Effect of Temperature on the Development of melanura (Diptera: Culicidae) and its Impact on the Amplification of Eastern Equine Encephalomyelitis Virus in Birds

FARIDA MAHMOOD AND WAYNE J. CRANS Department of Entomology, Rutgers the State University of New Jersey, 180 Jones Avenue, New Brunswick, NJ 08901-8536

J. Mrd. Ento.nol. 35(6); 1007-1012 (199S) ABSTRACT Eastern equine enceplialomyelitis virus (EEE) is perpetuated in maintenance cycle tliat involves Ciilif.rlii incliiiiiira (Coquillett) as the enzootic vector and passerine birds as the amplifying liosts. Amplification of virus in any given year requires influx of nulliparous Cs. niclfiiiiini in tlie presence of susceptible avian liosts. We conducted laboratory experiments at constant temperatures from 10 to 3-1C to develop thermal heat summation models to predict emergence in nature. Embryonic development progresses slowly at 10C, and the time to eclosion decreased significantly temperatures increased to 28C. High temperatures were letlial and eggs failed to hatch at 32C. Tlie thermal minimum (<) for embryonic development was 9.38C, and 38.46 degrees-days (DD) were required for egg hatch. Tlie time for larval development decreased witli increasing temperatures. Cs. iiirlnniirii larvae develop in subterranean habitats (crypts) wliere water temperatures remain below 20C throughout tlie summer. Under controlled conditions, egg hatch to emergence look 8 mo at 10C, 3 mo at 16C, and at 22C. Tlie thermal minimum for larval development () was 8.5C, and 467.29 DD required from eclosion to adult emergence. Our findings indicate tlial Cs. iiicliiniira is well suited to develop in cold water crypts wliere tlie larvae collected most frequently. Tlie appears to be bivoltine in tlie northeast with an overwintering generation of larvae that emerges spring brood of adults and generation of larvae tliat emerges in fall. Higher tlian normal water temperatures hasten development of the summer generation and increase the probability for amplification of EEE by bringing large numbers of nulliparous mosquitoes into contact witli recrudescing virus for subsequent transfer to (lie growing population of susceptible juveniles. Water temperatures in tlie crypts also may regulate tlie northern limit for virus amplification each year.

KEY WORDS Ciilisi'ln mcliiniim, temperature, larval development, eastern equine enceplialomy- elitis, amplification, birds

Ciilisctci mclamira (COOUU.I.ETT) is the enzootic vector lected from resting boxes as late as the 3rd wk of of eastern equine encephalomyelitis virus (EEE) November (Cusciora et al. 1972) when nighttime tem- throughout much of the Atlantic coast of North Amer- peratures are close to freezing. Mahmood and Crans ica (Morris 1988, Scott and Weaver 1989). Tlie mos- (1997) showed that Cs. nu'lanura females can com- quito has an eastern distribution tliat ranges from plete their gonotrophic cycle and lay eggs at temper- southern Florida to Quebec, Canada (Darsie and atures as low as 10C. Ward 1981), and is exposed to a wide range of tem- Developmental rates at fall temperatures have a peratures in the northern half of its range. Q., mrlii- direct impact on the age composition of overwintering wira larvae develop in a variety of subterranean cav- larval cohorts. Overwintering success determines ities, collectively known as crypts (Pierson and Morris adult population size in spring, which directly affects 1982). Common habitat in northeastern United States amplification of EEE in local bird populations. Crans includes recesses formed by the root balls of upturned et al. (1994) found evidence for a cryptic cycle in birds trees, underground pockets formed by rotting stumps, that may make EEE available for amplification during or spaces within the root systems of trees growing in the spring nesting season. However, limited informa- saturated soil (Moussa 1966, Joseph and Bickley 1969). tion is available to explain the emergence patterns of reproduction is continuous in the southern United vernal cohorts of C'.s. nwlanwci that transmit EEE to States, but the species overwinters as larvae in areas resident birds. wliere temperature and photoperiod initiate diapause Mahmood and Crans (1997) developed a heat sum- (Burbutis and Lake 1956, Hayes 1961, Joseph and mation model to predict the duration of tlie gonotro- Bickley 1969). phic cycle over a wide range of ambient temperatures. Culisela mrlanura remains reproductively active The current investigation extends that research by ^el] into tlie fall. Blood fed females have been col- presenting heat summation models to calculate im-

