JournaL of the American Mosquito Contrctl Association, l9(3):2ll-219,2OO3 Copyright O 2003 by the American Mosquito Control Association, Inc.
PRE- AND POSTPRANDIAL MOSQUITO RESTING BEHAVIOR AROUND CATTLE HOSTS
NOBUKO TUNO. YOSHIO TSUDA. MASAHIRO TAKAGI AND WANNAPA SWONKERD
Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, N agasaki 852 - I 52 3, Japan
ABSTRACT, Spatial distribution of mosquitoes around a bovine host was studied in November 1997 ln northern Thailand (17"38'N, 99"23'E). Forty bamboo stakes were arranged I m apart, in 4 rays of 10, around a cow tethered in an open field. AII mosquitoes found resting on the stakes were collected by aspiration between 7:00 p.m. and l1:00 p.m., sexed, and identified to species; and f'eeding status was categorized as fed or unfed. Collections were repeated over 8 nights, with and without the host cow. A total of 1,566 mosquitoes from 25 species (5 genera) was collected. Anopheles aconitus was the most abundant species (643 individuals), followed by An. peditaeniatus, Culex vishnui, and Cx. pseudovishnui. We found that the number of mosquitoes collected from the stakes was related to the presence of the cow host; the number of mosquitoes collected was unrelated to the compass point location of the bamboo stakes, with the exception of Mansonia uniformis; unf'ed mosquitoes preferred bamboo-stake resting sites that were closer to the host; the daily fed to unfed ratio of the dominant species was negatively correlated with the daily total number of mosquitoes collected; and fed and unf'ed mos- quitoes clustered in interspecific heterogeneous groups around the host cow. Cluster analysis separated the species into 2 groups. The lst consisted of 5 species with higher proporlions of fed mosquitoes, whereas the 2nd, represented by 7 species, aggregated around the host within a distance of 1-4 m with lower proportions of fed mosquitoes. The interspecific variation in the distribution of unf'ed females was presumed to be due to a lack of feeding success. We discuss the significance of prebiting resting. In cases in which large numbers of mos- quitoes are present, prebiting resting can be adaptive to avoid host defensive behavior triggered by attacking mosquitoes.
KEY WORDS Mosquito, bloodfeeding behavior, resting, Anopheles aconitus, Culex vishnui species group
INTRODUCTION study, we examined the spatial distribution of mos- quito species around the host before and after feed- The bloodfeeding behavior of mosquitoes is an ing. important aspect of the epidemiology of mosqui- toborne diseases. Mosquito bloodfeeding behavior consists of several phases: the search for potential MATERIALS AND METHODS hosts, attraction to hosts, attacking, feeding, and resting. Of these phases, much attention has fo- Study site: Studies were conducted in the coun- cused on preattack behavior because of its practical tryside near Ban Den Udom, Amphoe Thoen, importance. The interval between the appearance of Changwat Lampang, northern Thailand (17'38'N, a mosquito and when it alights on the host is called 99"23'E) on November lO-17, 1997. Ban Den the prebiting rest (Reid 1968) or preattack resting Udom is located in a wide valley (Huai Mae Mok) (Clements 1999). A prebiting rest has been reported covered by paddy fields, which were dry at the time in several taxa (Service 1993), including the of the study. A 25-m2 grassy plot (approximate Anopheles leucosphyrus group (Colless 1956a, height of the grass, 50 cm) was chosen as the study 1956b), An. dirus Peyton and Harrison (Scanlon site. Neither human dwellings nor animal sheds and Sandhinand 1965), An. gambiae s.l. (Smith were located near the site. The nearest human res- 1958), Culex quinquefascialus Say (De Meillon and idence, a temple, was several hundred meters away. Sebastian 1967), Cx. tritaeniorhync&as Giles (Wada Methods: We modified the sampling method 1969), and, Mansonia spp. (Wharton 1962, Service used by Wada (1969). Figure I illustrates the study 1969). Females subsequently rest while digesting design. After clearing the grass, 40 bamboo stakes the blood meal, presumably because the added (0.7 m high x 4 cm wide) were pushed