. ., -, ii L .

C , ,,

J. N a t n . Sci. Coun. Sri L a n k a 1995 23(3): 115-129

A GUIDE TO THE IDENTIFICATION OF THE ANOPHELINE

MOSQUITOES OF SRI mmw I n . PUPAE

F.P. AMERASINGHE
Department of Zoology, University of Peradeniya, Peradeniya.

(Received: 19 April 1995; accepted: 11 August 1995)

Abstract: An Illustrated key is provided for the identification o f the pupal

stages o f 21 o f the 22 species of anopheline mosquitoes occurring in Sri Lanka,
for which this lifestage has been previously described. The species discussed are:
Anopheles (Anopheles) aitkenii James, An. (Ano.) barbirostris V a n der W u l p ,
An. (Ano.) barbumbrosus Strickland & Chowdhury, An. (Ano.)gigas Giles var.
refutans Alcock, An. (Ano.) interruptus Puri, An: (h.) nigerrimus Giles, An.
(Ano.) peditaeniatus Leicester, An. (Ano.) peytoni Kulasekera, H a m s o n &
Amerasinghe, An. (Cellia) aconitus Dijnitz, An. (Cel.) annularis V a n der W u l p ,
An. (Cel.)culicifacies Giles,An. (Cel.)elegans James&. (Cel.)ja~msiiTheobald,
An. (Cel.) karwari (James),An. (Cel.) maculatus Theobald, An. (Cel.) pallidus
Theobald,An. (Cel.)pseudojamesi Strickland & Chowdhury,An. (Cel.)subpictus
Grassi, An. (Cel.) tessellatus Theobald,An. (Cel.) varuna Iyengar, anpan. (Cel.)
vagus Donitz. The pupa o f one species, Anopheles (Anopheles) reidi Harrison, is
unknown.

K e y w o r d s : Anopheles, key for identification, pupae, Sri Lanka.

INTRODUCTION

Mosquitoes of the genus Anopheles are vectors of human and animal malaria,
and are also involved in the transmission cycles of filarial nematodes and
v i r ~ s e s .Recent
~ . ~ researchin Sri'Lanka has.shown that althoughAn. culicifacies
Giles (species B ) is the major vector of malaria, several other species may be
involved in seasonal or local transmission in different parts of the ~0untr-y.~"
Thus, accurate identification of anopheline species is becoming increasingly
important in the context oflocal malaria entomology. As part of an ongoingstudy
on the taxonomy of Sri Lankan mosquitoes,illustrated keys for the identification
of adult and larval stages of local anophelines have been published previo~sly.~,~

Anopheline pupal taxonomy, however, has received relatively little atten-

tion in the Indian subcontinent. Pupae possess stable taxonomic characters that
are readily visible under high power on slide-mounted exuviae. However, these
characters are more difficult to visualize on whole pupae under dissecting
microscopes, due primarily to pupal shape, colouration, and the generally
inconspicuous nature of taxonomically important setae. Pupae often constitute
the smaller proportion of field-collected immatures (relative tolarvae), and field
workers usually go through the laborious process of rearing out adults from
collected pupae in order to establish species identity. However, pupae that die
during rearing and those directly killed and preserved in the field cannot be
identified by such means. The routine identification of field collected pupae is
of some importance, as apart from purely taxonomic considerations, quantita-
tive data based on this life stage can provide better estimates of adult production
from breeding habitats than those derived from larval stages alone. This is
 116 F.P. Amerasinghe

because mortality within different larval stages, and in the larval-pupal trans-
formation can be extremely Mgh. Following upon the publication of keys for the
identification of adults and lar~ae,"9~
the present paper provides a n illustrated
key to the pupae of Sri Lankan anophelines, so as to complete the series on the
taxonomy of the local anopheline species.