0022-2."iS5 ")S110(17-1012SD2 (10'0 IMS Eiitoinoloeic.il Suripli of Ampric'.i 1008 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 35, no. 6 1 | mature developmental rates from oviposition to adult Data Analysis. Differences in the duration of im- emergence. We discuss how water temperatures in Cs. mature development among temperatures were tested mclamira larval habitats regulate mosquito contact by a 1-way analysis of variance (ANOVA) andDuncan with susceptible bird populations and directly affect new multiple range test (Sokal and Rohlf 1981). Sur- amplification ofEEE in spring. vival values for each instar were calculated as the percentage of the original number of larvae that molted successfully to the next instar. Survivorship Materials and Methods from 1st instar to adult emergence was calculated as Strains. We used a colony of Cs. mclaniira in the the total number of adults divided by the total number F]3-F|- generation originally established from field of 1st instars. Because larval development spanned 3-8 stock collected from southern New Jersey (Mahmood mo at 10-16C, larval survivorship was low and the and Crans 1994). Adults and larvae were maintained data were combined for all 5 replicate pans at each in an insectary at 24 +/- 2C under a photoperiod of 16:8 temperature. Heat summation models of the form V (L:D) h. A 1-step 1.5-h dusk and 1.0-h dawn period was (t to)/k were estimated from the rate of embryonic, provided by a 15-W incandescent bulb. Larvae were larval, and pupal development over the temperature reared in uncovered pans containing deionized water range of 10-32C using linear regression (Mahmood with finely ground rat chow as food. Newly emerged and Crans 1997). V, the rate of embryonic develop- adults were held in a 0.6-m3 cage and provided with ment in eggs, was calculated as V 1/E, where E 10% sucrose solution. mean time in days from oviposition to egg hatching. A Embryonic Development. To determine the effect linear regression analysis was performed to obtain the of temperature on embryonic development, 6- to 9-d- values of V a + ht, where a the rate of develop- old females were offered a restrained bobwhite quail ment or intercept when temperature 0 and h was (Coliniis cirginiami.'i) at dusk for 3 h.1 The following the slope of the regression line. In the above model, k morning, groups of (20-45) fresh blood fed females was the thermal constant or the number of degree- were isolated in 3.8-liter paper cages and transferred days above (, the empirical thermal minimum below to environmental chambers at a photoperiod of 16:8 which embryonic development was halted. From the (L:D) h. Females were checked daily and late fed above, k 1/h and ( -(u/h). females were transferred individually to oviposition The mean age at molting for each instar was calcu- vials containing deionized water. The vials were lated in days. From these values, the mean duration plugged with cotton soaked in 10% sucrose solution as (D) of each instar (t) was calculated as D, t, (,.,, a source of nutrition for the gravid females. where (, the mean age at molting. The percentage Oviposition vials were checked daily for egg rafts, of immature life spent in each stage was calculated as and sugar solution was added to the cotton plugs as L, D,lt^, X 100, where (^ the mean time to adult needed. Females that oviposited were removed from emergence regardless of sex. All statistical procedures the vials and egg rafts were checked daily for hatch. followed Reisen and Siddiqui (1979) and Reisen et al. The time required from oviposition to egg hatch was (1982). Heat summation models for immature devel- determined at 10, 16, 22, 28, and 32C. opment were calculated using the time required by Cs'. Larval Development. Egg rafts were obtained from mvlanura to develop from 1st instar to adult at 10- colony adults and allowed to hatch at 24 +/- 2C. Five 32C. Because all larval stages overwinter in nature replicates of 50 first instars, within 18 h of eclosion, (Joseph and Bickley 1969), we calculated separate the were placed in white enamel pans (26.5 by 15.5 cm) heat summation models for individual instars using 1st- containing liter of deionized water and reared in an mean duration of time spent in each stadium (i.e., environmental chamber at 10, 16, 22, 26, 28, 32, and 2nd instar, 2nd-3rd instar, 3rd-4th instar, and 4th 34C; 80% RH; and a photoperiod of 16:8 (L:D) h. instar-pupa. The models predicted age structure in Larvae were fed a premeasured amount of finely larval populations and the transition from oviposition ground rat chow every 2nd d at 22-34C and every 4th to emergence when environmental temperatures d at 10-16C. The total amount of food required for were known. complete larval development was recorded for each temperature. Bacterial surface scum was removed Results with a paper towel and cohorts were transferred to new pans whenever scum accumulation was excessive. Embryonic Development. Egg hatch decreased sig- Deionized water was added to replace water lost by nificantly (F < 0.005) as temperatures increased from evaporation, and pans were checked each morning for 10 to 28C (Table 1). Eggs hatched in 2 d at 28C, but dead larvae. Cadavers and exuviae were removed and required nearly 20 d at 10C. No eggs hatched at 32C. recorded by pan and date. Cohorts of pupae from each The egg development rate in Cs. melanura slowed as pan were transferred into mesh-covered 2.50-ml glass temperatures decreased (K2 0.96). The thermal beakers containing 100 ml deionized water. The date minimum (to) below which embryonic development of pupation, date of emergence, and sex of emerging ceases in Cs. melanura was 9.38C. The thermal con- adults was recorded by pan for each temperature stant k or the number of degree-days above to required regime. for completion of embryonic development was 38.46 DD. The heat summation model for embryonic de- velopment in days (E) for Cs. melanura is E 1/V, November 1998 MAHMOOD AND CRANS: EFFECT OF TEMPERATURE ON Cs. melanura 1009