into the weight makes flight both difficult and dangerous. ground to serve as resting substrates. They were Field observations suggest that many mosquito spe- located at the 4 points of the compass in rays of l0 cies approach the host and remain nearby for some stakes, with l-m spacing between stakes. From 6:00 time before attacking. The biological significance p.m. until the following morning, I cow was teth- of this prebiting rest is unknown and preattack dis- ered in the center of the plot. Mosquitoes resting tribution has been poorly studied. Understanding on the sides of the stakes were collected by 2 peo- the spatial distribution of mosquitoes around hosts ple by usingaspirators for 20 min at 7:00, 8:00, can provide practical information for vector con- 9:OO,1O:OO, and 11:OOp.m. The collected specimens trol. This preattack period possibly provides an op- were kept alive in separate bottles, which were sort- portunity for the mosquito to distinguish among ed by compass point and distance from the host host species and become stimulated to bite. In this cow. The following morning, the mosquitoes were
211 2t2 JounNel- oF tHs AN4snrcAN Moseulro CoNrnol AssocrerroN Vor-. 19, No. 3
-ae:t -specles A -fed or unfed -dlstancc from the ',ftd celtet -compass dlrectlon
A cattle host -pregene6 hla.i -absence {controll lJ1m
Fig. l. Illustration of the study design. The parameters recorded are the number, sex, engorgement state, and species of moquitoes resting at the 40 stakes along arrays, in the presence or absence of a cattle host.
identified by sex, species, and state of engorgement and 14, to differentiate those mosquitoes that were (categorized as fed or unfed). Collections were attracted to rnosquito collectors, if any. made on 8 nights. A control set without the host Spatial distribution: Canonical correspondence cow was replicated over 2 nights, on November 13 analysis (CCA) was used to determine how mos- quito abundance on the stakes relates to presence or absence of the host cow among the 4 compass Table l. Speciescomposition of mosquitoescaptured directions and distance (Program CANOCO ver. at bamboostakes durins November 10-17. 1997. 4.0, Center for Biometry, Wageningen, Nether- Species Females Males Total lands). To increase statistical reliability, only spe- cies that were represented by more than 2O females Anopheles aconitu,s 638 5 643 were included in the analysis. The number of mos- An. peditaeniatus 113 173 quitoes was not transformed, and the number An. nivipes 98 98 of Ar. subalbatus 38 3B permutations was 199. An. minimus -l -l -f -) Correlation of proportion fed and mosquito An. splendidus 12 12 abundance: By using regression analysis, we also An. barbirostris l0 l0 determined the effect of mosquito abundance on the An. kochi 7 7 '1 ratio of the fed mosquitoes. We defined mosquito An. tesselatus 7 abundance as daily total catch of all mosquito sp€- An. sawactwongporni J -l cies, whereas proportion fed was calculated as daily An. willmori J 3 An. annularis 1 I value of each species. For statistical strength, only Culex pseudovishnui 152 152 species with samples of at least 50 females were Cx. vishnui IJJ 133 included in the analysis. Cx. tritaeniorhynchus 57 158 Patterns of mosquito distribution around the 'lo Cx. gelidus )t 138 host: clarify the general pattern of mosquito at- Cx. whitmorei l8 l8 tack behavior, we clustered the species according Cx. l5 t5 fuscocephala to the ratio of fed mosquitoes to the total collected Cx. bitaeniorhynchus t 12 Cx. quinquefasciatus I I in each distance category. Because of low sample Aedes vexans 5l 51 sizes at some stakes, data from the stakes were Ae. lineatopennis 2l 2l combined into 3 categories: l, 2-4, and 5-lO m Ae. albopictus 2 z from the tethered cow. Again, only those species Mansonia uniformis 26 20 46 for which more than 2O females were sampled were Ma. crassipes I I analyzed. The value pp : ns,lN was calculated for Total | 51R 28 I,566 each species, where hrd is the number of fed or Serreveen 2OO3 PnrsrrrNc RESTINc or. Moseurroas 2t3
Table 2. Number of female mosquitoes captured and fed ratio during the study period of November lO-17 lgg7.
