LARGE-VOLUME GRAVID TRAPS ENHANCECOLLECTION OF CULEX VECTORS

DAVID A. POPKO AND WILLIAM E. WALTON1

Department of Entomology, University of California, Riverside, CA 92521

ABSTRACT. Gravid mosquito collections were compared among several large-volume (infusion volume$35 liters) gravid trap designs and the small-volume (infusion volume 5 6 liters) Centers for Disease Controland Prevention (CDC) gravid trap used routinely by vector control districts for vector and pathogensurveillance. The numbers of gravid Culex quinquefasciatus, Cx. tarsalis, and Cx. stigmatosoma collected bylarge gravid traps were greater than by the CDC gravid trap during nearly all overnight trials. Large-volumegravid traps collected on average 6.6-fold more adult female Culex mosquitoes compared to small-volumeCDC gravid traps across 3 seasons during the 3 years of the studies. The differences in gravid mosquitocollections between large- versus small-volume gravid traps were greatest during spring, when 8- to 56-foldmore Culex individuals were collected using large-volume gravid traps. The proportion of gravid females incollections did not differ appreciably among the more effective trap designs tested. Important determinantsof gravid trap performance were infusion container size and type as well as infusion volume, whichdetermined the distance between the suction trap and the infusion surface. Of lesser importance for gravidtrap performance were the number of suction traps, method of suction trap mounting, and infusionconcentration. Fermentation of infusions between 1 and 4 wk weakly affected total mosquito collections,with Cx. stigmatosoma collections moderately enhanced by comparatively young and organically enrichedinfusions. A suction trap mounted above 100 liters of organic infusion housed in a 121-liter black plasticcontainer collected the most gravid mosquitoes over the greatest range of experimental conditions, and a35-liter infusion with side-mounted suction traps was a promising lesser-volume alternative design.

KEY WORDS Arbovirus, mosquitoes, gravid trap, oviposition

INTRODUCTION

Gravid mosquito sampling is a key componentof vector surveillance that can enhance pathogendetection rates and improve the precision ofpredictive models used to guide vector controlstrategies (Williams and Gingrich 2007, Ginsberget al. 2010). Gravid adult mosquitoes that havetaken a blood meal are more likely to harborblood-borne pathogens than are nongravid mos-quitoes collected by other methods such as lighttraps and carbon dioxide–baited suction traps(Meyer et al. 2003).

Gravid traps used for Culex spp. surveillancehave included a variety of trap designs (Allan andKline 2004, Braks and Carde 2007, Irish et al.2013) and oviposition attractants (Reisen andMeyer 1990, Allan and Kline 2004, Irish et al.2013). The Centers for Disease Control andPrevention (CDC) gravid traps and traps ofsimilar design can effectively collect mosquitoesin the Culex pipiens L./Cx. quinquefasciatus Sayspecies group that are important vectors ofarboviruses in urban environments (Reiter 1983,1987; Cummings 1992; Braks and Carde 2007),but standard gravid traps containing #6 liters ofhay/grass infusion provide variable gravid femaleinterception rates due to interactions amonginfusion volume, trap surface area, and container

color characteristics (Allan and Kline 2004, Irishet al. 2012).

Small-capacity gravid traps often fail to attractthe diverse range of mosquito species capable oftransmitting arboviruses. Despite different bait-ing strategies (Reisen and Meyer 1990, Meyeret al. 2003, DiMenna et al. 2006), importantvectors such as Culex tarsalis Coquillett are rarelycollected using standard gravid trap methodolo-gies, even when substantial populations of host-seeking individuals (and presumably gravidindividuals) are present (Walton and Workman1998). Mosquito oviposition is modulated bya variety of physical as well as chemical factors(Clements 1999). Culex tarsalis is often associatedwith comparatively large, structurally complexhabitats and responds to different habitat cuesthan does Cx. quinquefasciatus, the larvae ofwhich occur often in comparatively structurallysimple and organically enriched environments(Isoe and Millar 1995, Walton et al. 2009).Whereas both mosquito species colonized 1-m2

microcosms, larvae of each species were associat-ed with different water quality and vegetationregimes (Nguyen et al. 1999, Su and Mulla 1999,Duguma and Walton 2014).

Here we report the results of field trialscomparing mosquito collections among 3 designsfor large-volume (.35 liters) gravid traps as wellas for large-volume gravid traps versus a small-volume gravid trap traditionally used in mosquitosurveillance programs. The effects of container

1 Author to whom correspondence should beaddressed. Phone: 951-827-3919; Fax: 951-827-3086;E-mail: william.walton@ucr.edu.

Journal of the American Mosquito Control Association, 32(2):91–102, 2016Copyright E 2016 by The American Mosquito Control Association, Inc.

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architecture, suction trap number and mounting,and infusion volume, age, and concentration ongravid mosquito collection were assessed. Theperformance of each trap design was evaluatedusing the abundance and reproductive status ofadult female mosquitoes in suction trap collec-tions and by the prevalence of egg rafts on theinfusion surface.

MATERIALS AND METHODS

Field site

Gravid traps were tested at the University ofCalifornia at Riverside (UCR) Aquatic andVector Control Research Facility. The study areawas surrounded by eucalyptus trees and citrusgroves. Traps were located in an open, sunlit areawithin the facility. Four 32-m2 wetland areascontaining alkali bulrush (Schoenoplectus [5Scir-pus] maritimus [L.] Lye) were adjacent to thestudy site.

