\
VECTon!
PATIIOCEN / HO:;T I!'<TEH.-\cno:\'.
Tll"'N:;~ n:;:;ION
llost Feeding Pattern of Japanese Encephalitis
Virus Vector
Mosquitoes (Diptera: Culicidae)
fron1 Kuttanadu, Kerala, India
P. PHILIP SAMUEL,. N. ARUNACHALAl\L J. HIRIYAN,
Centre
flu' Research
in Medical Entomology (Indian Council of Medical Research),
Madurai. 625002 Tamil Nadll. India
AND
B. K. TYAGI
4. Sarojini St.. Chinna Chokkiklllam.
J. Med. Entomol. 45(5): 927-932 (2008)
ABSTRACT
Identification of blood meals of vector mosquitoes is an important tool in the epidemiological investigations of vector-borne diseases. The blood meals of three mosquito species involved
in the transmission of Japanese encephalitis virus (JEV) from the Kuttanadu area, Kerala, were
determined using the agarose gel diffusion technique. A total of 4,959 blood smears belonging to ClIlex
(ClIlex) tritacniorhynchus Giles (3,273), Cx. (ClIlex) gclidlls Theobald (64), Mansonia (Mnd.) indiana
Edwm-ds (735) ,and Ma. (Mnd.) uniformis (Theobald)
(887) were tested. Cx. tritaeniorhynchus had
predomimmtly fed on bovids (46.4%), mId a good proportion (29%) had fed on more than one host.
Cx. tritaeniorhynchus was highly zoophagic, and human feeding accounted for only 1.5% of those
individuals successfully tested. Cx. gelidlls showed bovid feeding at 36% and pig feeding at 12.5%. The
test results showed 42.3% Ma. indiana and 12.2% Ma. uniformis had fed on humans. Multiple feeding
was observed in Ma. indiana and Ma. tl11ifonnis, and most of the double feedings were from bovids
and ovids (7.9 and 20.1%, respectively).
Pig feeding accounted
for 4.8% of the feedings by Cx.
tritaeniorhYllchus, 5.3% of Ma. indiana, and 6.4% of Mo. unifonnis. This study is significant because
of the role played by these mosquitoes in the transmission of JEV in the Kuttanadu area of Kerala,
India.
KEY WORDS
Japanese encephalitis, feeding pattern, Mansonioides, Culex
Mosquito bloodmeal identification is an important tool
to study the host-feeding patterns of vector species, to
give information on proportion feeding on different
hosts, and to find out seasonal variations in the feeding
pattern. Feeding behavior of a vector species helps to
assess its capacity to transmit a given pathogen with
regard to a particular situation. The feeding pattern is
pivotal information in the epidemiological
investigation of vector-borne diseases for the understanding of
host-vector relationships and the dynamics of disease
transmission.
Sporadic cases of Japanese encephalitis (JEV; Flaviviridae, Flavivirus) have been reported from 1996
onward in Kerala state, India. During preliminary
studies, JEV was isolated from Culex (Cux.) tritaeniorhynchus Giles, Mansonia (Mnd.) indiana Edwards and
Ma. (Mnd.) unifonnis (Theoba\d)
(Dhanda et aI.,
1997). Cx. tritaeniorhynchus was determined
as the
primary vector of JE, whereas Ma. indiana and Ma.
unifarmis were identified as secondary vectors in this
region (Arunachalam et aI., 2002). A longitudinal entomological study was carried out to study the host
feeding pattern of vectors and suspected vectors. The
blood meals of the mosquito species Cx. tritaeniorhynchu~, Cx. (Cux.) gelidus Theobald, Ma. indiana, and
I Correspondinj!;
author, e-mail: [email protected].
Ma. unifarmis collected from the Kuttanadu area,
Kerala, were examined to identify bloodmeal sources.
Materials
and Methods
Study Area. The Kuttanadu region of Kerala in India
forms the interface of marine, estuarine, and fluvial
systems representing a highly complex ecosystem with
a deltaic formation of four major rivers together with
the low lying areas in and around Vembanad Lake.