Host (cow) present Host (cow) absent
Nov. l0 Nov. 11 Nov. 12 Nov. 15 Nov- 16 Nov. 17 Nov. 13 Nov. 14 Fed ratiot o.241 0.200 0.198 0.335 0.303 0.440 o.ff)O 0.o00 No. fed females 84 65 67 56 81 33 0 0 No. females 348 32s 338 t67 267 75 8 10 I Fed ratio is defined as number of fed mosquitoes divided by total numb€r of female mosquitres collected.
unfed females collected at distance d. measured Spatial distribution from the center of the plot (i.e., where the cow was tethered), and N is the total number of female mos- The CCA showed that the distribution of mos- quitoes of that species collected. The resulting data quito species among the 40 stakes was significantly matrix represented each mosquito species as a row affected by the presence of a cow, followed by the of p' values for each distance category (the col- distance from the cow and orientation of west to umns), and different clustering analyses were ap- east but not north to south (P : 0.010, 0.005, plied to the matrix of multispecies prr. We used 7 0.005, and 0.135, F-ratio : 4.2O, 3.87, 2.7t, and clustering strategies (weighted and unweighted av- 1.56, respectively). erage, complete, Ward minimum variance, single, An average of 253 (range 75-348) mosquitoes centroid, and median) to examine the Euclidezrn was collected daily on the bamboo stakes surround- distance matrix. The distributions of obvious clus- ing the tethered cow, whereas only 8 females were ters around the host were compared. Bonferroni collected on November 13 and l0 female mosqui- corrected /-tests were used to compare the average toes were collected on November 14 without a host distribution of groups in the 3 distance categories. (Table 2). Although it was impossible ro eliminate The ratios of fed to total females also were tested the effect of collectors on the species and numbers for significance with the t-test. Proportional data of resting mosquitoes, the results showed that most were arcsine-transformed before parametric analy- of the collected mosquitoes were attracted to the ses were applied. These statistical analyses were cattle host. The presence of the cow explained performed with STATISTICA Professional (1998 52.5Vo of cumulative percentage variance of the Edition, StatSoft Co., Tulsa, OK). species--environment relationship, whereas the 2nd east-west factor (if the number of mosquitoes col- lected differed north vs. south and west vs. east) RESULTS was 75.7Vo and the 3rd distance factor (if the num- Species abundance and sex ratio ber of mosquitoes collected was related to the dis- fance from the center) was 93.8Va. A total of 1,566 mosquitoes belonging to 25 spe- A significant difference was noted among the cies in 5 genera was sampled (Table 1). Anopheles numbers of mosquito species collected at the com- aconitus Ddnitz was the most common (643 indi- pass points of west to east but not north to south. viduals), followed by Anopheles peditaeniatus This was because of Ma. unifortnis, more of which (Leicester), Culex pseudovishnui Coless, and Cx. were collected in the east row. Because the other vishnui Theobald (Table l). Most of the mosquitoes species did not show such a tendency, they were were females (98.27a). Twenty of the 28 males col- presumed to be randomly distributed before ap- lected were Mansonia uniformis (Theobald), and proaching the host. only this species had an even sex ratio (female: We found significant correlations between the : male 1.3; Thble l). number of resting female mosquitoes and the dis-
Table 3. Spatial distribution of the number of mosquitoes resting at the stakes at l-lo m from the center with and without a cattle host.