Gravid traps

The CDC gravid trap (Model 1712; John W.Hock Company, Gainesville, FL; Fig. 1) contain-ing 6 liters of oviposition infusion in a light-colored (beige) plastic dishpan (maximum volume5 15 liters, height 5 20 cm, length 5 39 cm,width 5 32 cm) was considered a small-volumedesign. The CDC gravid trap (Reiter 1983), ora modified version of the trap (Cummings 1992),is widely deployed in mosquito surveillanceprograms. A single-suction trap was used witheach gravid trap, except in a subset of studiesduring spring 2013, when 2 suction traps wereplaced over the basin holding the organicinfusion.

Large-volume gravid traps were built using 2types of containers (Fig. 1). Yellow, semitrans-parent fiberglass cylinders (diameter 5 60 cm,height 5 100 cm, maximum volume 5 225 liters)were used during the 1st and 3rd years of thisstudy. A dark-colored, plastic garbage can(RoughneckH; Rubbermaid, Fairlawn, OH; di-ameter 5 56 cm, height 5 72 cm, maximumvolume 5 121 liters) was the 2nd container typetested. The surface area of infusion reservoir inthe large-volume gravid traps was approximately2.3 (fiberglass cylinder with 150 liters of infusion)and 2.0 (plastic garbage can with 100 liters ofinfusion) times larger than the CDC gravid trap.

The number and orientation of suction trapson the large-volume gravid traps differed amongthe experiments. Either single-suction or multi-ple (2 or 3)–suction traps (height 5 44 cm, innerdiameter 5 8 cm) with collection nets fromCDC gravid traps were used in the trials. Whensuction traps were oriented vertically above theinfusion in the large-volume gravid traps, the

wooden rails (length 5 40 cm) holding thesuction trap were mounted on top of 2 woodenfurring strips (1.9 cm 3 3.8 cm 3 61 cm)customized with shallow grooves to fit into thetops of the large-volume containers and placed30 cm apart. When multiple suction traps weredeployed on top of each large-volume gravidtrap, they were placed equidistantly from eachother along the length of the customized rails. Insome experiments, infusion volume in the largegravid traps was reduced to 35 liters, andsuction traps were mounted at approximately60u through container walls to maintain a dis-tance of 10 cm between suction traps and theinfusion surface (Fig. 1). An additional suctiontrap mounting system composed of trapssecured to wooden rails halfway down theinsides of the large plastic containers was tested;however, the air flow (i.e., fan exhaust) withinthe gravid trap presumably caused this design toperform poorly, and it will not be discussedfurther. Each fan motor was powered bya rechargeable 6-V battery and ran continuouslyovernight (average duration 5 18 h). Photocellson the suction traps were disabled.

Infusions

The infusion concentration and incubation timediffered among the experiments. In order to attractspecies such as Cx. tarsalis that prefer ovipositionsites containing less organic enrichment than doesCx. quinquefasciatus (Walton et al. 2009), theorganic infusions used in most of the experimentswere less concentrated than recommended for theCDC gravid trap (,4.3 g hay/liter tap water;http://johnwhock.com/products/mosquito-sandfly-traps/cdc-gravid-trap/): 0.1 g of alfalfa rabbitpellets (NutriphaseH; Pacific Coast Distributing,Inc., Phoenix, AZ)/liter of water was used tomake low-concentration (LC) infusions. A high-concentration (HC) infusion used 1.0 g of alfalfarabbit pellets/liter of water. Infusions using 2 g or4 g of rabbit pellets/liter of water also were testedin some experiments. Infusions were covered andfermented for 1 to 4 wk in the large-volumegravid traps at the study site.

Large-volume fiberglass gravid traps

Two series of experiments tested performanceof the large-volume fiberglass gravid traps. In the1st series of 3 experiments, 8 large-volumefiberglass gravid traps were equally spaced acrossa 5 m 3 2 m concrete slab during September–October 2011. Two treatments were randomlyassigned to the gravid traps in each experiment.The 1st experiment compared the number ofmosquitoes collected by a single-suction trapabove 2 LC volumes: 50 or 150 liters. The 2ndand 3rd experiments compared the efficacy of 1

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or 2 top-mounted suction traps above the 2 LCinfusion volumes (50 vs. 150 liters).

The 2nd series of experiments assessed theeffect of infusion age on the number of mosqui-toes collected by large-volume fiberglass gravidtraps relative to small-volume CDC gravid trapsat 4 sites spaced an average of 30 m apart duringSeptember–October 2011. Low-concentration in-fusions were fermented in covered fiberglassdrums for 1, 2, 3, or 4 wk. Within each site, eachbait–age treatment was assigned randomly tolarge-volume fiberglass gravid traps that wereseparated by 2 m. On the 1st evening of theexperiment, the large-volume gravid traps wereuncovered, and 6 liters of infusion (1.5 liters fromeach age treatment) were transferred to a pairedsmall-volume gravid trap. A single-suction trapwas top-mounted on both large and small gravidtraps.

Large-volume plastic gravid traps

Gravid mosquito collections by large-volumeblack plastic gravid traps containing 100 liters ofLC fermented for 10 days were compared tosmall-volume gravid traps during May–June2012. Two top-mounted suction traps were usedon each large-volume gravid trap. Pairwisecomparisons of mosquito collections by large-and small-volume gravid traps at 5 replicate siteswere made.