Most of the region lies below mean sea level and was
reclaimed from the surrounding backwater for rice
cultivation. As a result, the area is almost waterlogged
throughout the year. Rice is grown in these wetlands
called the "Punja lands." Six villages where at least one
con6rmed case of JE had occurred in 1996 and 1997
were selected as index villages for longitudinal epidemiological studies. Cow, goat, fowl, and ducks are the
common domestic animals in Kuttanadu. Pigs are
reared in backyards of some houses only.
Kuttanadu is a warm humid region with fairly uniform temperature
throughout the year ranging from
21 to 35°C (9°29' 28" N, 76°19'35" E). Humidity in
general is very high all through the year. The annual
average rainfall received is -300 cm, of which 8.3% is
received during the monsoon months of July through
October. Four distinct seasons, postmonsoon season
()()22-25H.'5/08/0927-()9.12$O4.00/0
<C>2()()8 Entomological
Socidy
of Arnerk:a
928
JOURNAL OF MEDICAL
January-March,
summer season April-June,
southwest monsoon season July-September,
and northeast
monsoon during October-December,
are experienced.
Mosquito Collection. Outdoor resting mosquitoes
were collected from vegetation and bushes by using
drop nets and also collected from indoor areas by
aspirator. Mosquitoes were identified using the keys of
Barraud (1934) and Christopher
(1933). A key prepared by Reuben et a1. (1994) was used for identification of the Cx. vishnui subgroup. The animal census
of this region was collected by the Collectorate Office,
Alleppy, Kerala.
Bloodmeal Identification. Engorged females were
placed on ice for transport to the laboratory. Midgut
contents were smeared on Whatman no. 1 filter paper
i
trips, dried at room temperature, and stored at 4°c.
~Afterward,
the filter papers were transported
to
CRME, Madurai, for further processing. The agarose
gel diffusion method (Collins et al. 1986) with minor
modifications as described in Reuben et ill. (1992) was
used to identify blood meals from wild-caught mosquitoes. Antisera to cow, pig, duck, goat, fowl, and
human were obtained from the Serologist, Government of India, Kolkata, India.
Forage ratio was calculated on the percentage of
engorged mosquitoes, which had fed on a given vertebrate host, divided by the percent, which the species
of host comprises of the total population of hosts
available in the mosquito habitat (Hess et ill. 1968,
Washino and Tempelis 1983, Kay et al.1985). A forage
ratio of one indicates neither preference nor avoidance of the indicated host animal. Forage ratios significantly greater than one indicate selective preference and values less than one indicate avoidance in
favor of other hosts.
Statistical Analysis. ¥" tests were carried out using
~ -:<:pi Info (CDC, Atlanta, GA) to determine the stati~ tiscal significance of observed differences in indoor,
outdoor, and seasonal feeding patterns.
Results
A total of 4,959 blood meals belonging to Cx. tlitaelIiorhynchlls (3,273), Cx. gelidus (64) Ma. indiana
(735), and Ma. uniformis (887) were tested, of which
79% ~ere identified. For Cx. tlitaeniorhynchus, 84% of
the blood smears were identified, of which human
accounted for 1.5%. Cx. tritaeniorhynchlls was predominantly zoophagic, having fed mostly on cows (46.4%)
and to a lesser extent on ducks (0.1%), fowls (0.3%),
goats (0.7%), and humans (1.5%). Unexpectedly, 29%
reacted to both bovids and ovids, whereas <1% reacted to a different combination of any of the antisera
tested. Triple feeds like cows, humans, and goats
(0.1%) and cows, humans, and pigs (0.03%) were also
found in the bloodmeal analysis. Pig feeding accounted for 4.8% of total blood meals of Cx. tlitaelliorh1/lIchlls (Fig. 1). The respective feeding pattem of
the outdoor and indoOl- collections showpd 47 and 35%
bm'id feeding. 0.5 and 16% human feeding. and 5.~ and
0..')% pig feeding. Mixed bovid/ovid feeding was 29%
ENTOMOLOCY
Vol. 45, no. 5
in outdoor and 36% in indoor feeding. Seasonal variation showed more cow feeding throughout the year
(38 -56%), peaking during the wet season (56%). Similarly, pig feeding ranged from 3 to 11% and showed
peak biting (11%) during the dry season. Human feeding patterns ranged from 0.1 to 3.8% (Fig. 2).