Distance from Mosquito Total the center (m) sex Engorgement Host catch l0 Female Fed Present 386 1759357241836361 Fed Absent o 0000000000 Unf'ed Present 1,134 466 335 171 75 40 20 12 9 3 3 Unfed Absent 18 64r0111121 Male Present 22 6162211210 Absent 6 o002to2lo0 JounNnr- or tHE Auenrce.N Mosqurro CoNrnol AssocrerroN Vor-. 19,No. 3
Ae. aconitus (N=638)
P=0 01I
'3 o.zs
A e. pedi I aen iat ur (N=l 73)
0.50 i
? ozs
0.00 mosquitodensity
Cx. pseudovishnu i (N=l 52)
P=ooo7 0.60
€ o.:o
0.00 100 200 lm 2m 3m 4m 5m 6m 7m 8m 9m 10m mosquitodensity distance (reter)
Fig.2. Regressions with probability values (analysis of variance) of 7 dominant culicine species among daily fed ratio of each species (ver.tical axis) and daily total catch of all species (horizontal axis) with their spatial distribution fiom the host. tance from the host. The distribution of mosquitoes resting mosquitoes collected vs. the daily ratio of fed around the host is shown in Table 3 and Fig. 2. It to total number are shown in Fig. 2. In 4 of the 7 shows both fed and unfed resting mosquitoes con- species (An. aconitus, An. peditaeniatus, Cx. pseu- centrated within 5 m from the host. dovishnui, and Cx. vishnui), the regression was sig- nificantly negative (analysis of variance [ANOVA], P = 0.011, 0.019, 0.007, and 0.021, respectively). No of proportion that had fed and Correlation significant correlation was found for the other 3 spe- number of resting mosquitoes cies (Anopheles nivipes (Theobald), Culex tritaenior- Seven species were represented by more than 50 hynchus Giles, and Aedes vexans (Meigen)) (ANO- females (Thble l). Regressions of the daily number of VA, P : O.O74,0.148,and 0.123, respectively). Seprerraasn2003 PREBITINGRESTTNG ot Moseurrors 215
o E 0.50
F .st g € o.z: o E
0.00 3m 4m 5m 6m ?m 8m 9m lom
distflce (meter)
Ae. nivipes Gr{=98\
o 1 148 c P=0 0.50
d 1 o P € o.zs E 0 E
0. 0.00 lm 2n 3m 4m 5m 6m 7m mosquitodensity distance (meter)
o 0.50
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o E 0.50 o
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Fig- 2. Continued. 216 IounNal oF THE AMERTcIN Moseurro CoNrnor_ Assocrarroll Vor-. 19, No. 3
all - | I I l><= Patterns of mosquito distribution around v tl lltl: I I I t') t. Ftl the host otl otl etl Table 4 summarizes the ratio all ,o,t-| | | | | r of fed to total fe- males and the data on distance from the cow for , d- l^lt^t^l the l2 species (2 Aedes, 4 Anophele.s, I Armigeres, il tvttetvl Etl l:Y I o tl I Mansonia, and 4 Culex species) that were suffi- b0 ll ciently numerous to cluster. Four clustering meth- o tl Etl I tErd- r r I r I ods (weighted and unweighted average, complete, " ll o tl o and Ward minimum variance) gave very similar re- o tl 'il sults; the other 3 methods resulted in uninformative tl HiHtHQ t I tQt I o tl ,!) chains. A representative phenogram (constructed by cd ll using the Ward Etl , Qcrt al nn* minimum variance method) showed etl t:: I iY i t-Y- | 2 distinct groups (Fig. 3): I of 5 species (Armigeres tl D !tl subalbatus (Coquillett), An. peditaeniatus, Ae. lin- etl 49L-nc.)N , -$6$ O------.-.-::ttYYYY | r-^^A eatopennis (Ludlow), Ae. vexans, and gelidus = Culex ll Theobald : rapid group) and a Znd of 7 species O ctnOc- @ctno.ta.t d i , (An. aconitus, An. nivipes, An. minimus, Theobald ll 'ddodt-**o to-oli+ d rooddd Ma. uniformis, Cx. vishnui, Cx. pseudovishnui, and atltl ktl Cx. tritaeniorhynchus : slow group). The cluster o , Or-O6-\OnN-n ll ; | -NNe{-O--oc{- analysis suggested ?, ll 9 r;:i::;::::: that females of some species u'--ll * waited longer before feeding (we termed this group e"lltr O€t.A01 m€O\o*nv) E:II A **omoio-i-*.i.") the