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Larval Environmental Temperature and the Susceptibilityof Aedes albopictus Skuse (Diptera: Culicidae)

to Chikungunya Virus

Catherine J. Westbrook,1 Michael H. Reiskind,1,2 Kendra N. Pesko,1,3

Krystle E. Greene,1 and L. Philip Lounibos1

Abstract

A key feature in the recent widespread epidemic of the mosquito-borne alphavirus chikungunya virus (CHIKV)

was the important role ofAedes albopictus, formerly regarded as a secondary vector, compared to the presumedprimary vectorAedes aegypti.Ae. albopictus, a container-inhabiting mosquito, is an invasive species that occurs overa wide geographic range spanning tropical and temperate latitudes. In this study we examine the effects of a broadrange of larval rearing temperatures on CHIKV infection, dissemination, and viral titer in Florida F1 Ae. albopictus.Adults from larvae reared at 188C, 248C, and 328C differed significantly in size, development time, and CHIKVinfection rate. Adult females with the largest body size were produced from the coolest temperature, took thelongest to mature, and six times more likely to be infected with CHIKV than females reared at 328C. There was alsoa significant effect of rearing temperature on viral dissemination, resulting in an increase in population dissem-ination at the coolest temperature. This study indicates that climate factors, such as temperature, experienced atthe larval stage, can influence the competence of adult females to vector arboviruses.

Key Words: Arbovirus(es)ClimateDengueEntomologyVector-borne.

Introduction

Climate is one of the principal determinants of thedistribution of vector-borne diseases (van Lieshout et al.2004). In particular, vector development and survival, andarbovirus replication are greatly influenced by temperature.In mosquito vectors, temperature can influence larval devel-opment time, larval and adult survival, biting rates, gono-trophic cycle lengths, and vector size (Madder et al. 1983,Rueda et al. 1990, Scott et al. 1993). Ambient temperature canaffect arboviral dynamics within the mosquito vector by al-

tering the length of the extrinsic incubation period (EIP),which is the time between ingestion of an infectious bloodmeal and when transmission to a subsequent host is possible(Chamberlain and Sudia 1955, Reeves et al. 1994, Patz et al.1996). Further, temperature often defines the latitudinal andaltitudinal ranges of a vector. Species range may limit the

distribution of disease when pathogens are species specific.Temperature may also limit viral transmission in areas wherethe vector is present, but the temperature precludes efficienttransmission (Purse et al. 2005).

Vector competence, which is the capacity of an arthropodto acquire an infection and transmit it to a subsequent host,can greatly vary among individuals and between populations(Lorenz et al. 1984, Turell et al. 1992) and is influenced bygenetics (Mercado-Curiel et al. 2008) as well as by climatevariables, such as temperature (Davis 1932, Turell 1993,Dohm et al. 2002). An increase in environmental temperature

for adult mosquitoes reduces the EIP (Davis 1932, Chamber-lain and Sudia 1955), most likely due to an increase in themetabolism of the adult mosquito and the replication speed ofthe virus.

Although the majority of research on mosquitovirus in-teractions has focused on adult mosquitoes, temperature

1Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences,University of Florida, Vero Beach, Florida.

2Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma.3School of Medicine, University of New Mexico, Albuquerque, New Mexico.

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changes experienced in the immature stages of holome-tabolous vectors before infection may affect vectorvirusinteractions by changing physical and physiological charac-teristics of midgut and salivary gland barriers, which couldhave direct consequences on viral infection, replication, andtransmission. Previous studies have shown that larval rearingtemperature can effect mosquito competence for several ar-boviruses, including Murray Valley encephalitis virus (Kay

et al. 1989b), Japanese encephalitis (Takahashi 1976), andwestern equine encephalitis (Hardy et al. 1990) viruses. In aspecific study withAedes taeniorhynchus, mosquitoes reared at198C had higher infection rates for Rift Valley fever and Ve-nezuelan equine encephalitis viruses than counterparts rearedat268C (Turell 1993). In view of global climate change models,which predict changes in temperature that will directly im-pact larval mosquito habitats, this study, which investigates apreviously unexplored relationship between Aedes albopictuslarval environmental temperature and chikungunya virus(CHIKV) susceptibility, could help increase the predictabilityof disease transmission patterns and future outbreaks.