The impact of infusion and trap characteristicson trapping efficacy for the 3 gravid trap designswas examined in experiments carried out betweenMay and October 2013. The 1st series of experi-ments compared mosquito collections by large-volume gravid traps using different numbers ofsuction traps (1, 2, or 3), infusion volumes (6, 12,

35, 100, and 150 liters), and infusion concentra-tions (0.1, 1, 2, and 4 g rabbit pellets/liter). Becausea limited number of gravid traps was available foreach experiment, a subset of the total treatmentcombinations was examined in each test. Infusionswere aged an average of 9 days. When both largegravid trap designs were deployed simultaneously,an equal volume of infusion (3 liters) was trans-ferred from each large trap design into an adjacentsmall-volume gravid trap.

The presence–absence (to assess the inherentattractiveness of particular gravid trap designconfigurations to resting and egg-laying mosqui-toes) and orientation of suction traps wereexamined during September and October 2013.Egg raft abundance in gravid traps with func-tioning suction traps was compared to gravidtraps without suction traps or with suction trapsthat were present but turned off. Mosquitocollections by the large plastic gravid traps thathad been modified for side-mounted suction trapswere compared to gravid traps with the standardtop-mounted suction traps in a 2nd group ofexperiments. Standard infusion-bait volumeswere used for small (6 liters), large plastic (100liters), and large fiberglass (150 liters) gravidtraps. Thirty-five liters of infusion were used inthe large plastic gravid traps with side-mountedsuction traps to homogenize trap-to-infusionsurface distance (,10 cm). An HC infusionfermented for 9 days was used in these experi-ments.

Trapping sites for the 2012 and 2013 experi-ments were separated by an average of 15 m. Thedifferent gravid traps were placed in a circle(separated by ,2 m) on the aforementionedconcrete slab and at 3 sites northwest of nurserywetlands containing S. maritimus.

Fig. 1. Examples of gravid trap designs tested at the University of California at Riverside Aquatic and VectorControl Research Facility: (a) large plastic gravid trap with top-mounted suction traps; (b) close-up of top-mountedsuction traps; (c) side-mounted suction trap in a large plastic gravid trap; and (d) 3 gravid trap designs: large-volume plastic gravid trap with side-mounted suction traps (left), large-volume fiberglass trap (right), and small-volume Centers for Disease Control and Prevention (CDC) gravid trap (center).

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Sample processing

Adult mosquitoes collected by gravid trapswere transported to the laboratory and frozen at220uC. Specimens were sorted on a chill table bygender and reproductive state (gravid, bloodfed,nongravid, unknown), and to species using theclassification of Meyer and Durso (1998). Re-productive state was not assessed in specimensfrom 2011. Females containing a blood meal andvisible eggs were lumped in a single “gravid”category in 2012, but were placed into 2 distinctcategories in 2013. Egg rafts, when present, werecounted and removed on the morning of trap netcollection.

Statistical analysis

Gravid trap data were pooled across suctiontraps when multiple suction traps were presenton, or in, a gravid trap and across dates fora replicate trap when gravid mosquitoes werecollected across multiple nights during a particularexperiment. Data that met the assumptions ofparametric statistical analyses were log10 (x + 1)transformed, and the significance of differences ingravid mosquito collections among trap designswas analyzed by analysis of variance (ANOVA).A post-hoc Tukey’s honest significant difference(HSD) test was carried out if the ANOVA wassignificant (P , 0.05). Data that did not meet theassumptions of ANOVA were analyzed usinga nonparametric Kruskal–Wallis ANOVA (morethan 2 variables) or Mann–Whitney U-test (2variables). Experiments comparing infusion fer-mentations and seasonal variation in mosquitocollections during 2012 required separation ofsample dates in the statistical analysis; a repeated-measures multivariate ANOVA (MANOVA) wascarried out. Date effects were tested using Pillai’strace statistic (SYSTAT version 9.0; SPSS Inc.,Chicago, IL; SPSS 1998).

To assess the relative importance of thedifferent trap design components on gravidmosquito collection, a standardized effect sizewas calculated using Cohen’s d for pairwisecomparisons (Cohen 1977). The number ofmosquitoes collected nightly was log10 (x + 1)transformed. The standard deviation of thedistribution of difference scores for pairedobservations at each sampling site was used incalculations. Gravid trap size (large vs. small),number of suction traps per gravid trap, suctiontrap mounting (top-mounted suction traps above100 liters of infusion vs. side-mounted suctiontraps above 35 liters of infusion in large plasticgravid traps), infusion bait volume in large gravidtraps (effect of distance between the suction trapand the infusion surface), duration of infusionfermentation (age), and infusion concentrationwere evaluated. Pairwise comparisons of mos-

quito collections were made among all infusionages (older vs. younger) tested on a particulardate. Effect size for 6 trap design components wasbased on the differences in the total number ofCulex females and for each of the 3 Culex speciesin gravid trap collections. A trap component hada significant impact on gravid mosquito collec-tion if the 95% confidence limits for mean effectsize of multiple experiments investigating a par-ticular trap component did not overlap zero.

