2013 NORTHEASTERN NATURALIST 20(2):363–371 Color and Container Size Affect Mosquito (Aedes triseriatus) Oviposition Gary Joseph Torrisi1,2,* and W. Wyatt Hoback3 Abstract - This investigation compared color and artificial container size as attractants for the gravid female Aedes triseriatus (Eastern Tree-hole Mosquito) oviposition site selection. Three colors (white, green, and black) were investigated in combination with two container sizes (3672 ml and 12,000 ml). Presence of mosquito larvae was used to determine percent occupancy of containers. Black containers were selected in 70% of trials, compared to 63% for green containers and 41% for white containers. Large containers were selected more frequently (72% of trials) than small containers (44% of trials). Results suggest that container size may serve as an important selective cue for A. triseriatus and may take precedence over color when gravid female mosquitoes are given the choice. Our results provide further insight into the complexity of cues that influence mosquito oviposition behavior. Introduction Natural tree-hole habitats serve as ovipositional sites for gravid female mosquitoes. These habitats are common in the tropics and are also found in temperate climates although these habitats are not as varied or abundant as those found in the tropics (Kitching 1971). Artificial tree-holes created using plastic containers infused with leaf litter and rainwater attract many of the same mosquito species found in natural tree-holes including Aedes triseriatus (Say) (Eastern Tree-hole Mosquito), which we examine in this paper (Yanoviak 2001). Extensive research suggests that color, primarily dark, light-absorptive backgrounds of black or brown that are similar to the linings of natural treehole cavities serve as the main attractant for oviposition site selection for most mosquitoes (Collins and Blackwell 2000, Frank 1986, Jones and Schreiber 1994, Yanoviak 2001). There are some exceptions where lighter-colored tropical bromeliads are attractants to ovipositing Wyeomyia mitchelli (Theobald) and Wyeomyia vanduzeei Dyar and Knab. In addition, pitcher plants found in sphagnum bogs associated with temperate forests are attractive to Wyeomia smithii Coquillett (Pitcher Plant Mosquito). Frank (1985) demonstrated that Pitcher Plant Mosquito response to color differs dramatically from other mosquitoes in their selection of light-colored oviposition sites. Other cues in addition to color are used by gravid female mosquitoes in selecting sites for the deposition of eggs. Past studies have tested variables including: location of tree holes in illuminated gaps versus darker understory ¹Department of Entomology, University of Nebraska at Lincoln, Lincoln, NE 68508. 2Current address - 6 Jennifer Court, Saratoga Springs, NY 12866. 3Department of Biology, Bruner Hall of Science, 905 West 25th Street, University of Nebraska at Kearney, Kearney, NE 68849. *Corresponding author - gjtorrisi@aol.com. 364 Northeastern Naturalist Vol. 20, No. 2 cover (Yanoviak 2001), vertical distribution of tree holes (Jones and Schreiber 1994), form and color of containers (Frank 1986), leaf-litter quality and stability of habitats (Minakawa et al. 2005, Yanoviak 1999), light intensity (Rodriquez-Tovar et al. 2000), and the role of conspecifics as attractants and interspecific competitors as deterrents (Braks et al. 2004, Onyabe and Roitberg 1997, Yee et al. 2004). In addition, investigations have examined predation (Etiam and Blaustein 2004), the influence of preexisting eggs (Allan and Kline 1998), precipitation and temperature (Alto and Juliano 2001), size and surface area of habitats (Lester and Pike 2003, Minakawa et al. 2005) and crowding, confinement, starvation, and infection (Zahiri and Rau 1998). These studies point to the variety of variables that influence mosquito oviposition behavior. Approximately seventy recognized mosquito species reside in New York State including many species that serve as vectors of disease (NYSHD 2012). Examples of emergence of vector-borne disease include the 1999 outbreak of West Nile virus (WNV) in New York City leading to a subsequent epidemic throughout the United States (Hayes and Gubler 2006). Furthermore, epidemiological data from the Center for Disease Control (CDC) report the emergence of dengue fever (CDC 2010a) in the Americas since the 1950s and significant under reporting and under diagnosis of LaCrosse encephalitis (CDC 2010b) especially in the southeastern United States. In addition, there has been an increase in the incidence of eastern equine encephalitis (EEE) and WNV in horses in the northeastern United States (Kurt Lutgens, Doctor of Veterinary Medicine, Saratoga Springs, NY, pers. comm.). Discovery of mosquito larvae in large, white vessels exposed to direct sunlight (G.J. Torrisi, pers. observ.) seemed to be at odds with published results on mosquito habitat selection. Based on these observations, we tested whether the combination of color and container size serve as attractants for gravid mosquitoes. We hypothesized that if both color and container