METHODS AND MATERIALS

The following 21 species are included in the key: Anopheles (Anopheles) aitkenii
James,An. (Ano.) barbirostrisVanderWulp,An. (Ano.) barbumbrosus Strickland
& Chowdhury, An. (Ano.) gigas Giles var. refutans Alcock, An. (Ano.) interruptus
Puri, An. (Ano.) nigerrimus Giles, An. (Ano.)peditaeniatus Leicester, An. (Ano.)
peytoni Kulasekera, Harrison & Amerasinghe, An. (Cellia)aconitus Donitz, An.
(Cel.) annularis Van der Wulp, An. (Cel.) culicifacies Giles, An. (Cel.) elegans
James, An. (Cel.) jamesii Theobald, An. (Cel.) karwari (James), An. (Cel.)
maculatus Theobald, An. (Cel.) pallidus Theobald, An. (Cel.) pseudojamesi
Strickland & Chowdhury, An. (Cel.) subpictus Grassi, An. (Cel.) tessellatus
Theobald, An. (Cel.) uaruna Iyengar, andAn. (Cel.) uagus Donitz. The pupa of
An. (Ano.) reidi Harrison is unknown, and is not included in the key.

The primary taxonomic literature references used in constructing the key

were the works of Harrison & Scanlonl (for subgenus Anopheles), Reid3 and
Christophersg (for subgenera Anopheles and Cellia), and Harrisonl0 (for the
Myzomyia series of subgenus Cellia). These works contained taxonomic descrip-
tions of pupae, as well as species-group related keys for the southeast Asian or
Oriental regions, that allowed the formulation of key steps for most ofthe species
occurring in Sri Lanka. In addition, Mendis et al. l1 and Kulasekera et al. l2were
consulted for detailed descriptions ofAn. elegans and An. peytoni, respectively.

Pupal stages (slide-mounted exuviae with associated larval and adult

stages) of local Anopheles collected by the Smithsonian Institution's Medical
Entomology Project (MEP), as well as field matefial collected by the author and
deposited'at the Department of Zoology, University of Peradeniya, were also
examined during the study. These includedAn. barbirostris, An. nigerrimus, An.
peditaeniatus, An. peytoni, An. aconitus, An. annularis, An. culicifacies, An.
elegans, An. jamesii, An, subpictus, An. varuna, and An. uagus. Specimens ofAn.
tessellatus were obtained from a colony maintained a t the Institute of Funda-
mental Studies (IFS), Kandy. This material was used to check taxonomic
characters used in the key, as well as to formulate the key characters used for
the identification of An, subpictus and An. uagus (pupae of which have been
incorrectly separated previously)" as well as An. elegans and An. tessellatus
which have not been treated in previous pupal keys.3

Pupal chaetotaxy designations used herein follow Harbach & Knight.13 The
illustrations were drawn by the author partly from original material (in the case
of species available for direct examination) and partly adapted from previous
works.ls3 They are diagrammatic representations of characters used in the key,
meant as a guide to those unfamiliar with the morphology and chaetotaxy of
Key to Anopheles Pupae 117

mosquito immature stages, and follow the standard conventions established f ~ r

mosquito taxonomic publication^.'^ The following points of clarification are
made for those unfamiliar with aspects of the modern terminology and conven-
tions that may be found in the key and illustrations: (i)Pupal setal numbers are
denoted by arabic numerals and abdominal segment numbers by roman numer-
als; (ii)The meatal cleft of the trumpet is always located on the inner (mesal)side,
and the secondary cleft (if present) located on the outer (lateral) side; (iii) The two
borders of the paddle are referred t o as lateral (outer) and mesal (inner)borders;
(iv) The refractile border of the paddle refers to the region of the lateral border
that usually bears marginal serrations ("paddleteethn),andis refractile to light;
(v) the term "mesad refers to the inner (mesal) side, and "laterad" to the outer
(lateral) side of any structure.

KEY TO ANOPHELINE PUPAE

1. Trumpet with longest axis more or less vertical to stem; rim of

trumpet simple, without secondary cleft (Fig. l a ) .............................. 2

Trumpet with longest axis transverse to stem; rim of trumpet

with or without secondary cleft (Fig. lb) (SubgenusAnopheles;
Laticorn section; Myzorhynchus series; barbirostri's and
hyrcanus gps.) .....................................................................................
18

2(1). Trumpet very broad and rounded, transverse axis about as

long as vertical; a very large species (Fig. 2a) (Subgenus
Anopheles; Angusticorn section; Anopheles series;
lindesayi gp.) ..............................................:.................................... gigas