RESULTS

Large-volume fiberglass gravid traps

The numbers of gravid mosquitoes collected bylarge-volume fiberglass gravid traps averaged 3females per trap-night during autumn 2011 andwere the lowest of all studies (Fig. 2). Despite lownumbers of mosquitoes in collections, large-volume fiberglass gravid traps collected signifi-cantly more mosquitoes than did small-volumegravid traps (LC: total Culex: F1, 59 5 40.41,P , 0.001; Cx. quinquefasciatus: U 5 705.5,P , 0.001) and collected Cx. tarsalis, whereas thesmall-volume gravid traps failed to do so. Veryfew adult female mosquitoes (,0.1 individualsper trap night) were collected, and egg rafts werenot observed in small-volume gravid traps.

The number of mosquitoes collected by large-volume fiberglass gravid traps was affected byinfusion volume, but not by the number ofsuction traps per gravid trap (1 or 2) or byinfusion age (LC fermented for 1, 2, 3, or 4 wk).Large-volume gravid traps with 150 liters ofinfusion collected 3-fold more adult Cx. quinque-fasciatus females (U 5 20.00, P , 0.05) and 5-fold fewer Culex spp. egg rafts than did fiberglassgravid traps with 50 liters of infusion (Fig. 3; U 514.0, P , 0.05). The number of suction traps pergravid trap did not significantly alter the numberof female mosquitoes collected per gravid trap(Mann–Whitney U-tests, P . 0.65); however, thenumber of egg rafts in gravid traps with 1 suctiontrap was 5-fold greater compared to fiberglassgravid traps with 2 suction traps (U 5 48.0,P , 0.05). Infusion age did not significantlyinfluence adult mosquito or egg raft collections(infusion age: F3, 59 , 0.03, P . 0.8; trap type 3infusion age: F3, 59 , 0.1, P . 0.98).

Large-volume plastic gravid traps

Trap size: Mosquitoes were at intermediateabundance during spring 2012 compared to otheryears, and large-volume gravid traps collectedapproximately an order of magnitude moremosquitoes than did small-volume gravid trapsusing LC as the oviposition attractant (Fig. 2;F1, 8 5 97.34, P , 0.001). The large-volume,dual-suction trap design collected at least an

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order of magnitude more Cx. quinquefasciatus(F1, 8 5 111.9, P , 0.001) and Cx. tarsalis (U 5

293.5, P 5 0.002) females than did the small-volume, single-suction trap design.

Culex quinquefasciatus was most abundant ingravid trap collections (86% of total), followed byCx. tarsalis (7%), Cx. stigmatosoma Dyar (4%),and unidentified damaged specimens (3%) inexperiments during 2012. About 79% of femalemosquitoes were gravid (included bloodfed speci-mens), 15% were nongravid, and 6% were ofunknown reproductive state (i.e., specimen de-stroyed by the fan; Fig. 4). The percentage of thefemale mosquitoes that was gravid did not differby mosquito species, gravid trap design, site, orsample date (F test or Kruskal–Wallis test,P . 0.10).

Mosquito abundance during 2013 was thehighest of all years in our study, and large-volume gravid traps collected 4-fold greaternumbers of gravid mosquitoes compared tosmall-volume gravid traps (Fig. 2). The relativedifference in collection size between trap typesvaried by sample season, mosquito species, andexperimental modifications.

During spring (Fig. 2), the large-volume plasticgravid trap with dual-suction traps and aninfusion (LC) volume of 100 liters collected atleast 8-fold more adult females of the 3 pre-dominant Culex species compared to the small-volume gravid traps (F1, 18 5 35.21, P , 0.001).The difference in the number of Cx. tarsaliscollected between the 2 gravid traps was evenmore striking (56-fold; U 5 83.00, P , 0.005).Later in the year, the large-volume gravid trapswith dual-suction traps collected 2- to 3.6-foldmore Cx. quinquefasciatus (summer: F1, 11 5 6.03,P , 0.035; autumn: U 5 80.0, P 5 0.013) and Cx.stigmatosoma (autumn: U 5 80.5, P 5 0.011)females compared to the small-volume CDCtraps. Culex tarsalis abundance was comparative-ly low and did not differ between trap designsduring summer and autumn (Mann–Whitney U-tests, P . 0.30).

Egg rafts were collected from large-volumegravid traps in all trials. Egg rafts were present inthe small-volume gravid traps only during springtrials, but they were 40-fold fewer than in thelarge-volume gravid traps (U 5 89.0, P , 0.001).

Number of top-mounted suction traps

The total number of Culex females collectedper gravid trap was not influenced by the numberof suction traps per large-volume gravid trap(1 vs. 2 suction traps: U 5 24.0, P . 0.6). Large-volume gravid traps with a single-suction trap,however, collected 3-fold more egg rafts thandid the dual-suction trap design (U 5 56.0,P , 0.001).