Tnlile 1. Effect of tnnperulure the duration of embryonic number of days required for 1st instars to complete dcvelopineiil in Cs. mdfiiittra their development at temperatures between 10 and 32C in nature. No. egg Days to egg hatch Tables 3 and 4 present the necessary parameters to Temp, C rafts (mean +/- SD) calculate immature development in days over the 19.7 +/- 0.6a range of temperatures included in our studies. In Ta- 8.5 +/- 0.5b the for thermal heat 3.0 +/- 0.3c ble 3 we present parameters 2.0 +/- O.Od summation models for each larval instar to predict the No hatch number of days required to molt to the next stadium using values from mean days in stadium. Table 4 pre- Means with different letters .significantly different by the Dun- sents similar parameters to calculate duration between multiple range test (F < 0.005). stadia using mean age at molting. Table 5 presents data on the effect of temperature where V (I 9.38) /38.46. This mode] can be used on survival. More than 90% of 1st instars survived at to calciilnte the time from oviposition to egg hatch temperatures between 22C and 32C. Higher and under field conditions. lower temperatures produced significant mortality at Figure shows the number of degree-days above this instar. In later instars, low temperatures were the thermal limit (/) generated by average temper- tolerated, but mortality increased at water tempera- atures from 10.4 to 28.4C and the corresponding du- tures above 28C. Pupae survived over the widest ration of embryonic development of Cs. mclaniirn in range of temperatures and succumbed only at the egg rafts over tliose temperature ranges. The graph highest temperatures used. Overall emergence suc- can be used to determine the time required for egg cess from 1st instar to adult was highest at 26C. Higher hatching after oviposition at any point in the breeding and lower temperatures produced fewer adults. season when temperatures range between 10 and 28C. Discussion Larval Development. Mean age at molting and du- ration of each stadium increased significantly as tem- Adaptations for Overwintering in the Larval Stage. peratures increased from 10 to 26C (Table 2). Tem- Cs. inrltiniirt! is a mosquito with exceptionally slow peratures above 28C accelerated development in 1st development. Development from egg hatch to pupa- >3 instars but slowed the growth of later instars. The heat tion required mo at water temperatures of26C, summation model for immature development (/M) in mo at 16C, and >7 mo at 10C. In contrast, Ardcs days from st instar to adult was IM 11 V, where V WMW, (Meigen), a temperate floodwater species, (t ?o)/fc. The thermal minimum (