Trumpet not so broad, vertical axis longer than transverse;

~ s i - v d .......................
, m ~ w h ~ h~ ~m~ ~ -~hf wxgrk0h) ~ .?o

3(2). Paddle broad, 1.5 or less times as long as wide; seta 5-V-VII
usually no stouter than 1-V-VII;male genital lobes with
apical knobs (Fig. 3a) (Subgenus Cellia).............................................. 4

Paddle elongate, 1.6 or more times as long as wide; if not elongate

then seta 5-V-VII much stouter than 1-V-VII; male genital
lobes not ending in knob (Fig. 3b) (Subgenus Anopheles;
Angusticorn section; Anopheles and Lophoscelomyia series) ...........16

4(3). Seta 1-Pa short, 0.15.or less length of lateral margin of paddle;
9-V-VII usually less than 0.35 length of lateral margin of
their respective segments (Fig. 4a) (Neomyzomyia series) ...............5

Seta 1-Pa long, 0.25 or more length of lateral margin of paddle, ,

c u ~ e dsinuate
, or hooked at tip; 9-V-VII usually 0.35 or more ,

length of lateral margin of their respective segments (Fig. 4b) .........6

 F.P.Amerasinghe

5(4). Seta 1-VI,VII 3-5 branched; seta 9-V-VII, 0.15 or less

length of lateral margin of respective segments; genital
lobe with clearly defined knobs (Fig. 5a) .............................tessellatus

Seta 1-VI,VIPusually single; seta 9-V-VII 0.25 or more

length of lateral margin of respective segments; genital
lobe with indistinctly defined knobs (Fig. 5b)........................... elegans

6(4). Seta 9-1 simple, rarely branched, long, usually twice or more
length of lateral margin of segment I (Fig. 6a)
(Pyretophorns series).......................................................................... 7

Seta 9-1 s h p l e or branched, shorter to slightly longer than lateral

margin of segment I (Fig. 6b) ............................................................... 8

7(6). Seta 9-V-VII with distinctly blunt tips (Fig. 7a) ........................subpictus

Seta 9-V-VII with distinctly sharp pointed tips (Fig. 7b) ................ vagus

$(6). Seta 9-IV usually 0.67 or more length of 9-V, with same tapering
sharp pointed shape as 9-V; 1-11with 8 or more branches
(Fig. 8a) (Myzomyia series) .................................................................. 9

Seta 9-IV 0.15 to 0.67 length o f 9-V, broader with more rounded
apex than 9-V; 1-11with 2-10 branches, usually less than 8
(Fig. 8b) (Neocellia series) ................................................................ 1 1

9(8). Seta 1-V-VII simple; seta 7-VI,VII same length or slightly shorter
than 9-VI,VII, approximately 0.35 to 0.70 length of segment
VI, WI lateral margins; paddle fringe not extending
mesad of seta 1-Pa (Fig. 9a) ............................................. .culicifacies

Seta 1-V-VII usually 2-5 branched; seta 'I-VI,VII much longer

than 9VI,VII, equal to or slightly longer than segment VI,
VII lateral margins; paddle fringe extending mesad of seta
1-Pa (Fig. 9b)...................................................................................
10

lO(9). Paddle Ginge spicdes mesad of seta 1-Pa short, widely spaced,
approximately 0.5 length of spicules just laterad of 1-Pa,
mesa1 spicules not extendingto mesa1 angle of paddle; trumpet
pinna distally rounded, venter convex at apex; sum of
branches of both setae 1-III,14- 32; sum of branches of
both setae 5-111, 9-22 (Fig. 10a) .............................................aconitus
Key to Anopheles Pupae

Paddle fringe spicules mesad of seta 1-Pa long, closely spaced,

equal in length to spicules just laterad of 1-Pa, mesal spicules
extending to mesal angle of paddle; trumpet pinna distally
flattened, venter concave at apex; sum of branches on both
setae 1-111, 31-39; sum of branches on both setae 5-III,22-37
(Fig. lob) ................................................................................. varuna

ll(8). Seta 9-IV usually less than 0.1 length of lateral margin of
segment, less than 0.25 length of 9-V (Fig. l l a ) ................... karwari