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Infusion concentration

Adult female numbers in traps above HC bait(2 g/liter) were 2- to 4-fold more abundant than intraps above LC bait (0.1 g/liter), and thisdifferential was significant for Cx. quinque-fasciatus and Cx. stigmatosoma (Mann–WhitneyU-tests, P , 0.01). Egg raft numbers in gravidtraps were not related to infusion concentration(Mann–Whitney U, P 5 0.665)

Infusion volume

Large-volume and small-volume traps withsingle-suction traps placed over 6 liters of in-fusion trapped similar adult numbers (F1, 17 5

1.29, P . 0.27), even though egg rafts wereabundant in the large-volume gravid traps andwere absent in the small-volume gravid traps(U 5 90.0, P , 0.001). During autumn 2013, thelarge-volume plastic gravid trap with a top-mounted suction trap (100 liters of HC infusion)was the most effective mosquito collector of 5gravid trap designs tested (all top-mountedsuction traps: large-volume plastic trap with 12or 35 liters of infusion, large-volume fiberglasstraps with 150 liters of infusion, CDC gravidtraps) and averaged 2- to 6-fold more adultfemale mosquitoes than the small-volume gravidtraps (F4, 55 5 21.94, P , 0.001). Significantlygreater numbers of each of the 3 Culex specieswere collected by the large-volume traps ascompared to the small-volume gravid trap (Cx.

quinquefasciatus: F4, 55 5 23.20, P , 0.001; Cx.tarsalis: H4 5 12.8, P 5 0.012; Cx. stigmatosoma:H4 5 25.7, P , 0.001).

Egg raft abundance in large-volume gravidtraps was overall higher than in small-volumegravid traps during 2013 (Fig. 3). Large-volumeplastic gravid traps with 35 liters of infusion andtop-mounted suction traps contained 4-fold moreegg rafts compared to the large-volume gravidtrap design with the next highest number ofegg rafts (100 liters of infusion; H4 5 32.67,P , 0.01).

Inherent structural attraction

When suction traps were absent, large-volumeplastic gravid traps containing 35 liters of in-fusion also collected the most egg rafts (ANOVA,P , 0.05; Fig. 5); however, gravid traps withnonoperational suction traps (top-mounted) over35 liters of infusion collected similar numbers ofegg rafts compared to gravid traps holding 100liters of infusion (Tukey’s HSD, P . 0.05).Nonoperational side-mounted suction traps didnot significantly alter oviposition (data notshown).

Suction trap mounting

Side-mounted suction traps above 35 liters ofinfusion and top-mounted traps above 100 liters ofinfusion captured similar quantities of adult mos-quitoes in experiments during October–November

Fig. 3. Abundance (mean 6 SE) of female mosquitoes and egg rafts per sample night among years, infusionvolumes, and gravid trap designs. Sm 5 small-volume gravid traps. Lg 5 large-volume plastic gravid traps withtop-mounted suction traps. Lg-S 5 large-volume plastic gravid traps with side-mounted suction traps. FG 5 large-volume fiberglass gravid traps. Letters indicate significant differences of gravid mosquito collections: Capital lettersindicate Kruskal–Wallis analysis of variance (ANOVA) P , 0.05, and lowercase letters indicate Tukey’s honestsignificant difference (HSD) test P , 0.05.

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2013 (Fig. 3). The numbers of total Culexfemales, Cx. quinquefasciatus, and Cx. stigmato-soma females in both large-volume trap designsexceeded the small-volume traps by more than7-fold (total Culex: F2, 15 5 28.94, P , 0.001; Cx.quinquefasciatus: F2, 15 5 30.91, P , 0.001; Cx.stigmatosoma: H2 5 12.94, P , 0.01); however,the abundance of Cx. tarsalis did not differstatistically among the 3 trap designs (H2 5 2.38,P . 0.30). The number of side-mounted suctiontraps (1, 2, or 4) per container did not affectthe numbers of adult mosquitoes or egg raftscollected per gravid trap (U-tests, P . 0.13).Culex quinquefasciatus was the predominantspecies (78% of total), with lesser abundanceof Cx. stigmatosoma (9%), Cx. tarsalis (7%),Culiseta spp. (2%: Cs. inornata [Williston] and Cs.incidens [Thomson]), and unidentified damagedspecimens (4%).

Side-mounted suction traps reduced the num-ber of egg rafts in the gravid traps by more thanhalf (H2 5 10.88, P 5 0.004) compared to thetop-mounted suction traps on the large-volumegravid trap. Gravid traps with .2 suction trapswere nearly devoid of egg rafts, while gravid trapswith 1 suction trap contained an average of 2 eggrafts per night. Egg rafts were absent in the small-volume gravid traps during this series of experi-ments.

Side-mounted suction traps collected twiceas many adult males compared to the trap designwith top-mounted suction traps (ANOVA:F2, 78 5 93.76, P , 0.001; Tukey HSD: P 50.026). Males were rare in small-volume gravidtraps.

Reproductive state of females

During 2013, the reproductive profile oftrapped female mosquitoes was 56% gravid,10% with recent blood meal, 15% nongravid,and 19% of unknown reproductive status. Ingeneral, small- and large-volume (.6 liters ofinfusion) gravid traps collected similar propor-tions of gravid and bloodfed Culex mosquitoes(Fig. 4) and the dominant species, Cx. quinque-fasciatus (Mann–Whitney U-tests, P . 0.40);however, the proportions of gravid and blood-fedCx. stigmatosoma and Cx. tarsalis in collectionsfrom the large-volume gravid traps were between2- and 4-fold higher compared to small-volumegravid traps (Mann–Whitney U-tests, P , 0.02).Interestingly, the proportions of gravid plusbloodfed Cx. quinquefasciatus females collectedby large-volume gravid traps declined markedlywhen the infusion volume was 6 liters (Fig. 4;40% vs. 73%; Mann–Whitney U-tests, P , 0.01).Regardless of infusion volume, the numbers ofnongravid Culex mosquitoes, as well as each ofthe 3 Culex species, were 2 to 3 times higher inlarge-volume gravid traps compared to the small-volume traps (Mann–Whitney U-tests, P , 0.02).