of inimali *ta(<*1

3rd instar 4tll in.star Pupa Temp, C 1st instar 2 1(1 in-star ------~^^ Mran ,>HP at ting (d. ys +/- SD) 13.0 +/-0.4 23.4 +/- 1.3 22.7 +/-1.7- 3.0 +/- 0.0 7.3 +/- 0.2 32 Ki.9 +/- 0.:) 19.2 +/-0.4 +/- 0.2 10.9 +/- 0.1 28 4.7 +/- 0.2 7.9 32.4 +/-0.3 12.2 +/-0.5 29.9 +/- O.S +/-0.9 26 4.1 +/-0.0 7.1 15.4 +/- 0.2 21.1 +/-0.1) 27.7 +/- 0.5 '5.9 +/- 0.0 10.8 +/- 0.5 22 68.1 94.5 101.1 18.8 39,3 16 164.3 244.3 10 44.1 100.6 M<';in days ii stadium +/- SD 10.6 +/- 1.5 1.8 +/- 0.2 4.2 +/- 0.3 5.7 +/- 0.4 32 3.0 +/- 0.0 +/-0.1 2.3 +/- 0.1 3.2 +/-0.1 3.0 +/-0.1 6.1 28 4.7 +/- 0.2 +/- 4.8 +/- 0.5 17.8 +/- 1.0 2.5 0.2 4.1 +/-0.0 3.3 +/- 0.2 26 9.0 +/- 0.1 3.3 +/- 09 4.9 +/- 0.5 4.6 +/- 0.4 22 5.9 +/- 0.0 6.6 20.5 28.8 26.4 16 18.8 17.3 56,5 63.7 62.8 10 44.1

by Morris et al. (1976). There appears to be an northern New Jersey ranged froin a winter low of 1C gested overwintering generation of larvae that emerges as a in January to a brief high of20C in August. Temper- to .spring brood of adults and a summer generation of atures remained below 10C from mid-November larvae that emerges as a fall brood of adults. The mid-May, the time that Cs. mdanwa overwinters in rarely exceeded offspring of these 2 generations appear to play very the larval stage. Water temperatures ofEEE. September, the time when different roles in the epidemiology 16C between July and of larvae emerge as would be developing at the fastest rate. Joseph The overwintering generation larvae May and in New Jersey and blood Bickley (1969) recorded temperatures in cavity adults during June and on adult birds that nest in the vicinity within the Pocomoke Cypress Swamp on the feed primarily habitat habitat et al. 1994). Eggs produced shore of Maryland. They found that temper- of the larval (Crans eastern that emerged in spring produce the reached 10C in April, "\ mo earlier than levels by the mosquitoes atures of larvae that emerge as nulliparous adults measured in northern New Jersey. Surprisingly, larval generation in late July, August, and September. The early summer in Maryland remained cold throughout the habitats ofCt. mpliiniira adults makes contact with res- summer season, ranging from 14 to 18C from June to cohort ident bird populations that includes juveniles as well September. cohorts emerge during migration and temperatures, eggs laid in late May in as adults. Later At these with transient populations. New Jersey would require at least 3 mo to make contact primarily northern from the combined cohorts of the July, August, develop and would not produce adults until late Au- Eggs and September brood of adults produce larvae that gust or early September. Eggs laid in midsummer make up the overwinteringgeneralion and emerge the would not produce adults until late fall. Larvae from Cypress Swamp, MD, may undergo accel- following spring. Pocomoke of;> Bi-Voltinc Life Cycle on Amplification erated development during the warmest portion of the Impact ofEEE in Birds. Bird populations in the northeastern season and emerge as adults mo earlier. This suggests United States vary markedly in age structure and spe- that Cs. mvlaniira is probably bivoltine throughout cies composition over the course of full season most of the northeast rather than trivoltine as sug- (Pough 1949, Berthold 1993). In May, summer resi- dents (i.e., wood thrush, gray catbird) move north- ward from wintering sites in the tropics to join per- manent residents (i.e., Carolina chickadee, tufted titmouse) that remain year round in New Jersey (Boyle 1994). Both groups establish nesting sites in wooded habitats during May to raise their young. The fledglings of both permanent and summer resident