Seta 9-IV 0.2 or more length of lateral margin of segrne~t,

0.25 or more length of 9-V (Fig. Ilb) .............................................. 12

12(11). Seta 1-Pa, unstraightened, about 0'.33 or more length of

paddle; fringe spicules extend mesad of seta
1-Pa (Fig. 12a) .................................................................... maculatus

Seta 1-Pa, unstraightened, less than 0.33 length of paddle;

fringe spicules do not extend mesad of seta 1-Pa (Fig. 12b) .........13

13(12). Seta 2-VII usually simple; trumpet meatus about 0.33

trumpet length (Fig. 13a) .............................................
pseudojamesi

Seta 2-VII usually 2-4 branched; trumpet meatus about 0.25

trumpet length (Fig. 13b)........................................................... 1 4

14(13). Seta 9-1 usually triple; lower refractile border of paddle

with non-filamentous spicules (Fig. 14a)........................... annularis

Seta 9-1 simple or bifid; lower refractile border of paddle with

filamentous spicules (Fig. 14b).......................................................
15

15(14). Seta 1-Pa strongly coiled (Fig. 15a) ..............................................

jamestz
..

Seta 1-Pa hooked, but not coiled (Fig. 15b) ...............................pallidus

16(3). Trumpet without meatal cleft; seta 1-Pa with 2-5 branches
from midpoint; phytotelmic habitats (Fig. 16a)
(Lophoscelomyia series; asiaticus gp.).............................interruptus

Trumpet with deep meatal cleft; seta 1-Pa simple; ground water
habitats (Fig. 16b) (Anopheles series, aitkenii gp.) ....................... 17
 F.P. Amerasinghe

17(16). Seta 1-IV with 2-5 branches; seta 9-IV with blunt, rounded tip;
..
paddle refractile margin long (0.6) (Fig. 17a) ....................... aithnzz

Seta 1-IV with 5-9 branches; seta 9-IVwith sharp tip; paddle
refractile margin short (0.4-0.5) (Fig. 17b) ............................ peytoni

18(1). Opening of trumpet narrow, very transverse when viewed

from above; seta 1-VII a strong tuft of 15-50 branches
(Fig. 18a) (barbirostris gp.).......................................................... 19

Opening of trumpet more expanded when viewed from above;

seta 1-VII a weaker tuft of 1-7 branches
(Fig. 18b) (hyrcanus gp.) .................................................................20

19(18). Trumpet without secondary cleft, but with thickened seam;

abdominal seta 9 dark brown to black; seta 9-VII 6-8
times a s long as thick (Fig. 19a) .................................barbumbrosus

Trumpet with secondary cleft, without seam; abdominal seta 9

yellow to light brown; seta 9-VII 4-6 times
a s long as thick (Fig. I9b) ................................................ barbirostris

20(18). Trumpet with thickened saw-toothed areas on rim; seta 9-VIII

branching reduced or absent (Fig. 20a) .......................peditaeniatus

Trumpet without thickened saw-toothed areas, but with dark

border.&ea delineating thin, uniform rim; seta 9-VIII
with well developed branches (Fig. 20b) ..........................nigerrimus

(The pupa of An. (Ano.) reidi of the barbirostris group is unknown. However,
based on group characteristics it should key out to the barbirostris group a t step
18).

Figures 1-20 are diagrammatic representations of key characters, numbered in

accordance with the key steps. Abbreviations are as follows: GL = genital lobe,
MC = meatal cleft, MP = metanotal plate, Pa = paddle, S = seam, SC = secondary
cleft, Tr = trumpet.
Key to Anopheles Pupae

FIG l a

Figure la,b: Lateral view of angusticorn and laticorn trumpets.

FIG 2a FIG 2b

Figure 2a,b: Lateral view of two angusticorn trumpets.

FIG 3a FIG 3b

Figure 3a,b: Dorsal view of abdominal segments V-VII to show seta 1,5-V-WI,
and ventral view of genital lobe.
 122 F.P. Amerasinghe

FIG 4 a FIG bb

Figure 4a,b: Dorsal view of abdominal segments V-VII to show seta 9-V-VTI,
and of paddle to show seta 1-Pa.