Analysis of blood meals of 64 Cx. gelidus with 56
positive reactions (87.5%) showed more preference
toward cows (36%) and pigs (12.5%) and lesser preference toward humans (6.25%), ducks (1.5%), and
goats (1.5%). Only two types of double feeds like
bovid/ ovid (25%) and goat/ human (4.7%) were identified. Outdoor
and indoor collected
mosquitoes
showed equal blood feeding on cows (36.8 and 35.5%).
Human-fed mosquitoes were found indoors (21.4%).
Similarly, pig feeding was recorded only in the outdoor resting collection (12.5%). More mixed feeding
of cow and goat was shown in indoor mosquitoes
(28.9%) rather than outdoors (15.8%). There was no
significant difference for different seasons in the total
feeding for cows, humans, and pigs.
Mansonia indiana and Ma. uniformis were comparatively more anthropophagic
than Cx. tritaeniorhynchus, with 42.3% of Ma. indiana and 12.2% of Ma.
uniformis feeding on humans. Double feeding was also
seen in Ma. indiana and Ma. uniformis, and most of the
double feedings were from cows and goats (7.9 and
20.3%). Pig feeding accounted for 5.3% of Ma. indiana
and 6.4% of Ma- uniformis blood meals. Ma. indiana
apparently fed more on humans throughout the year
(20-61 %) and had a low preference for cows (2-15%)
and pigs (0-11%) (Fig. 1). Feeding patterns of Ma.
indiana and Ma. uniformis showed 21 and 34% cow
feeding in the outdoor collections and 3.4 and 10% cow
feeding in the indoor collections. Human feeding
showed 1.1 and 1.9% for outdoor and 65.5 and 61% for
indoor feeding for Ma. indiana and Ma. IIniformis,
respectively. Similarly pig feeding accounted for 15
and 7.8% of the feeding in the outdoor collections of
Ma. indiana and Ma. uniformis, but no pig feeding was
recorded in the indoor collections of either species
(Figs. 3 and 4). Mixed bovidl ovid feeding occurred in
17 and 24% of outdoor ,collections and 2.8 and 4% of
indoor feeding for Ma: indiana and Ma. IInifonnis.
Seasonal feeding patterns of Ma. uniformis showed
more preference for cattle (12-32%), followed by humans (3.4-17.7%) and pigs (1.3-11.4%).
K- values showed significant differences within the
seasons (P < 0.05) in the outdoor and indoor feeding
behavior for cow and human for Ma. IIniforlllis except
for January to March for cow and also showed no
significant difference between the indoor and outdoor
feeding habitat for pig in all seasons (P > 0.05). Similarly, the K- value showed a significant difference
between the feeding behavior of Ma. indialla in the
indoor and outdoor feedings on cows, humans. and
pigs during the rainy season from July to December
but showed no difference for cows during the AprilJune season and for cows and pigs during January-March. Howt'ver. the,-e W.Lo;
a significant ditlt'n'nce li.ll"
human feeding between the indoor and outdoor n'stin~
habitats
in all the se.Lo;ons for Cr. friflll>lIio,-hullcllIIs. Dii--
,/
I'"
//
September
2008
PHILIP SAMUEL IT AI..: H~'T
929
PA"ITEHN
. Indoor
Outdoor
Ma. Indiana
Cow &Gool
17%
FEEDING
Pig
15%
Human
1%
Negative
26"'<'
Others
43%
Ma. uniformls
Cow
10%
Negative
23%
Pig
8%
Others
3%
Human
60%
4%
Cx. tritaeniorhynchus
Negative
7%
Negative
17%
Others
4%
Others
2%
Pig \
5%
Pig
1%
Human
0%
Fig. 1. Host feeding pattern of Mansonia and Culex mosquitoes
ferences between cow, human, and pig feeding occurred
during the transmission season, January-March. In total,
Ma. indiana, Ma. unifarmis, and Cx. tritaeniorhynchus
showed significant differences for cow, human, and pig
feeding in outdoor and indoor habitats. Between seasons
in the outdoor habitat, Cx. tritaeniorhynchus showed a
in Kuttanadu,
significant difference in pig feeding. Similarly, between
seasons in the indoor habitat, pig feeding for Ma. uniformis and human and pig feeding for Ma. indiana showed
significant differences.