Table 3. Purnnirters for llir In-ill aiinimulion mo-iiiprraliir<- (I)

Immature to k R2 stadium (Thermal min.) (dpRrpc-days above t,,) 0.94 1st instar 10.03 71.27 0.79 2nd instar 8.83 71.41 average temperature, num- 0.62 Fig. 2. Relationships among 3rd instar 8.25 8.5.78 required 0.51 ber of degree-days above the thermal minimum (to) 4th instar 5.51 210.57 Cs. mela- 44.63 0.99 for immature development, and number of days for Pupae 8.32 nura to develop from 1st instar to adult. melons 1011 EFFECT OF TEMPERATURE ON C. November 1998 MAHMOOD AND CRANS.. are locally numerous W, and when susceptible juveniles for llu- l"-al ,u,n,nBtion mo.lcl [IM temperatures m T..M,. 1. PBrarncK-ri. of.l^re,,u,re nJ. bet.e.-n ... the transient ^ enough to have the main emergence contact k in the process of Immature '11 R2 of birds that are (de-pree-days above o) populations migrateto the staRe ('rhernial min.) rather than juveniles that remain close 0.94 ing south 10.03 71.27 lst-2nd instar 0.92 9.24 148.94 is correct, water temperatures in lst-3rd instar 241.19 0.85 "Tour'h^pothesis instar S.SO the northern limit Ist-lth 450.73 0.76 Cs. meknwa habitat also regulate 1st mstar-pupa 467.29 0,84 each year. In the extreme north- i.ll 8.50 for virus amplification 1st instar-ad 396.35 0.75 melanw-a, emergence clult 7.98 of the range of Cs. 2nd inslar-ai 319.79 0.72 ern portion kilt 7.99 probably takes place too 3rd inslar-ai 231.16 0.74 of the summer generation instar-a(lull 7.46 mosquitoes to 4th 44.63 0.99 for large numbers of nulliparous Pupa-adult 8,32 hte contact with either recrudescing virus or juve- make result, epiormtics nile birds that remain on site. As a entirely absent near the north- the month of June in New Jersey of EEE are sporadic or species appear during is illustrated in the work of the fall. Migration generally ern limit of the range. This on site into where antibody levels in Sremam with transients that nest further Emord and Morris (1984) b gi, r n late August declined over a 3-yr period after moving through the area, birds progressively north continuouslythe activity at a study site m in any local area In September, docun-iented EEE upper^New ing populations water in subterranean habitats resident species south to then- preferred York State. Warmer summer yellow-rumped south would hasten development of eggs laid Ser range. Winter residents (i.e., further likelihood nuthatch) join permanent Mav or June and increase the ofnulhp^ warSer red-breasted res^and interfacing conditions favorable for New Jersey during the month of October arou.s mosquitoes rdentsn; This phenomenon is illustrated by until spring (Boyle 1994). virus amplification. remain mtroduction d et al. (1994) at a site in southern Amplification of EEE requires the he work of Crans of suscep ibl was isolated from Cs. mc'h- virus into large populations New Jersey, where EEE infectious (oc, and antibody levels in resident Adult birds that nest at en.ootic c. ery'year for 6 yr birds unsuitable for h^h^gamplinca remained relatively cons ant. levels of immunity and arc bird populations func- numbers during June the summer cohort is probably a fcn Fledglings appear in large The size of the susceptible hosts for rapid ^ feeding success in mosquitoes wave of tion of andana PproMO ^le t that turn is thatformed o of EEE. Uans et ilai. (1994) showed May-June emergence. This in amplification ^'^l l60% of of the late fall cohort of egg laying June to amplification Besides bird nopulation late in the season. important factor in the ofvirus_ that recrudescmg cohort of larvae from late et al. (1994) hypothesized sTmd 4th instars, a large virus availa Ie in 1st or 2nd mstar adult birds with latent infections make season egg hatch would overwinter for amplification in to make contact with nestlings to Cs. ^,,n, susceptible^ InTemerle injun^lfthe that epiornitic cycling was dependent were producing recrudescing virus, lings. Tliev felt adult birds pop- influx of nulliparous 0. to as^c would be set for amplification in Juvenile upon an mchn^ by fema e stage melanw-a adults. that recrudescing virus would be acquired by the fall emergence of Cs. for he vn to ulations to explain why mosquitoes that lived long enough We have proposed a mechanism susceptible a method to predic replicate and be transmitted to juvemi^ FEE becomes epiornitic as well as in lanal mode s as Our data indicate that water temperatures of amplification usingheat summation be a kc> onset information habitats from mid-April to August might tools. Considerable additional A '^clen e 1 Jnostic over the geo- factor in the amplification process. to validate this hypothesis in the crypts wou d has- rSred in Cs. melannra and crease in water temperatures c^ange where EEE cycles overwintering generation o ^S be monitored m the ten development of the h W^temperatures should emergence of a iT where C. ^lan.ra Cs. mcknun, and permit ""'1Pal.o ro.1 ray of avity habitats in adult buds cohort when the virus is recrudescing