The intercontinental dispersal of invasive arbovirus vec-tors, such as the Asian tiger mosquito Ae. albopictus, is ac-

companied by an increase in vulnerability to the exoticdiseases vectored by these invaders (Juliano and Lounibos2005). In 20052006 CHIKV, a single-stranded positive senseRNAenvelopedAlphavirus in thefamily Togaviridae emergedas an important pathogen in the Indian Ocean Basin. On theisland of La Reunion alone, 241,000 clinical cases of chi-kungunya fever, representing 31% of the population, werereported (Paquet et al. 2006). A sylvan transmission cycle ofCHIKV involving mosquitoes, such asAedes furcifer andAedesluteocephalus, and wild primates is limited to tropical Africa,and epidemic transmission of the virus is sustained thoughinfection of the mosquitoes Aedes aegypti and Ae. albopictus inurban and peridomestic environments (Jupp and McIntosh1990, Diallo et al. 1999).

A unique feature of the South West Indian Ocean CHIKVoutbreak was the increased importance of Ae. albopictus as avector. The enhanced role ofAe. albopictus as a CHIKV vectoron La Reunion was in part due to the absence of the primaryvectorAe. aegypti (Delatte et al. 2008); however, an amino acidsubstitution in La Reunion CHIKV isolates from alanine tovaline at the 226 position of the E1 envelope structural proteinpresent has been shown to increase Ae. albopictus, but not Ae.aegypti susceptibility to the virus in the laboratory (Tsetsarkinet al. 2007). After the Indian Ocean Basin epidemic, a strainof CHIKV nearly identical (99.9% nucleotide identity) to iso-lates from La Reunion emerged in India, where 1.3 millionhuman cases were reported in 13 states in 20052006 (Ara-nkalle et al. 2007). This widespread epidemic was not re-

stricted to the tropics, with autochthonous transmissionreported in Italy in 2007 (Rezza et al. 2007). The epidemiccontinued to spread in Indonesia, Sri Lanka, and Singapore,where cases were reported through 2008 and into 2009(Seneviratne et al. 2007, International Society for InfectiousDiseases 2009).

In this study we explore how variation in temperatureduring larval development affects the susceptibility ofAe. al-bopictus for CHIKV by measuring infection and disseminationrates, and viral titer. We also assess how larval temperatureaffects growth and survivorship by measuring larval mortal-ity, development time to adulthood, and adult body size.

Materials and Methods

Mosquitoes and viruses

Ae. albopictus used in this experiment were generated fromfield collections of approximately 4000 larvae and=or eggsmade from June to August 2007 in Palm Beach County,Florida. This population of Ae. albopictus was previouslyshown to be highly susceptible to the La Reunion (LR2006-

OPY1) CHIKV strain (Reiskind et al. 2008). Females rearedfrom field-collected immatures were given 20% sucrosead libitum, blood fed weekly on live chickens, and kept incages at 14L:10D, 268C (18C SD) and >80% relative hu-midity (rh). Chicken care followed federally mandated animaluse and care policies (University of Florida, IACUC ProtocolVB-17). Eggs (F1) were hatched in tap water, and, within 24 hafter hatching, individual larvae were placed in 50 mL Fal-con conical tubes with 35 mL of tap water and 0.0105 g 1:1yeast:albumin food. Based on preliminary studies, 0.0105 g offood at the beginning of the experiment was adequate for thecompletion of individual development but did not increasemortality at the higher temperature. Larvae were reared at188C, 248C, and 328C with a 14L:10D cycle in a Percival

(Percival Corporation, Perry, IA) incubator. The experimentaltreatment units in this study were different incubators thatwere identical in all respects except for rearing temperature;thus, it was assumed that differences in treatments werecaused by rearing temperature. These temperatures arewithin the temperature range of the treehole environmentoccupied by Ae. albopictus in Florida (Lounibos 1983). Larvaein each temperature treatment were from the same cohort ofeggs whose hatch was staggered to synchronize adult emer-gence among all three temperature treatments. After the finallarval instar, pupae were removed from rearing tubes, sexed,and stored in groups of 10 in water-filled vials to record adultemergences. After emergence, all adults were held at 248C,9599% rh with a 14 L:10 D cycle in a biosafety level-3 facility

and given 20% sucrose ad libitum.The LR2006-OPY1 CHIKV strain (GenBank accession

number DQ443544) was isolated in France from a febrile pa-tient who had been infected on the island of La Reunion(Parola et al. 2006). This recently emergent strain of CHIKVcontains the alanine to valine substitution at the 226 positionof the E1 envelope structural protein that has been identifiedas a feature in many current CHIKV epidemics (Rezza et al.2007) and has been shown to increase Ae. albopictus suscepti-bility (Tsetsarkin et al. 2007). To produce virus for infectiousblood meals, a T-75 cm2 flask with a confluent monolayer ofVero cells in 10 mL of cell culture medium ( M199 mediumsupplemented with fetal bovine serum, antibiotics, and anti-mycotics; Invitrogen, Carlsbad, CA) was inoculated with

150 mL of previously frozen stock virus, and allowed to in-cubate in a 5% CO2 and 358C atmosphere for 24 h.