The results of the forage ratio showed that these
three species are zoophilic mosquitoes, preferring
80
n=3O8
70
60
C> 50
c:
'0
~
40
0
30
~
n =57
20
10
n=3
n=O
0
Cow
n = total
number
Fig. 2.
of blood
Human
feeding
Outdoor
I-
Kerala.
Outdoor
Pig
Ellndoorl
and indoor feeding pattern
of Ma. imliana.
~):30
JOURNAL OF MEDICAL
Vol. 45, no. 5
ENTOMOJ .OGY
80
70
n=94
60
50
'"
c
'g
40
2
:I?
030
20
10
n=O
0
Cow
n = total number of blood feeding
Human
Pig
I_Outdoor Ellndoorl
Fig: 3. Outdoor and Indoor feeding'pattem
of Ma. uniformis.
~
in the absence of cattle (Colless 1958). In Gambia,
Snow and Boreham (1973) found that 88.2% of the
specimens had fed on cattle and concluded that there
was no difference in the feeding habits of Cx. tritaeniorhynchm in West Africa and the eastern part of its
range. In Senegal, Cx. tritaeniorhynchus
showed an
opportunistic
feeding pattern because considerable
variation was observed from location to location and
seemed to be closely related to host abundance in a
particular area (Gordon et al.1991). The same behavior was observed in Dibrugarh, where pig feeding was
found to be much higher than 40% in Cx. tritaeniorhynchus, which was because of the considerable number 'of pigs present (Bhattacharyya
et al., 1994). It
shows the opportunistic habit of Cx. tritaeniorhynchus
because the feeding pattern changes according to local host availability.
Culex tritaeniorhynchus yielded the greatest number
of JEV isolates in Tamil Nadu, Karnataka, and Kerala
(Philip Samuel et ill. 1998). In Kuttanadu also, more
virus infections were detected in Cx. tritaeniorhynchlls
(Dhanda et al. 1997). Higher abundance of Cx. tritaeniorhynchus during paddy season/ JE transmission season might have facilitated the transmission of JE in
Kuttanadu. This species is considered a major vector
for JEV in Kuttanadu based on epidemiological data
pigs, cows, and goats, even when other hosts were also
available. The forage ratio was always <1 for humans
except in Ma. indiana. All these four mosquitoes
showed more preference for pigs (Table 1).
Discussion
Culex tritaeniorhynchus has been recognized
for
many years as the major vector and plays an important
role in the epidemiology of JEV in India. Most isolations of JEV in nature have been made from Cx. tritaeniorhynchus (Phillip Samuel et al. 1998). Bloodmeal
analysis of Cx, tritaeniorhynchus showed that this species was principally a cattle feeder in Vellore (Christopher and Reuben 1971), in villages near Madurai
(Mani et al. 1991), and in the Cuddalore district (Reuben et aI.1992). The preponderance
of blood feeding
on cattle by Cx. tritaeniorhynchus is clearly evident in
Pakistan (Reisen and Boreham 1976).
.~
A low human feeding rate of Cx. tritaeniorhynchus
~ observed by us in this study was comparable to results
obtained in the Cuddalore district (Reuben et al.,
1992). However, in Senegal, Cx. tritaeniorhynchus was
found to have fed more frequently on human than on
cattle (Gordon et aI.1991). In Okinawa, pigs were the
pt'eferred host (Pennington and Phelps 1968). In Singapore, Cx. tritaeniorhynchus was attracted to humans
60
5OJ
'"
c
'0
n = 1444
40
3O
'if.
n =33
20
10
n= 1
0
Cow
Human
~Outdoor
Fig. 4.
EIIndoor
OlltdoD!' and Indoor f"t'ding pall..rn
Pig
I
of Cx friflll'//;orhU//'-/lI/s
S('pt('mber
Tn"\" I.
2008
PHILIP SAMUEL E1' AL.: HOST FEEDING
I""'n~" ruli.. ..f ",',"""ru'"
Host
A
Cow
Human
Pi
Cow and Goat
A, % mosqnitoes
46
5
4.9
29
1...,1, f
Ihre,' ,liff,'n'nl """';".