FIG 5a FIG Sb F I G 6a F I G 6b

Figure. Sa,b: Dorsal view of segments Figure. 6a,b: Dorsal view of abdominal
'VI-VIII to show seta segment I, to show seta 9-1.
1-VI-MI,and ventral view
of male genital lobe.

F I G 7a
/&

Figure. 7a,b: Dorsal view of abdominal segments V-VII, to.show seta 9-V-VII.
Key to Anopheles Pupae 123

FIG 8 a FlCi8b

Figure. 8a,b: Dorsal view of abdominal segments I1 and IV-V, to show seta 1-11
and 9-IV-V,respectively.

FIG 9 a FIG 9 b

Figure. 9a,b: Dorsal (left)and ventral (right)view of abdominalsegmentsV-VII

to show seta 1,7,9-V-VII,and dorsal view of paddle to show distri-
bution of fringe spicules.

F I G 1Oa FIG 'iOb

Figure. 10a,b: Lateral view of trumpet to show shape of pinna, dorsal view of
paddle to show distribution of fringe spicules, and dorsal view of
abdominal segment I11 to show seta 1,5411.
 F.P. Amerasinghe

FIG l l a FIG I l b

Figure lla,b: Dorsal view of abdominal segments IV,Vto show seta 9-N,V.

F I G 12a F I G 12b

Figure 12a,b: Dorsal view of paddle t~ showseta 1-Paand distribution of fringe

spicules.

FIG 13a -FIG 13b

Figure 13a,b: Dorsal view of abdominal segment VII to show seta 2-VII, and
lateral view of trumpet to show relativemeatal length.
Key to Anopheles Pupae

FIG I4a F I G 14b F I G 1%

FIG 1Sb

Figure 14a,b: Dorsal view of abdominal Figure 15a,b: Dorsal view

segment I to show seta 9-1, of paddle to
and of paddle to show fringe show seta 1-Pa.
spicules.

FIG l6b

Figure 16a,b: Lateral view of trumpet to show meatil cleft, and dorsal view of
paddle to show seta 1-Pa.
F I G 17a F I G 17b ,

Figure 17a,b: Dorsal view of abdominal segment IV to show seta 9-IV, and of
paddle to show refractile border.
 F.P. Amerasinghe

F I G 18a FIG l 8 b

Figure 18a,b: Dorsal view of trumpets to show transverse and expanded

nature, and of abdominal segment VII to show seta 1-MI.

. .
F I G 19a FIG 19.11

Figure 19a,b: Lateral view of trumpet to show nature of secondary cleft, and
dorsal view of abdominal segment VII to show seta 9-VII.

@-9
pX%<
F I G 20a

:..,. ., .....,
.... ..., ,.....
... .. . .....

,
.
::
.' .....,
' . ,.'..::I.::..',...,. . ., . ..

. . . .. .., .
. . .: .
:. .
'I
FIG 20b

9h
VIII .

Figure 20a,b: Lateral view of trumpet to show nature of rim, and dorsal view
of abdominal segment VIII to show seta 9-VIII.
Key to Anopheles Pupae

NOTES ON THE KEY

1. Much of the emphasis in the present key is on characters of the pupal

trumpet, paddle, and the more prominent abdominal setae which can be
visualized without too much difficulty even on whole pupae. In most instances,
however, the magnification provided by dissecting microscopes will be insuffi-
cient, and it will be necessary to make a temporary mount of the pupxe on
microscopical slides and view a t magnifications upto lOOx under a transmitted-
light microscope for a definite identification to be made.

2. The key does not include all possible characters useful in identifications, as
this would make it unwieldy and time consuming to use. The characters selected
for use in the key provide the most rapid andunambiguous means of separating
the different species. Additional characters that can be used in the separation of
some species pairs are listed below.

3. The pupae of An. elegans and An. tessellatus have not been separated
previously. The characters used in the key (key step 5) are based on the
examination of Sri Lankan material of both species. Whilst An. elegans report-
edly occurs only in South India outside ofSri Lanka,An. tessellatus is widespread
throughout the Oriental region and the variability i n its pupal characters on a
regional scale is unknown. Thus, the characters used i n the key should be
regarded as provisional, and applicable only to the local populations of these two
species.