Vector competence

Groups of fifty 5- to 7-day-old mosquitoes were placed in1-L cylindrical, waxed cardboard containers (Dade Paper,Miami, FL) with mesh screening. Mosquitoes were starved for24 h before being offered an infectious blood meal of 1:1000dilution of freshly propagated CHIKV in citrated bovineblood (Hemostat Laboratories, Dixon, CA) supplementedwith ATP (5 mM) as a phagostimulant. Water-jacketed glass

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membrane feeders (Rutledge et al. 1964) covered with Edi-coll collagen film (Devro, Sandy Run, SC) and connected toa Haake Series F water circulator (Thermo Haake, Paramus,NJ) were used to maintain the blood meal at 378C. Mosquitoeswere given 30 min to feed. Low feeding success ofAe. albo-pictus made it necessary to conduct three consecutive days offeeding for individuals from all three temperature treatments.The blood meals from the three consecutive feeding days were

assayed using quantitative real-time polymerase chain reac-tion (qRT-PCR), with copy number standardized to plaqueforming units by plaque assay performed on 10-fold serialdilutions of virus stock (Bustin 2000). Virus titers in bloodmeals were log10 4.7, 4.5, and 3.4 plaque forming units=mL for the three feeding days, respectively. After feeding,mosquitoes were cold anesthetized, and fully engorged mos-quitoes were removed, placed in new cages, and given 20%sucrose ad libitum.

After a 10-day EIP at 248C, surviving Ae. albopictus femaleswere killed by freezing. Females were stored at 808C; afterthawing, wings were removed for measurements, bodieswere assayed to determine infection status and titer, and legswere tested to check for a disseminated infection (Turell et al.

1984). Wing length was measured in millimeters as an indi-cator of body size (Blackmore and Lord 2000) from alula towing tip of digital images using a computer imaging andmeasurement program (i-Solution lite; AIC, Princeton, NJ).

Bodies and legs were triturated separately in 2 mL micro-centrifuge tubes containing 900mL of BA-1 medium (Lanciottiet al. 2000) and two zinc-plated beads. Samples were ho-mogenized at 25 Hz for 3 min using a Tissuelyzer tissuehomogenizer (Qiagen, Valencia, CA) and then clarified bycentrifugation (3000g for 4 min). Viral RNA was extractedfrom 250mL of the sample with Trizol-LS (Invitrogen) fol-lowing the manufacturers instructions and using a finalelution volume of 50mL in diethylpyrocarbonate (DEPC)-treated water.

One-step qRT-PCR was used to determine infection anddissemination status and body titer by previously establishedprotocols (Reiskind et al. 2008). Primers from the CHIKV E1gene were designed with the following sequences: forward,50-ACC CGG TAA GAG CGA TGA ACT-30; reverse, 50-AGGCCG CAT CCG GTA TGT-30. The probe sequence was:50-=5Cy5=CCG TAG GGA ACA TGC CCA TCT CCA=3BHQ_2=-30 (IDT DNA, Coralville, IA).

Statistical analysis

Kruskal-Wallis tests followed by Dunns pairwise multiplecomparisons (family a 0.05) were used to determine differ-ences among treatments in distributions of wing lengths and

development times to adulthood (SAS 9.1 for Windows, 2003;SAS Institute, Cary, NC). Males and females were analyzedseparately because of sex-specific sizes and developmentaltimes in this species. Chi-squared tests of independence wereused to determine the effect of temperature on survivorshipto adulthood. If significant effects were detected, post hocpairwise comparisons were made with an alpha-adjusted(Bonferroni) correction to account for multiple comparisons(Minitab 15.1 for Windows, 2006; Minitab, State College, PA).