Cx. iri!a(,lliOt-h!l"clltls
B
A/B
4.1
40.4
0.01
5.7
fed on that particular
11.3
0.04
490
5.08
vertebrate
..f "",.qui
9:31
P.-\T1'EHN
,
:\{a.illd;alla
A
10
42
5.3
29
host; B, % total population
showing that its peaks of population coincide with
increased transmission of JE to humans.
Culex gelid liS was present in all collections but was
less abundant than Cx. tritaeniorhynchus. Adults were
found closely associated with humans and domestic
animals, and most of them feed on bovids in the North
Arcot district, Tamil Nadu (Christopher and Reuben
1971) and pigs in the Cuddalore district, Tamil N adu
(Reuben et al. 1992). It is an important vector of JE,
and isolation of JE virus has been reported from India
(Reuben et ai. 1994). In the Cuddalore district, Cx.
gelidus was observed to be highly zoophagic and
poorly anthropophagic
(Reuben et aI., 1992). Although this mosquito was only seasonal, because of its
feeding habit and infection rate (MIR), this mosquito
has an important role in amplifying JEV transmission
in the zoonotic cycle (Gajanana et al. 1997).
In this study, the host feeding pattern of Ma. indiana
was more anthropophilic than Ma. uniformis. Ma. uniformis have shown preference for cattle and pigs. In
Dibrugarah, Assam, Ma. uniformis showed the highest
percentage of feeding on cows (Bhattacharyya
et ai.
1994). Studies of the host feeding patterns of mosquitoes in the subgenus Mansonioides
from Orissa
showed a higher feeding proportion
on humans
(Hazra et aI., 2002). A higher proportion of human
blood meals were detected for Ma. uniformis collected
inside houses (85%) compared with those collected
outdoors (5.3%) in Kenya (Beier et al. 1990) as observed in this study.
Multiple feeding is defined as two or more blood
meals fed from different vertebrate hosts, the last of
which has been taken before the first has been digested
(Boreham and Garrett-Jones 1973, Reisen and Boreham
1976). In this study, the most common combination of
double feeding was bovidl ovid (0.7-27.7%), followed by
humans and goats (8.1-1.5%), and cows and humans
(0.2-0.8%), and the rates of multiple feeding in individual species ranged from 0.1 to 28% in Cx. tritaeniorhynchus. Human and pig feeding (0.2-0.3%) was observed only in Ma. uniformis and Ma. indiana. Triple
feedings on cows, goats, and humans were noticed in
Cx. tritaeniorhynchus and Ma. uniformis. Triple feeding
on cows, humans, and pigs by Cx. tritaeniorhynchus
was also observed. Edman and Downe (1964) reported the incidence of multiple feeding ranged from
9.7 to 61.8% in 17 culicines in Kansas. Mixed human
and cow meals were detected for three Ma. uniformis
in Kenya (Beier et al. 1990). Any increase in the
number of host contacts as a result of multiple feeding,
however modest, may increase disproportionately
the
:\fa. III/ironll;"
B
A/B
A
B
A/B
4.1
40.4
0.01
5.7
2.4
1.05
530
5.08
29.3
12.2
6.4
20.3
4.1
40.4
0.01
5.7
7.1
0.33
640
3.6
of host available in that habitat; A/B. Forage ratio.
rate at which virus transmission occurs (Anderson and
Brust 1995).
Undetermined
blood meals of Cx. tritaeniorhynchliS
(15%), Ma. unifonnis (29%), and Ma. indiana (32%)
may be because of the narrow host range selected for
testing or the advanced digestion of the blood meal.
There were many other common birds (including
ardeids) reported in that area. Sera of these species
could not be prepared because of the practical difficulties faced in the mode of collection of the live birds.
Among the characteristics of a species of mosquito
that increase its vector potential are that the population should be high and stable during the epidemic
season; that the species has a long survival rate so that
the extrinsic incubation period of the virus is completed; that the mosquito has a catholic feeding habit,
basically zoophilic in nature having occasional contact
with humans and repeated isolation of the virus from
wild-caught
specimens
(Sehgal and Bhatia 1955,
Okuno et ai. 1975, Philip Samuel et al. 2000). Biting on
cattle at one side diverts the vector population and
also act as dampening agent in the multiplication of JE
virus, thus reducing the probability of disease transmission to humans; however, it also serves as an alternative means of survival and multiplication of the
mosquito population and also multiplication
of the
pathogens present in them.
