 1P seta with 4 or more branches Figure 4b
Figure 4a Inner clypeal (2-C) hair
Inner clypeal (2-C) hair
long stout & simple branched Outer clypeal (3-C) hair Post clypeal hair (4-C) simple or bifid branched from the base
3
Inner clypeal hair simple, outer clypeal (3C) hair branched. 4A & 11C branched 4
A
1 3 2
2
4
C
An. peytoni
hairs simple
C
2
10 9
Figure 5
Meso (M) & Metathoracic (T) plural hairs simple
7
6
5
4
3
with many branches. Root large and dark
21 P
6
An. tessellatus
7 5
4
3
2
6
Post clypeal hair (4-C)
with 2-5 branches
P
An. elegans
8
C
An. jeyporiensis
Palmate hair on abdominal segment I rudimentary
6
Palmate hairs with sharp
pointed filaments Abdominal seta 6 in segment V, VI with 3-6 branches
6
II
An. maculatus
8 3
3
3 2
5 4
1
6
M
Thoracic palmate hair rudimentary; roots of shoulder (M) hairs small & pale
Palmate hairs with blunt filaments Abdominal seta 6 in segment V, VI
with 6-16 branches
1-1 8 6
4
35
2
O
V
7
6
76 3 5
5 4
3
21
Outer (3-C) and post (4-C) clypeal hairs very
short, situated posteriorly between the inner clypeals (2-C) Base of the post clypeal (4-C) hairs wide apart & length of the outer clypeal (3-C) hair is about 1/3 of the length of inner clypeal hair (2-C)
An. karwari
Figure 5. Palmate hairs on abdominal segments.
5 4
6
9
8
10
1
2 3 1
Jamesii Group
54 76
2 V 5 4 1
C
Figure 6
developed;
P
O
8
Palmate hairs on abdominal segment no I well
Mesothoracic Pleural hairs not both simple; one branched
7 6
9
An. pallidus
An. annularis
a: Thoracic palmate hair well developed; b: Abdominal tergal plates small, transverse.
21
10
8 9 10
Figure 4. Thoracic palmate hair and abdominal tergal plates.
5 4
simple or split at the tip
Palmate hair on abdominal segment Palmate hair on abdominal segment I rudimentary without obvious leaflet I, with well developed leaflets
Inner shoulder (1-P) hair well developed,
1
Inner sutural (8-C) hair
branched from the base
C
Palmate hair on abdominal segment I & II rudimentary
branches. Root small and pale
hairs strongly branched
Inner sutural (8-C) hair
32
3
An. interruptus
Inner shoulder (1-P) hair weak with 2- 5
Inner (2-C) & outer (3-C) clypeal
2
3 2 4
II
C
inconspicuous fraying
Inner (2-C) & outer (3-C) clypeal
1
1
An. aitkenii
Inner (2-C) & outer (3-C) clypeal hairs with
C
423
P
2 1
M
5 3
Thoracic palmate hair moderately developed; roots of shoulder (M) hairs strong and dark P
1-1
M
7 65 4 O 3 6 7
An. culicifacies
Outer (3-C) and post (4-C) clypeal hairs longer, situated posteriorly below and outside the inner clypeals (2-C) Base of the post clypeal (4-C) hairs close together & length of the outer clypeal (3-C) hair is about 1/2 of the length of inner clypeal hair (2-C)
An. vagus 3 2
3
3
2 2 3 4 C 4
An. subpictus
On abdominal segment II always well developed; hairs on segment I sometimes rudimentary; Meso and Metathoracic Pleural hairs not all simple; one or more branched.