A generalized linear mixed model (PROC GLMMIX, SAS9.1, Cary, NC) was used to describe relationships amongtemperature (fixed effect), feeding day (random effect), in-

fection (# with virus=# fed), dissemination (# with virus intheir legs=# with virus), and population dissemination (# withvirus in their legs=# fed) specifying a logistic link function anda binomial error distribution. Odds ratios (OR) and 95%confidence intervals (CI) for infection at each temperaturetreatment were also calculated (SAS9.1 for Windows, 2003). Alinear mixed model (PROC MIXED, SAS 9.1, Cary, NC) wasused to test for effects of temperature treatments (fixed) and

feeding day (random), which were both class variables onbody titer, a continuous variable. Titer data did not fit themodel assumptions of normality, but approximate normalitywas achieved through a natural log transformation of titervalues (SAS 9.1 for Windows, 2003). Wing length compari-sons, pooled across all temperature treatments, betweenbinomial variables, infected versus uninfected and dissemi-nated versus nondisseminated, were analyzed with t-tests(Minitab 15.1 for Windows, 2006).

Results

Growth and mortality

Wing lengths and development times to adulthood were

significantly affected by temperature for both females (winglength: H 417.5; df 2; p< 0.0001; development time: H1279.6; df 2;p< 0.0001) and males (wing length:H 2127.4;df2; p< 0.0001; development time: H 1697.3; df 2;p< 0.0001) (Table 1). There was an inverse relationship be-tween wing length and temperature with the largest adultmosquitoes produced at 188C and the smallest mosquitoesproduced at 328C. Mosquitoes reared at the lowest tempera-ture (188C) took over two times longer to develop than thoseat 248C and 328C. Juvenile mortality rates at 188C, 248C, and328C were 9.16% (n 1454), 7.34% (n 1520), and 16.90%(n 1473), respectively. There was a significant relationshipbetween survivorship to adulthood and temperature (w254.123, df 2, p< 0.0001). Ae. albopictus reared at 328C were

significantly more likely to die as larvae when compared withindividuals reared at the 188C and 248C, with no differencesbetween the two lower temperatures.

Chikungunya infection and dissemination

There was a significant temperature treatment effect on thepercentage of females that developed CHIKV infections(F 16.92, df 2, p< 0.0001) (Fig. 1). Infection was six timesmore likely in adult females reared at 188C than at 328C(OR 6.052; 95% CI 3.2211.373), and females reared at 248Cwere 2.7 times more likely to be infected than those reared at328C(OR 2.722; 95% CI 1.3855.351).Females reared at 188Cwere 2.2 times more likely to be infected than those reared at

248C (OR 2.223; 95% CI 1.3283.722). Among the infectedindividuals the number of females that developed dissemi-nated infections did not vary significantly among larvalrearing temperatures (F 0.85, df 2, p> 0.4293); however,there was a significant temperature effect on population dis-semination (F 6.20, df 2, p< 0.0022) (Fig. 1). Populationdissemination was approximately five times more likely inadult females reared at 188C than at 328C (OR 4.905; 95% CI1.81413.271) and 2.3 times more likely in females reared at188C than at 248C (OR 2.291; 95% CI 1.0884.827). Therewas no difference in population dissemination between the248C and 328C treatments.

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After the 10-day EIP, no significant variation in body titerof virus-positive mosquitoes was observed among the tem-perature treatment groups (F 1.14, df 2, p> 0.4300). Ae.albopictus females that were positive for CHIKV infection in allthree temperature treatments were significantly larger thanuninfected females (t 3.59, df 327, p 0.0004) as mea-sured by mean wing length. There was no significant differ-ence in mean wing lengths of females with disseminated andnondisseminated infections (t0.41, df 94, p 0.6830).

Discussion

Due to the impact of climate on vector ecology, mosquito-borne diseases will be sensitive to projected changes in globaltemperatures. In this laboratory study we demonstrated thatlarval rearing temperature can influence survival, develop-ment time, and wing length, and may directly impact diseasetransmission by influencing the likelihood of infection withCHIKV. Although the proportion of infected females thatdeveloped disseminated infections did not differ significantlyamong the three larval rearing temperatures, the populationdissemination rates were significantly higher at 188C, whencompared to the two higher temperatures of 248C and 328C.Disseminated infection is generally accepted as a measure ofa mosquitos ability to transmit a virus through biting (Turellet al. 1984). The rate of dissemination, when expressed as a