4. The separation ofAn. subpictus anddn. vagus pupae (key step 7) should be
regarded as provisional as it is based on a single character seen in Sri Lankan
material. Reid3usedthe length and branching of seta 6-IV (short and double in
An. subpictus, long and single in An. vagus) and the nature of the lower paddle
spicules (tips hooked inAn. subpictus, not hooked inAn. vagus) to separate the
two species.

However, Reid's keys were based on descriptions andlor the examination of

very few specimens of these species from the Southeast Asian region. I have
found the appearance ofthe paddle spicule tips and the lengthbranching of seta
6-IV to be unreliable for the separation of Sri Lankan An. subpictus and An.
vaqus. Indeed, contrary to Reid, most local An. subpictus have 6-IV single, and
An.wagus have seta 6-IV double. In the present work, seta 9-V-VII (blunt tip in
An. subpictus, sharply pointed tip inAn. vagus) has been used to separate the two
species, based on an examination of local material.

5. An. interruptus can be separated from the aitkenii group (key step 16)on the
basis of the following additional characters seta 9-V-VII hooked a t tip, seta 5-
V-VII with long, strong axis and short branches along its length, and seta 1-V-
VII very small and weak in An. interruptus, contrasted with seta 9-V-VII not
hooked a t tip, seta 5-V-VII with weaker axis and long branches along its length,
and seta 1-V-VII well developed in the aitkenii group.'
128 F.P. Amerasinghe

6. An. barbumbrosus and An. barbirostris can be separated on the basis of a n

additional character a t key step 19. The sum of the branches of both setae
5-111 less than 30 in An barbumbrosus, whilst this sum is greater than 30 in
An. barbir0stris.l

7. An. peditaeniatus and An. nigerrimus can be separated by an additional

character a t key step 20.An.peditaeniatus has seta 1-Vwith 1-6branches whilst
An. nigerrimus has seta 1-V with 8 or more branches.l

Acknowledgement

I thank Dr. Manthri Ramasamy of the Institute of Fundamental Studies (IFS),

Kandy, for providing specimens of An. tessellatus.

References

1. Harrison B.A. & Scanlon J . E . (1975). Medical entomology studies - 11. The
subgenus Anopheles in Thailand (Diptera: Culicidae). Contributions of the
American Entomological Institute 12: 1-307.

2. Peiris J.S. M., Amerasinghe P.H., Amerasinghe F.P., Calisher C.H., Perera
L.P., Arunagiri C.K., Munasingha N.B. & Karunaratne S.H.P.P. (1994).
Viruses isolated from mosquitoes collected in Sri Lanka. American Journal
of Tropical Medicine & Hygiene 51(2):154-161.

3. Reid J.A. (1968). Anopheline mosquitoes of Malaya and Borneo. Studies

from the Institute for Medical Research Malaysia. Government of Malaysia.
No.31. 903pp.

4. Amerasinghe F.P., Amerasinghe P.H., Peiris J.S.M.& Wirtz R.A. (1991).

Anopheline ecology and malaria infection during the irrigation development
of a n area of the Mahaweli Project, Sri Lanka.American Journal of Tropical
Medicine & Hygiene 45: 226-235.

5 . Mendis C., Herath P.R.J., Rajakaruna J., Weerasinghe S., Gamage-Mendis

A.C., Mendis K.N. & de Zoysa A.P.K. (1992). Method to estimate relative
transmission efficiencies ofAnophelesspecies (Diptera: Culicidae) in human
malaria transmission. Journal of Medical Entomology 29: 188-196.

6. Ramasamy R., De Alwis R., Wijesundere A. & Ramasamy M.S. (1992).

Malaria transmission a t a new irrigation project in Sri Lanka: the emergence
of Anopheles annularis as a major vector. American Journal of Tropical
Medicine and Hygiene 47: 547-553.

7. Amerasinghe F.P. (1990). A guide to the identification of the anopheline

mosquitoes of Sri Lanka. I. Adults. Ceylon Journal of Science (Biological
Science) 21: 1-16.