The number and position of suction trapsaffected the distribution of Culex females amongthe reproductive status categories collected bylarge-volume gravid traps. The large-volumegravid trap with a 100-liter (LC) infusion anda single top-mounted suction trap collectedproportionally fewer gravid Cx. quinquefasciatusand a greater abundance of nongravid mosqui-toes compared to the same trap with 2 suction

Fig. 4. Percentages (mean 6 SE) of gravid, nongravid, and unknown fecundity for female mosquitoes in gravidtrap collections from 2012 and 2013. Gravid numbers included females with visible eggs or blood meals. Sm 5small-volume gravid traps. Lg 5 large-volume plastic gravid traps with top-mounted suction traps. Lg-S 5 large-volume plastic gravid traps with side-mounted suction traps. FG 5 large-volume fiberglass gravid traps.

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traps (Mann–Whitney U-tests, P , 0.03). Theopposite trend was found for large-volume gravidtraps with 35-liter infusions and side-mountedsuction traps. Single side-mounted suction trapscollected larger numbers of gravid Cx. quinque-fasciatus and smaller numbers of nongravidmosquitoes than did traps with 2 side-mountedsuction traps (Mann-Whitney U, P , 0.05). Theproportions of mosquitoes in the categories ofreproductive status did not differ significantlywhen either 1 or 2 suction traps were used onsmall-volume gravid traps (Mann–Whitney U, P. 0.40). Infusion concentration did not affect theproportions of gravid and nongravid mosquitoesin large-volume gravid traps with top-mountedsuction traps (Mann–Whitney U, P . 0.10).

Relative importance of gravid trap components

Large-volume gravid traps collected moreCulex mosquitoes, and more of each of the 3Culex species, than did small-volume gravid trapsacross experiments during the 3 years of in-vestigation, with effect size ranging between 1.00and 2.45 (Table 1: Size). Infusion volume in thelarge-volume gravid traps had the largest effecton the total number of Culex mosquitoes incollections and relates to the distance between thesuction trap and the infusion surface. Suctiontraps positioned approximately 10 cm above theinfusion surface collected more gravid mosqui-toes than when the distance between the infusionsurface and the opening to the suction trap was.10 cm. Collections of total Culex females for

the 2 best gravid trap designs (top-mountedsuction traps above 100 liters of infusion vs.side-mounted suction traps above 35 liters ofinfusion) did not differ; although top-mountedsuction traps collected markedly more Cx.stigmatosoma mosquitoes (Table 1: Suction trapmounting). Averaging effect size across multipleexperiments, large gravid traps with 1 suctiontrap collected more Culex mosquitoes than didgravid traps with multiple suction traps, but theseeffects were based on comparatively few experi-ments (Table 1).

Comparatively concentrated infusions (2 g/litervs. 0.1 g/liter) strongly attracted Cx. stigmato-soma. A large effect of infusion concentration wasalso observed for Cx. quinquefasciatus; however,despite a response of gravid females that differedfrom random (black plastic gravid trap infusionconcentration: Mann–Whitney U), the 95% con-fidence limits of effect size barely overlapped 0.

Gravid trap size was the most important trapdesign component influencing the size of Cx.tarsalis and Cx. quinquefasciatus collections. ForCx. stigmatosoma, organic matter in the infusionand top-mounted suction traps on large-volumegravid traps had larger effects on mosquitocollections than did gravid trap size, whichnevertheless was highly significant. The numbersof Culex (species combined), Cx. tarsalis, and Cx.stigmatosoma females collected above youngerinfusions were larger than above comparativelyolder infusions, but these effects were weakcompared to gravid trap size.

Fig. 5. Abundance (mean 6 SD) of egg rafts and/or female mosquitoes during autumn 2013 when suction trapswere absent (left panel), were present and turned off (middle panel), and were turned on (right panel) withindifferent gravid traps and infusion volumes. Sm 5 small-volume gravid trap. Lg 5 large-volume plastic gravid trap.FG 5 fiberglass gravid trap. Letters indicate significant differences of gravid mosquito collections: lowercase (eggrafts) and capital (adults) letters indicate Tukey’s honest significant difference (HSD) test P , 0.05.

98 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 32, NO. 2

DISCUSSION

Large-volume gravid traps collected on average6.6-fold (based on back-transformed means)more adult female Culex mosquitoes comparedto small-volume CDC gravid traps during the 3years of these studies. Infusion container size andinfusion volume within large-volume gravid trapswere significantly associated with changes in thesize and species composition of mosquito collec-tions. Twice as many Cx. quinquefasciatus andCx. stigmatosoma females were collected abovecomparatively concentrated infusions (2 g vs. 0.1g rabbit pellets/liter). Suction trap number andmounting style, and infusion age, contributed lessto the observed differences in mosquito collec-tions between gravid trap designs.