percentage of the number of mosquitoes infected, may pro-vide information about the effect of a midgut escape barriermoderating whether gut infections are able to disseminateinto the hemolymph. In this study, individuals reared at 328Chad significantly lower infection rates, but no significant dif-ference was found in dissemination rate. Thus, it can bespeculated that there may be a reduced midgut escape barrierin mosquitoes derived from the higher rearing temperatures.On the other hand, the population dissemination rate, ex-pressed as a percentage of the total number of mosquitoestested, was greater for mosquitoes from 188C, and is epide-miologically more important in that it gives an estimate of the

vector competence or the transmission potential of a popu-lation. Body titers did not differ among temperature treat-ments for mosquitoes with disseminated infections. Althoughthis result was somewhat surprising, it is possible that afterthe 10-day EIP virus titers stabilized to the extent that treat-ment effects on titer were diminished. Additionally, onlya limited number of mosquitoes developed disseminatedinfections, and although the mean titer of disseminated

Table 1. Treatment Medians and Interquartile Ranges(25th Percentile75th Percentile) for Development

Time to Adulthood and Wing Length

188C 248C 328C

Female time toadulthood (days) 24.5a 11.0b 9.0c

Interquartile range 23.026.0 10.011.5 8.59.5

(n) (515) (556) (445)Male time toadulthood (days) 22.5a 10.0b 8.0c

Interquartile range 21.524.0 9.510.5 7.58.5(n) (817) (860) (791)Female wing

length (mm) 3.40a 3.14b 2.91c

Interquartile range 3.293.51 3.003.26 2.803.01(n) (347) (150) (207)Male wing

length (mm) 2.83a 2.61b 2.44c

Interquartile range 2.772.90 2.552.66 2.372.51(n) (745) (810) (740)

Medians with different superscripted letters are significantlydifferent with a measured variable.

FIG. 1. Bivariate plots of mean wing lengths (standarderror) and (A) percent infection, (B) percent dissemination,and (C) percent population dissemination grouped by treat-ment. Numbers within parentheses above symbols represent

the number of mosquitoes tested.

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mosquitoes from 188C was higher, it was not significantlydifferent from the other treatments.

The higher temperature of 328C decreased survivorship,when compared with 188C and 248C. This was unexpectedbecause results from preliminary experiments showed nodifference in survivorship between the three temperaturetreatments. The increased mortality could have been dueto the stress of high temperature or the interaction of high

temperature and excess nutritional resources resulting in theproliferation of detrimental microorganisms in the larval en-vironment. Larvae took longer to develop at cooler temper-atures, which produced larger adults. Mean wing lengthdifferences between successive temperature treatments wereapproximately 0.2 mm, confirming forAe. albopictus that adultbody size, within limits, exhibits an inverse relationship withlarval rearing temperature (Briegel and Timmermann 2001).Upper and lower thermal limits for the growth of Ae. albo-pictus larvae are approximately 118C and 358C, at whichtemperatures larval development is inhibited, eventually re-sulting in death (Hawley 1988, Monteiro et al. 2007).

Our data indicate that at lower larval rearing temperaturesthere is an increased likelihood of an adult female becoming

infected with CHIKV virus. This may have had a positiveeffect on CHIKV infection rates in locations such as highlandsin La Reunion, where entomological surveys recovered Ae.albopictus at elevations of 1200 m and temperatures as low as12.68C (Delatte et al. 2008). However, 12.68C is a lower larvalenvironmental temperature than what was investigated inthis study and therefore it is uncertain whether the relation-ship between reduced temperature and higher infection rateswould hold true at this temperature. The ability of Ae. albo-pictus to tolerate low temperatures and adapt to diverse eco-logical environments combined with their vector competencefor currently circulating CHIKV isolates may help to explainthe 2007 northern Italy CHIKV outbreak and increases thepotential for future epidemics in other temperate areas where

Ae. albopictus is abundant.This study focused only on the influence of larval temper-

ature, and adults were maintained at a common temperatureof 248C. How combinations of different adult and larvaltemperatures may affect vector competence was not ad-dressed. It is likely that adults maintained at the lower tem-perature will have decreased virogenesis and a longer EIPresulting in a decreased probability of transmission. There-fore, the maintenance of low adult temperatures may result ina reduction or elimination of any benefit low rearing tem-perature may have on increasing vector competence.

Results from this study are consistent with other systemswhere arboviral vector competence was reduced in femalemosquitoes that were reared at higher compared to lower

temperatures (Kay et al. 1989a, Hardy et al. 1990, Turell 1993).Unfortunately, none of the previous studies that exploredlarval temperature effects on adult arboviral susceptibilityreared mosquitoes individually to separate temperature anddensity effects, nor did they measure variables such as wingor body size and survivorship, as we have done in this study.