P M
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is characteristic of this group. This character should be checked carefully during identification. The pigmentation of seta I-II is an useful secondary character in separating barbirostris from barbumbrosus (pale or colorless seta). In addition, seta orientation of the outer clyepeal hair (3-C) can be used to separate these two species as the optional character in order to confirm the identification. It can be described as follows; 3-C of An. barbumbrosus has 1236 thin attenuated branches usually loose and separated out, whereas 3-C of An. barbirostris has more branches which are thick (19-95), usually stiff and broom like (Figure 3). The larval morphology of An. reidi has still not been reported. 4.1.2. Lophoscelomyia series An. interruptus is the only species in this series reported in the country. I t can be distinguished by the simple inner clypeal hair (2-C), and branched outer clypeal hair (3-C), 4-A and 11-C. In the previous key for Sri Lankan anopheline larvae, the identification was confirmed through the presence of reduced setae 5 , 6 and 7 - C , some or all of which were short and a few-branched or simple; seta 4-C simple or branched only in distal half[2]. These characters are rather difficult in identification and controversial. Therefore, the new character which has been included in this key is useful to minimize misidentification of this species (Figure 4). 4.1.3. Anopheles series There are three species in the country, namely: Anopheles gigas, An. aitkenii and Anopheles peytoni (An. peytoni). An. peytoni is an endemic species in Sri Lanka and had been confused with An. insulaeflorum; the larvae of An. peytoni has bases of setae 2-C nearly as wide apart as 2-C and 3-C on one side which is different from the larval morphology of An. insulaeflorum. In this key the following characters have been included to confirm identification of An. peytoni: 2-C long and simple, 3-C simple or bifid. These features facilitate the separation of An. aitkenii which has branched 2-C and 4-C (Figure 4). 4.2. Subgenus Cellia 4.2.1. Myzomyia series Separating Anopheles varuna and An. aconitus in the Myzomyia series of subgenus Cellia is sometimes difficult when the barbed nature of setae 2-C, 3-C and branching of 4-C in An. aconitus are observed by drift or the larval mouth brushes. I t is more difficult to distinguish An. jeyporiensis as it separates only by having a small tergal plate and 4-5 branches in 4-C (Figure 2). An. culicifacies, the main vector of malaria in S ri Lanka can be separated very easily among the members in Myzomyia series of subgenus Cellia by the presence of small tergal plates, unbranched 2 - C and 3 - C and moderately developed thoracic palmate hairs (Figure 5).
4.2.2. Pyretophorus series An. subpictus and An. vagus are the only two recorded species in this series in Sri Lanka. These two species can be easily separated from An. culicifacies by the moderately developed thoracic palmate hair and strong and dark roots of shoulder hairs. An. vagus has very short 3-C and 4-C, 4-C is situated between 2-C with the base of 4-C being wide apart, and the length of 3-C being about 1/3 of the length of 2-C; An. subpictus has longer 3-C and 4-C, and 4-C seta is situated below and outside 2-C; the base of the 4-C seta and length of the 3-C is about ½ of the length of 2-C (Figure 5). 4.2.3. Neocellia series Six species of Sri Lankan Neocellia can be divided into those with bushy setae 3-C (Anopheles jamesii, Anopheles annularis, Anopheles pallidus) and those with simple or barbed 3 - C ( Anopheles maculatus, Anopheles kawari, Anopheles pseudojamesi). A further character that separates the two groups is the length of the filaments in abdominal palmate setae IV –VII, these being equal to or greater than ½ the length of the blades in the former and 1/3rd or less in the latter (Figure 6). Inner (2-C) & outer (3-C) clypeal hairs
has short barbs
3
2
An. pseudojamesi
Inner clypeal (2-C) hair with short barbs
& outer clypeal (3-C) hair with brush like branches
3
2
An. jamesii
Figure 6. Palmate hair on abdominal segment I rudimentary without obvious leaflet.
4.2.4. Neomyzomyia series Two species, An. tessellatus and An. elegans (the latter belonging to the leucosphyrus group) have been reported in Sri Lanka. An. elegans is an endemic species in the country. Identification of An. tessellatus is important in malaria vector surveillance programmes in the Sri Lanka as it has been incriminated as a secondary vector of malaria transmission in the country. Identification of this species can be confirmed by the presence of 2-5 branches in the inner shoulder hair (1-P) with small and pale roots. Individuals with opposite morphological features can be regarded as An. elegans (Figure 4). Sri Lankan anopheline larvae can be differentiated easily by observing basic characters. However, this may become a difficult task when processing whole larvae in routine malaria entomological surveys.