Among the large-volume trap design compo-nent combinations tested, 2 black-plastic con-tainer designs, each with a different suction trapmounting style and infusion volume, were mosteffective at gravid mosquito monitoring. Top-mounted suction traps and 100 liters of infusionprovided the greatest yields for the combinedabundance of all mosquito species as well asgravid Cx. tarsalis and Cx. stigmatosoma females.This gravid trap configuration was the mosteffective at collecting mosquitoes across an order-of-magnitude increase in mosquito abundancebetween 2012 and 2013. The differences in thenumbers and species of mosquitoes collected bylarge-volume gravid traps and CDC gravid trapswere more pronounced in the spring compared tosummer and autumn. This evidence suggestslarge-volume gravid traps filled with infusionvolumes .6 liters have potential to enhancegravid mosquito collection and the detection ofinfected mosquitoes early in the annual activityperiod of vector mosquitoes. Early-season detec-tions of arbovirus-infected mosquitoes can aid inthe prediction of late-season severity of diseasessuch as those caused by West Nile virus (Gins-berg et al. 2010).

Black-plastic gravid traps with side-mountedsuction traps over 35 liters of infusion and withtop-mounted suction traps over 100 liters ofinfusion collected similar numbers of gravidCulex females. One likely reason for this resultwas that the side-mounted suction traps werepositioned at the same distance (about 10 cm)from the surface of the infusion as were the top-mounted traps placed above the surface of the100-liter infusion. Suction trap to bait surfacedistance was a critical factor influencing adultmosquito catches. When infusion volume wasreduced (,100 liters), gravid traps with top-mounted suction traps collected fewer gravidmosquitoes, a larger proportion of nongravidfemales, and more egg rafts. Increasing thedistance from the suction trap to the bait surfacereduced the suction force at the egg-layingT

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JUNE 2016 GRAVID MOSQUITO TRAP DESIGN 99

surface and presumably facilitated escape and egglaying.

Large-volume black-plastic gravid traps withintermediate volumes of organic infusion mayhave also created a humid microenvironmentwithin the trap that was favorable for mosquitoresting and egg laying. High humidity is com-monly preferred by resting adult mosquitoes andis associated with increased survival and fecundi-ty (Clements 1963, Yamana and Eltahir 2013).The interior of the large-volume gravid trap wasa region of comparatively high humidity. Apoststudy comparison indicated that the 35-literinfusion formed a 20-cm-tall zone of constantrelative humidity in the infusion container thatwas 10% higher than that measured concurrentlynear the surface of the 100-liter infusion (D.Popko, personal observation). Egg raft numbersin containers (suction traps absent) with 35 litersof infusion were about 10-fold larger compared togravid traps with 100 liters of infusion. A calmmicroenvironment may have further affected theplume signal of trap baits and created an enrichedzone of volatile chemical cues emitted during baitfermentation important in both long- and short-range mosquito oviposition behaviors (Isoe andMillar 1995, Cooperband and Carde 2006).

The number of suction traps per large-volumegravid trap was not directly related to the totalmosquito numbers in collections, such that totalmosquito catches by 2 suction traps regardless ofcontainer type and mounting design did notexceed that by a single-suction trap. However,suction trap number was inversely related to thenumbers of egg rafts on the infusion surface andthe relative abundance of nongravid mosquitoesin collections from the large-volume gravid traps.For gravid traps with top-mounted suction trapsand 100 liters of infusion, 2 suction trapsdecreased egg rafts and the proportion ofnongravid female mosquitoes, especially Cx.quinquefasciatus, in collections compared to 1suction trap. Dual-suction traps were probablymore efficient than a single-suction trap atintercepting gravid mosquitoes before oviposi-tion. Within particular experiments, suction trapnumber, however, was not linked to differencesamong collections of gravid Cx. tarsalis and Cx.stigmatosoma females. Yet, considering the effectsize for suction trap number based on a smallnumber of comparisons across the entire study,a single-suction trap appeared to perform betterthan did multiple suction traps per large-volumegravid trap for collecting gravid and nongravidCx. quinquefasciatus and Cx. stigmatosoma mos-quitoes. Further studies are needed.

To purposely target Cx. tarsalis, the infusionconcentration used for our tests was typicallylower than the 4 g/liter of organic matter/literrecommended for CDC-style traps; however, lessconcentrated baits were not more attractive, nor

did strong baits (#2 g/liter of organic matter)inhibit Cx. tarsalis collection. A possible conse-quence of using LC was to reduce the numbers ofdirty-water species such as Cx. quinquefasciatusand Cx. stigmatosoma, which responded toinfusion composition more readily than did Cx.tarsalis, as evident in this and other studies(Reisen and Meyer 1990, Rodcharoen et al.1997). Interestingly, the large-volume gravid trapwith the least concentrated bait (0.1 g/liter)collected 10-fold more Cx. quinquefasciatus mos-quitoes than did the CDC gravid traps with themost concentrated (4 g/liter) infusion that wastested. This trend was consistent with thedifference of gravid mosquito collections betweenlarge- and small-volume gravid traps and sug-gested infusion concentration played a relativelyminor role among different gravid trap designsunder field conditions.