Mosquitoes in the subgenus Mansonioides maintained higher densities, and the infection was observed at a lower level during the period between May
and September, coinciding with the rainy season and
propagation
of water plants. Cx. tritaeniorhynchus,
however, was relatively scarce during the rainy season
and achieved its highest abundance during the local IE
epidemic season. Thus, the predominant
species Cx.
tritaeniorhynchus and species in the subgenus Mansonioides probably served as vectors of JEV transmission
in a sequeI1tial manner. Cx. tritaeniorhynchus
fed
mainly on animals, and the Mansonioides species were
found feeding on humans and pigs. Apparently, Cx.
tritaeniorhynchus acts as a primary vector, and species
of Mansonioides act as secondary vectors in Kerala.
Acknowledgments
We thank the Director General, Indian Council of Medical
Research for providing the facilities. The authors are grateful
to SEARO/WHO New Delhi for financial support (Project
SN 1094). We thank the staff Shriyuts A. Veerapathiran, V.
Kodangi Alagan, and V. Rajamannar of Vector Biology and
training division of Centre for Research in Medical Ento-
932
JOURNAL OF MEDICAL
mology for exceIlent technical assistance. We appreciate the
excellent help rendered by A. Venkatesh and K. Venkatsubramani (CRME, Madurai) in preparation of this manuscript.
References
~
-
Cited
Anderson, A. R., and R. A. Brust. 1995. Field evidence for
multiple host contacts during blood feeding by Culex
tarsalis, Cx. restuans and Cx. nigripalpus (Diptera: Culicidae). J. Med. Entomol. 32: 705-709.
Arunachalam, N., P. P. Samuel,J. Hiriyan, V. Thenmozhi, A.
Balasubramanian, A. Gajanana, and K. Satyanarayana.
2002. Vertical transmission ofJapanese encephalitis virus
in Mansonia species, in an epidemic-prone area of southern India. Ann. Trop. Med. Parasitol. 96: 419-420.
Barraud, P. J. 1934. The fauna of British India, including
Ceylon and Burma, Diptera, Family Culicidae. Taylor and
Francis, London, United Kingdom.
Vol. 45, no. 5
ENTOMOLOGY
dance and infection frequency. J. Med. Entomo1. 34: 651659.
Gordon, S. W., R. F. Tammariello, K. L. Linthicum, R. A.
Wirtz,andJ. P. Digoutte. 1991. Feedingpatternsofmosquitoes collected in the Senegal river basin. J. Am. Mosq.
Cont. Assoc. 7: 424-432.
Hazra, R. K., N. Mahapatra, S. K. Parida, H. K. Tripathy, and
A. P. Dash. 2002. Host feeding pattern of Mansonioides,
vectors of Brugian filariasis in Orissa. In Proceedings, 6th
International Symposium on vectors and vector borne
diseases, 9-11 February 2002, Bhubaneswar, India.
Hess, A. D., O. R. Hayes, and C. H. Tempelis. 1968. The use
of forage ratio techniques in mosquito host preference
studies.
Mosq.
News
28: 386-389.
.
Kay, B. H., P.F.L. Boreham, and I. D. Fanning. 1985. Hostfeeding patterns of Culex annulirostris and other mosquitoes (Diptera: Culicidae) at CharleviIle, Southwestern
Queensland, Australia. J. Med. Entomol. 22: 529-535.
Mani, T. R., C.V.R. Mohan Rao, R. Rajendran, M. Devaputra,
Beier,
J. C., W. O. Odago, F. K. Onyango, C. M. Asiago, D. K.
Y. Prasanna, Hanumaiah, A. Gajanana, and R. Reuben.
Koech, and C. R. Roberts. 1990. Relative abundance and
1991. Surveillance for Japanese encephalitis in villages
blood feeding behavior of nocturnally active culicine
near Madurai, Tamil Nadu, India. Trans. R. Soc. Trop.
mosquitoes in Western Kenya. J. Am.Mosq.Cont. Assoc.
Med. Hyg. 85: 287-91.
6: 207-212.
Okuno, T., C. J. Mitchell, P. S. Chen, S. Hsu, and E. Ryu.
Bhattacharyya,D. R., R. Handique, and L. P. Dutta. 1994.