slow group), whereas other species had a short- r E ll.tr :::: i::::::: - tsil er preattack rest (the rapid group). The relationship sell? between distance from the tethered cow and the o,o ll o proportion of fed or unfed females to the total 4=il O | |I I t: t r r t | | num- -il < g ber of females is shown in Fig. 4 (unfed females in lll o tr:iln the upper figure, fed females below). The propor- lllll:il^q^+lltion of the rapid group that had fed was higher than - oil r*tl that of the slow group at every distance (r-test, otl Bonferroni adjusted P : 0.003, O.O0l, E .ll HHt t I=| | I | | r and O.OO6, at distances of l, 2-4, and 5-10 m, respectively; ;3ll ON +6 :: | | l:= | | | I I Fig. 4). The proportion of unfed females was higher o Ell in the slow group than in the rapid group at I and > i: tl 2-4 m (Bonferroni : I I I rEr rS r I 18 adjusted P 0.038 and 0.007, --llR9II tttt:tt:ttl'o E o'''o respectively), but the proportions did not differ at eEll ,sl 5-lO m. The rapid group also had higher overall -;tl n+O(, o-ni € ::i:: l--:: | | proportions fed (r-test, pooled variance t : 7.822, rr | t ihtl df : 10, P < 0.001) than the slow group. Sum- , d ll oil f 3 rE,35 rB r58 marizing these differences, the species in the rapid otl .- t-. iY- tY t- !il group attacked the host more successfully than 3tl :,tl n€-f,-o-€-o\+oo those in the slow group, so that unfed members of o tl the slow group accumulated near the host (l-4 m). -tlFll oil ?tl j3888 Ftl f f 3 r3lX .6 tl DISCUSSION tl rc ll O+l--O€.Oc{F Dendrogromof l2 culicidspecies Word minimumvorionce meihod Euclideondistonce Ae. vexons An. peditoeniotus Cx.gelidus Ae. lineotopennis Armigeressp. An. minimus Mo. uniformis Cx. tri* An. nivipes An. oconitus Cx. pseudovishnui Cx.vishnui 0.0 1.0 1.5 2.O 2.5 *Cx.triloeniorhynchus Fig. 3. Dendrogram for 12 culicine species resulting from cluster analysis (Ward minimum variance method with Euclidean distances) on the basis of Thble 4. The rapid group had a shorter preattack rest than the slow group. Ae., Aedes:, An., Anopheles; Cx., Culex; Ma., Mansonia. p<0.05 a0. . (l) ?0. g Eo.o NS F €0. Unfed Female o r______-^_\ q I slow group i0. r Rapidgroup ,oE + .o (u .9 FedFemale t o- " Slowgroup o o_ r Rapidgroup Distance(meter) Fig. 4. Comparison of distributions of fed and unfed mosquitoes among rapid and slow groups. Absolute numbers of fed and unfed females were changed into proportional values out of the total females (fed + unfed) of each species. The upper figure shows the distribution of unfed females and the lower figure shows that of fed females. The vertical axis shows the mean value of the slow (longer preattack rest) and the rapid (shorter preattack rest) groups. The horizontal axis shows the distance from the host. Error bars show standard errors ofthe groups. P-values above vertical lines indicate significant or nonsignificant (NS) difference of the proportion in the distances (1 2-4, and 5-10 m from the host) between the groups (Bonferroni corrected t-test). 218 JouRNeloF rHEAMERTcAN MosQurro CoNrnol AssocrerroN Vor-.19, No. 3 fed mosquitoes were those that presumably f'ed on large numbers of mosquitoes are present, prebiting the tethered cow. Fod mosquitoes might gradually resting can be adaptive to avoid host defensive be- distance themselves from the cow by slowly mov- havior triggered by attacking, If this negative rela- ing to the outer stakes. However, unfed mosquitoes tionship between mosquito density and mosquito may be unfed because of their recent arrival or be- feeding successis a general rule (Kelly 2001), why cause they are waiting to f-eedor have been unsuc- is prebiting