Bait fermentation age was not a significantfactor in the performance of fiberglass large-volume or small-volume gravid traps in 2011.This finding was unexpected considering additionof organic matter changed the abundance andspecies composition of Cx. quinquefasciatus, Cx.stigmatosoma, and Cx. tarsalis collections indevelopmental sites over the course of severalweeks (Beehler and Mulla 1995, Su and Mulla1999, Jiannino and Walton 2004, Walton et al.2009). Transient peaks in oviposition of theaforementioned studies were in open-water sys-tems with extensive top-down (e.g., consumptionby larval mosquitoes) and/or bottom-up (e.g.,reduction of nutrients across time) regulation ofmicrobial growth that could have altered thechemical signature of oviposition sites. The lackof a transition in mosquito abundance and/orspecies in our experiments may have been relatedto the predominately sealed, semitransparentnature of the fiberglass containers. Dense algalmats thrived (D. Popko, field observations) in thesunlight-exposed environment devoid of filter-feeding invertebrates such as mosquitoes. Theproximity of different treatments in each replicateblock of the experiment also may have interactedto reduce differences in the volatile signals relatedto bait age. Last, the overall low levels ofmosquito populations during autumn 2011 couldhave precluded the detection of bait age effects.Across the 3 years of our study, infusion age hada small to moderate effect on the number ofmosquitoes collected by gravid traps: Cx. tarsalisand Cx. stigmatosoma collections were larger overcomparatively younger infusions. Age-based dif-ferences in the performance profile of baits inlarge-volume gravid traps should be examinedfurther.

Color and water surface area were othervariables that may have altered the effectivenessof the gravid traps to collect different mosquitospecies. Gravid Cx. quinquefasciatus females are

100 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 32, NO. 2

known to prefer dark-colored bait to light-colored bait in the laboratory (Beehler et al.1993), and dark-colored habitat has been associ-ated with increased mosquito numbers in fieldmesocosms (Belton 1967). O’Meara et al. (1989)found that Cx. nigripalpus Theobald laid moreegg rafts in 0.25-m2 vats than in smaller jars (1-quart glass Mason jar; ,0.003 m2), whereas Cx.quinquefasciatus females preferred to lay eggs inthe jars. Allan and Kline (2004) reported a 3-foldincrease in Cx. quinquefasciatus adult abundancein dark-colored gravid traps with twice thesurface area and volume than light-colored traps,but they found no differences in the numbers ofmosquitoes collected by gravid traps with dark-colored (black or green) versus beige infusioncontainers. Irish et al. (2012) demonstrated nochange in Cx. quinquefasciatus adult numbers intraps of the same color and surface area thatdiffered 2-fold in volume. However, Lewis et al.(1974) found that gravid traps with a largerinfusion volume collected more Cx. quinquefas-ciatus mosquitoes than did traps with less in-fusion. In our study, the differences in color andsurface area between large-volume plastic andsmall-volume gravid traps were expected to berelated to differences in gravid mosquito collec-tions between designs; however, adult mosquitoabundance was similar when bait volume was thesame (6 liters) in both types of gravid traps. Theproportion of nongravid females and egg raftnumbers differed between the 2 gravid trapdesigns. Also surprising was that light-coloredfiberglass cylinders performed poorly comparedto the maximum-volume large plastic designdespite a 1/3 greater volume and nearly 20%greater surface area.

The gravid trap with side-mounted suctiontraps and 35 liters of infusion could be particu-larly useful for vector control monitoring pro-grams because it would be a more practicaloption than the gravid trap using top-mountedsuction traps and 100 liters of infusion, especiallyat sites where water supply is limited. The smallerinfusion volume was easier to store, transport,and manipulate than the larger infusion volume.Side-facing suction traps were more secure thanthe top-mounted suction traps.

In conclusion, large-volume gravid traps offera promising alternative to gravid traps usedtraditionally for mosquito-borne disease surveil-lance. The small-volume CDC gravid traps aremore portable and practical than are the large-volume gravid traps for collecting species such asCx. pipiens and Cx. quinquefasciatus. The large-volume gravid traps collect more gravid mosqui-toes and, at times during the annual activityperiod of Culex adults, collect significant num-bers of principal arbovirus vectors such as Cx.tarsalis, especially during the spring when mos-quito populations and arbovirus populations are

increasing. The effect of gravid trap size on Cx.tarsalis collections is even greater than indicatedby our study because there were sampling dateswhen Cx. tarsalis was collected by the large-volume gravid traps but not by the small gravidtraps; consequently, effect size could not becalculated for these sampling dates. The plasticgarbage cans are more portable than in-groundoviposition pools (Surgeoner and Helson 1978)and are relatively inexpensive and easier tomanage (i.e., stackable) than are the fiberglasscontainers. Box traps (Cummings 1992, Braksand Carde 2007) could be fitted across the top ofthe infusion reservoir to reduce the damage tomosquitoes by the fan blade. The disadvantagesof using large infusion volumes might be offsetby using infusions for multiple-week deploymentsand covering the infusion basin between gravidmosquito collections. Whether the benefits ofthe enhanced collections of gravid mosquitoesfor arbovirus surveillance provide a significantimprovement in early warning for disease out-breaks and outweigh the disadvantages of largeinfusion volumes and costs (capital and labor)associated with large-volume traps remains to beevaluated.

ACKNOWLEDGMENTS

This research was supported by the CoachellaValley Mosquito and Vector Control District, theMosquito Research Foundation, the CaliforniaDepartment of Transportation (Caltrans), andthe Agricultural Experiment Station at Universityof California at Riverside. This work wassupported by the US Department of AgricultureNational Institute of Food and Agriculture,Hatch project 1007869. N. McConnell providedhelpful comments on an earlier draft of thispaper.

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