1975. Experimental transmission of Japanese encephaliHost feeding patteros of Culex vishnui subgroup of mostis virus by Culex tritaeniorhynchus and C.fuscocephalus.
quitoes in Dibrugarh district of Assam. J. Comm. Dis. 26:
Ann. Trop. Med. Parasitol. 69: 203-206.
133-138.
Pennington, N. E., and C. A. Phelps. 1968. Identification of
Boreham, P.FL., and C. Garrett-Jones. 1973. Prevalence of
the host range of Culex tritaeniorhynchus mosquitoes on
mixed blood meals and double feeding in malaria vector
Okinawa, Ryukyu Islands. J. Med. Entomol. 5: 483-487.
(Anopheles sacharovi Favre). Bull. WHO 48: 605--614.
Philip Samuel, P., J. Hinyan, V. Thenmozhi, and A. BalasuChristopher, S. R. 1933. The fauna of British India, includbramanian. 1998. A system for studying vector compeing Ceylon and Burma, Diptera, vol. 4: Family Culicidae,
tence of mosquitoes for Japanese encephalitis virus. Indian J. MalarioL 35: 146-50.
Tribe Anopheleni. Taylor and Francis, London, United
Kingdom.
Philip Samuel, P., J. Hinyan, and A. Gajanana. 2000. JapaChristopher, S. R., and R. Reuben. 1971. Studies of the mosnese encephalitis virus infection in mosquitoes and its
quitoes of North Arcot District, Madras State, India. Part
epidemiological implications. ICMR Bull. 30: 37-42.
4: host preferences as shown by precipitin tests. J. Med. Reisen, W. K., and P.F.L. Boreham. 1976. Feeding patterns
Entomol. 3: 314-318.
of Punjab mosquitoes (Diptera: Culicidae): a short-term
Colless, D. H. 1958. Notes on the culicine mosquitoes of
survey. Biologia 22: 299-304.
Singapore. VI. Observation on catches made with baited
Reuben, R., V. Thenmozhi, P. Philip Samuel, A. Gajanana,
and unbaited trap nets. Ann. Trop. Med. Parasitol. 53:
and T. R. Mani. 1992. Mosquito blood feeding patterns
251-267.
as a factor in the epidemiology of Jap.mese encephalitis
Collins, R. T., B. K. Dash, R. S. Agarwala, and K. B. Dhal.
in Southern India. Am. J. Trop. Med. Hyg.46:654-663.
1986. An adaptation of the gel diffusion technique for
Reuben, R., S. C. Tewari, J. Hinyan, J. Akiyama. 1994. Ilidentifying the source of mosquito blood meals. Indian J.
lustrated keys to species of Culex (Culex) associated with
Malariol. 23: 81-89.
Japanese encephalitis in Sputheast Asia (Diptera: CulicDhanda, V., V. Thenmozhi, N. P. Kumar, J. Hinyan, and N.
idae) Mosq. Systematics 26: 75-96.
Arunachalam. 1997. Virus isolation from wild-caught
Sehgal, S., and R. Bhatia. 1955. Manual of Zoonoses. Namosquitoes during a Japanese encephalitis outbreak in
tional Institute of Communicable Dise.l~es. Delhi, India.
Kerala in 1996. Indian J. Med. Res. 106: 4-6.
Snow, W. F., and P.F.L. Boreham. 1973. The feeding habits
Edman, J. D., and A.E.R. Downe. 1964. Host-blood sources
of some West African Culex (Diptera: Culicidae) mos.md multiple-feeding habits of mosquitoes in Mansas.
quitoes. Bull. Entomol. Res. 62: 517-526.
Mosq. News 24: 154-160.
Washino, R. K., and C. H. Tempelis. 1983. Mosquito host
Gaj.mana, A., R. Rajendran, P. Philip Samuel, V.Thenmozhi,
blood meal identification methodology and data analysis.
Annu. Rev. EntomoL 28: 179-201.
T. F. Tsai, J. Kimura-Kuroda, and R. Reuben. 1997. Japanese encephalitis in South Arcot District. Tamil Nadu,
India: a three-year longitudinal study of vector-abunReceit'ed 26 FelJl"1lllry2007; accepted 21 March 2008.