in Cx. ini'^ni "iirvivor..li.l, ;lllll riiirrp'lK-r Tahl.. S. V.fSrrI "f lrl"liimilii Emergence, \p m Piupa in-,1 4lli insl Temp, C tar 3rd 6.0 )sl iiislar 93.7 50.0 7.6 54.3 45.1 1130.0 10 52.4 89.5 46.0 +/- 5.0 25.9 56.7 84,9 1.9 16 46.4 83.5 i 5,6 3.7 +/- 3.8 81.1 2.2 ^ 78.3 +/- 85.2 +/- 98.2 -S. 1.3 ^ 22 93.2 2.2 ^+/- 93.0 2.5 +/- 0.1 +/- 2.2 95.7 2.0 59.6 +/- 93.4 +/- 90.9 ^3.1 a) 94.7 3.0 +/- 91.8 1.9 1.0 +/- 0.7 +/- 2.1 89.1) 2.1 +/- 16,4 fc 92.0 +/- -1.0 87.6 )6.8i: 9.4 21.4 4.5 64.3 2.5 ND 93.0 +/- 1.2 78.4^ ND ND NC ND 1 34 0.0 1012 JOUBNAL OF MEDICAL ENTOMOLOGY Vol. 35, no/ overwinters in the Atlantic Hyway. Such information Joseph, S. R., and VV. E. Bickley. 1969. Culiwta melan^ could predict appearance of the summer generation (Coquillett) on the eastern shore of Maryland (Diptei-HI and likelihood for amplification in resident bird pop- Culicidae). Univ. Md. Agric. Exp. Stn. Bull. 161: 1-83 ulations. Mahmood, F., and VV. J. Crans. 1994. Laboratory coloniza^l tion of Cnli.seta melanura from southern New Jersey. Prop^| N.J. Mosq. Control Assoc. 81: 93-95. '3 1997. A thermal heat summation mode] Acknowledgments to predict the du-^ ration of the gonotrophic cycle of Ciilixrta melanura in The authors thank the Cape May County Mosquito Ex- nature. J. Am. Mosq. Control Assoc. 13: 92-94. termination Commission, their superintendent, Judy Hansen, Malnney, J. M., and R. C. Wallis. 1976. Response of colo. and her staff for the technical and logistical support provided nized Ciili.wta melaniira to photoperiod and temperature for this investigation. This is New Jersey Agricultural Exper- Mosq. News 36: 190-195. iment Station Publication No. D-40400-06-98 supported by Moussit, M. A., D. J. Gould, M. P. Nolan, Jr., ;>nd D. E. Hayes. 1966. 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