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Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements would like to thank Dr. Panduka Wijeyarathne, the of the Malaria Elimination Program of Tropical and Environmental Diseases and Health Associates (TEDHA) Pvt. Ltd for his encouraging support and financial assistance from the Global Fund for Aids, Tuberculosis and Malaria (GFATM) (Round 8) through TEDHA (Grant No. SRL809G11M). I
P rogram D irector
Comments Background Morphological taxonomic keys are indispensable tools in malaria vectors surveillance and control. They help field operators to easily and accurately identify anopheline species in the field. I n S ri L anka, the published keys are outdated and need to be revised as knowledge on anophelines have advanced. Research frontiers This paper is carried out to revise a larval morphological identification key of the anopheline species of Sri Lanka. Related reports This key has reported one more species than the guide to the identification of anopheline mosquitoes of Sri Lanka published by Amarasinghe in 1992. Innovations and breakthroughs This paper contains the first illustrated key that allows the identification of the immature of 22 anopheline species currently known in Sri Lanka. Applications This key will assist field workers involved in malaria surveillance and control in Sri Lanka to firmly identify the anopheline species currently known in this island. Peer review This study is very important because the identification of mosquito species is always the first step in all surveillance and control of malaria strategies. It describes an updated and easy way to use illustrated key that will be useful for Sri Lankan malaria control personnel and researchers interested in local anopheline fauna. References [1] C arter HF . C eylon mosquitoes list species and names of
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mosquitoes recorded from Ceylon. Ceylon J Sci Sect B Zool 1950;
24: 85-115.
[2] Amarasinghe FP. A guide to the identification of anopheline mosquitoes (Diptera; Culicidae) of Sri Lanka. II. Larvae. Ceylon J Sci 1992; 22: 1-13. [3] Amarasinghe FP. A guide to the identification of anopheline mosquitoes (Diptera; Culicidae) of Sri Lanka. I. Adult females. Ceylon J Sci 1990; 21: 1-16. [4] Christophers SR. The fauna of British India, including Ceylon and Burma. London: Taylor & Francis; 1933, p. 371. [5] Colless DH. Further notes on the systematic of the Anopheles leucosphyrus group (Diptera: Culicidae). Proc R Entomol Soc London 1975; 26: 131-139. [6] Reid JA. Anopheles mosquitoes of Malaya and Borneo. England: Staples Printers Limited; 1968, p. 518. [7] Harrison BA. Medical entomology studies–XIII. The Myzomyia series of Anopheles (Cellia). In Thiland, with emphasis on intrainterspecific variations (Diptera: Culicidae). Contrib Am Entomol Inst 1980; 17(4): 1-195. [8] Harrison BA, Scanlon JE. The subgenus Anopheles in Thailand (Diptera: Culicidae). Contrib Am Entomol Inst 1975; 12(1): 1-307. [9] Linton YM, Harbach RE, Chang MS, Anthony TG, Matusop A. M orphological and molecular identity of Anopheles ( Cellia ) sundaicus (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia. Syst Entomol 2001; 26: 357-366. [10] Junkum A, Komalamisra N, Jitpakdi A, Jariyapan N, Min GS, Park MH, et al. Evidence to support two conspecific cytological races of Anopheles aconitus in Thailand. J Vector Ecol 2005; 30: 213-224. [11] Linton YM, Dusfour I, Howard TM, Ruiz LF, Duc Manh N, Ho Dinh T, et al. Anopheles (Cellia) epiroticus (Diptera: Culicidae), a new malaria vector species in the Southeast Asian Sundaicus Complex. Bull Entomol Res 2005; 95: 329-339. [12] Rattanarithikul R, Harrison BA, Panthusiri P, Coleman RE. Illustrated keys to the mosquitoes of Thailand I. Background; geographic distributions; list of genera, subgenera, and species; and a key to genera. Southeast Asian J Trop Med Public Health 2005; 36(Suppl 1): 1-80. [13] Sallum MA, Peyton EL, Wilkerson RC. Six new species of the Anopheles leucosphyrus group, reinterpretation of Anopheles elegans and vector implications. Med Vet Entomol 2005; 19: 158199. [14] Rattanarithikul R, Harrison BA, Harbach RE, Panthusiri P, Coleman RE. Illustrated keys to the mosquitoes of Thailand IV Anopheles. Southeast Asian J of Trop Med & Pub Health. 2006; 37(Suppl 2): 1-128. [15] Knight KL, Stone A. A catalog of the mosquitoes of the world (Diptera: Culicidae). 2nd ed. Maryland: Entomology Society of America; 1977, p. 1-611. [16] Reinert JF. Mosquito generic and subgeneric abbreviations (Diptera: Culicidae). Mosquito Systematics 1975; 7: 105-110. [17] R einert JF . R evised list of abrreviations for genera and subgenera of Culicidae (Diptera) and notes on generic and subgeneric changes. J Am Mosq Control Assoc 2001; 17: 51-55. [18] Harbach RE, Knight KL. Taxonomists’ glossary of mosquito anatomy. Marlton: Plexus Publishing; 1980, p. 415-420.