In our work lower rearing temperature produced mosqui-toes that were not only more susceptible to viral infection butalso significantly larger. Blood consumption by females is afunction of size, and large females are known to imbibe morethan twice as much blood as smaller females (Briegel 1990).Thus, in smaller mosquitoes reared at the higher temperature,

the imbibing of a lower blood volume would decrease theinitial viral dose and, in combination with a low CHIKVtiter in the blood meal, limit the establishment of infection.Low initial exposure to virus may not affect dissemination,which process requires postinfection virus replication. If thisexplains why large mosquitoes derived from cooler temper-atures have higher infection rates than smaller mosquitoes,then we would expect that titers of freshly engorged mos-

quitoes will increase with body size, which will be tested infuture studies. We also predict that different-sized mosqui-toes derived from different temperatures and fed an infec-tious blood meal with a high virus titer would overcome athreshold infectious dose and may result in similar infectionrates.

Because the variation in size was achieved through differ-ent temperature treatments, it is difficult to separate the effectof temperature from the response variable body size. Theremay be other temperature-dependent phenotypic traits thatvary in a way so as to cause an increase in infection whenadults are subjected to a lower temperature larval environ-ment that we did not measure. Previous studies show a lack ofconsistency in relationships between vector size and patho-

gen transmission. Large adult Ae. aegypti females from low-density larval conditions showed higher rates of oral infectionwith dengue virus (DENV) compared to two other size classesfrom higher density larval conditions (Sumanochitrapon et al.1998), and similar findings were reported for Ae. aegypti andRoss River virus (Nasci and Mitchell 1994). In contrast, Ae.albopictus adults reared in competitive larval environmentswere smaller and had higher rates of infection and dissemi-nation for Sindbis virus and DENV, while within the samestudies a competitive larval environment did not have a sig-nificant effect on vector competence in Ae. aegypti for the twoviruses (Alto et al. 2005, 2008a). In Ae. aegypti, when size wasexamined independent of rearing conditions small adultswere more susceptible to DENV; however, the size range of

individual measures was extremelynarrow (Alto et al. 2008b).In contrast, larger Aedes triseriatus adults produced throughvariation in competitive treatments had higher infection anddissemination rates for La Crosse virus (LACV) (Bevins 2008).In nutritional deprivation studies with Culex tritaeniorhynchusand Ae. triseriatus, smaller mosquitoes derived from nutrient-deprived larvae were more susceptible than their well-fed,larger counterparts, for West Nile Virus (Baqar et al. 1980) andbetter transmitters of LACV to suckling mice (Grimstad andHaramis 1984, Grimstad and Walker 1991). Smaller Ae. tri-seriatus adults generated from field-collected pupae weremore likely to transmit LACV to suckling mice (Paulson andHawley 1991). However, nutritional deprivation that led tosmall mosquitoes had no effect on vector competence in Culex

annulirostris for Murray Valley encephalitis virus (Kay et al.1989c) and Ae. vigilax for Ross River virus (Jennings and Kay1999). These contradictions in the effect of size on vectorcompetence could be based on the intrinsic differences be-tween vectorviral systems; however, it is also possible thatlarval conditions, such as high temperature and low nutrientsor competition, produce small mosquitoes by different mech-anisms that differently affect their competence as vectors.

In summary, cooler rearing temperatures produced mos-quitoes that were larger, had higher survival, and were morelikely to become infected with CHIKV, emphasizing the im-portance of the mosquito larval environment in determining

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adult vectorvirus interactions. Future studies should explorethe connection between larval rearing temperature-infectionpatterns observed in the laboratory and patterns in the field,and how climate and climate change may continue to impactthe mosquito larval environment and the epidemiology ofCHIKV.

Acknowledgments

We thank Naoya Nishimura for rearing and measuringmosquitoes and Drs. Stephanie Richards and George OMearafor their critical reviews of earlier versions of this article. Thisresearch was supported by NIH Grant R01 AI-044793.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Catherine J. Westbrook

Florida Medical Entomology LaboratoryDepartment of Entomology and NematologyInstitute of Food and Agricultural Sciences

University of Florida200 9th Street SE

Vero Beach, FL 32962

E-mail: cwestbrook@ufl.edu

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