resting behavior only observed in some cessful at feeding; the experiment was not designed mosquito species? Our results showed that the den- to distinguish these possibilities. We attribute the sity-dependent ratio of fed to total numbers difl'ered interspecific differences in the distribution of unfed among species (Fig. 2). Waage and Davies (1986) mosquitoes to a lack of successin feeding by unf'ed reported that intra- and interspecific competition females. Two observations support this inf-erence. occurred for tabanid species, which was associated First, we noted different patterns of unfed mosqui- with parasite density and host defensive behavior. toes between the 2 groups (slow and rapid). The At higher overall rates of fly landing, the alighting unfed individuals in the slow group were closer to times on hosts of individual flies decreased as a the cow than the unfed individuals of the rapid result of increased host grooming. Prior experience group. On the other hand, correlation of f'ed mos- with these density-dependent selective pressures quito numbers in the rapid group with distance sim- might be a cause of observed preprandial differ- ply reflected the higher proportion that had fed. The ences of mosquito spatial distribution. other reason is that a negative correlation was ln conclusion, a prebiting rest was observed in found between the proportion that had f'ed and the several taxa of mosquitoes. We postulate that the total number of resting mosquitoes. This supports prebiting resting is a period that allows the mos- the postulate that mosquitoes remained near the quito to evaluate many aspects of its host and de- cow because of a lack of feeding success. Viewed cide whether to attack. Gillies (1980) showed that in this light, it is reasonable to conclude that the carbon dioxide generally attracts host-seeking mos- interspecific variation in the distribution of unfed quitoes. Most mosquitoes are attracted by general females was due primarily to a lack of feeding suc- host cues. such as carbon dioxide and heat, whereas cess. Collections were made only until I l:20 p.m., these general cues do not trigger biting in some and the unfed mosquitoes very likely fed on the species. Because they wait for their host in loca- host later in the night. This possibility does not tions where general cues would be strongest, they deny our claim, because we analyzed the compar- might need other cues from the host to initiate suc- ative spatial distribution of fed and unfed mosqui- cessful feeding. toes but not their abundance in number, which might increase later. Wada (1969) quantified the nocturnal biting ac- ACKNOWLEDGMENTS tivity of Anopheles sinensis Wiedemann and C.r. We thank Yoshito Wada, Motoyoshi Mogi, To- tritaeniorhynchus by various methods (counts of shihiko Sunahara, and Noboru Minakawa for pro- mosquitoes alighting on pigs, on plates, or on tape viding useful criticisms and correction of the man- set near a dry-ice bait) and found that the peaks of uscript. We sincerely thank Bruce F Eldridge and mosquito abundance differed among the methods. 2 anonymous reviewers who helped us to improve The number of mosquitoes counted at dry-ice-bait- the manuscript. ed tapes showed I or 2 peaks, whereas counts of mosquitoes alighting on pigs were almost constant. He attributed this difference to the preattacking REFERENCES CITED rest. The dry-ice-bait counts reflected the flight-ac- ClementsAN. 1999. The biology of mosquitoesVolume tivity rhythm of mosquitoes, which differed from 2 Sensoryreception antl behaviour. Cambridge, United pigs. 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