size are cues for oviposition site selection by gravid female mosquitoes, then large containers would be preferred regardless of color. Field Site Description This investigation took place in upstate New York, in the town of Wilton (43°18'N, 73°74'W), in a suburban housing development surrounded by parcels of patchy temperate forest. The dominant trees of the area are Quercus rubra L. (Red Oak), Acer rubrum L. (Red Maple), and Pinus strobes L. (White Pine). This investigation was conducted from mid-June to mid-August in 2007, 2010, and 2011. Methods Decaying leaf litter of oak and maple was removed from the forest floor and cut into 1–2 cm2 sections. An equal volume of fresh leaves was stripped from the branches of the dominant species of trees and cut into same-sized sections and mixed with the decaying litter. The leaf litter was then covered in rainwater 2013 G.J. Torrisi and W.W. Hoback 365 collected on site and the mixture was allowed to stand for three days to promote algal and microinvertebrate colony formations that would initially serve as the basic nutrients for mosquito larvae. Nine small, plastic, cylindrical containers measuring 12 x 17 x 18 cm and nine large, plastic, cylindrical containers measuring 20 x 24 x 25 cm served as replicates of artificial habitats. Three small and three large containers were left white or painted green or black (Rust-Oleum® hunter green and flat black) for six container color by size combinations per replication. Eighteen containers, three replications, were used in each of three trials per year (n = 54/year). Thus, across all three years, each container color and size combination was made available 27 times for ovipositing mosquitoes. Because the time of the year affected abiotic factors including temperature and rainfall and because weather patterns across years were different and likely to affect mosquito activity, percent occupancy of container type was calculated by year (n = 3). Holes were drilled into the sides of the containers half way up from the bottom to prevent weight overload and the potential for detachment of the containers from trees caused by heavy rainstorms or high winds. A water-repellant nylon cord was used to attach each container by its handle to a tree at a height of 1 m above the forest floor. Containers were placed no closer than 5 m and no further than 10 m from one another. Containers were affixed to oak and maple trees with a diameter greater than 20 cm. The leaf-litter mixture was placed in the bottom of the containers covering one-half of the bottom surface area. Additional rainwater was then added to the level of the pre-drilled holes. When rainwater was unavailable, untreated water from a well point was substituted. We inspected containers every 3–5 days to insure proper attachment of the artificial containers, to replace water loss as needed, and to assess whether earlystage mosquito larvae were present. Detection of any mosquito larvae resulted in the container being designated as selected by a gravid female mosquito, whereas absence of larvae resulted in the container being designated as not selected. The number of larvae was not quantified or compared. Detection of early-stage larvae, in any one container, initiated 21 consecutive days of observation, record keeping, and sampling of larvae. All containers were monitored during the 21-day period. A flashlight was used to search the bottom of the darker containers for wrigglers. Individual late-stage larvae were removed during each trial to identify species. Larvae were transferred to a 10-ml vial, labeled with date, container color, and container size. Captures were then stored in water and refrigerated. Samples were examined under magnification (400x) to identify species. In addition, samples of late-stage larvae were reared to adult and identified. At the conclusion of each investigation, all containers were emptied, cleaned, and reset with leaf litter and water in the manner described above. Results All collected larvae were identified as Aedes triseriatus. Across all trials and years, 94 containers out of a possible 162 (58%) were selected by gravid females. 366 Northeastern Naturalist Vol. 20, No. 2 Percent occupancy by container size was compared using a Student’s t-test (n = 9). Small containers were selected 44% of the time, while large containers were selected 72% of the time (Fig. 1), which was significantly more often (twotailed, unpaired t-test: t = 3.5, df = 8, P = 0.008). Percent occupancy by container color was compared using an analysis of variance (n = 18). Black containers were utilized significantly more frequently (Fig. 1) than white containers, while other comparisons were not significant (ANOVA: F =3.8, df = 17, P = 0.047). Across all three years (n = 3), mosquitoes oviposited most frequently in the large green (88% occupancy) and large black (78% occupancy) containers (Fig. 2). Small white and small green containers were occupied least frequently (33% and 37%, respectively). Of the 15 possible comparisons of occupancy between container color-size combinations (Table 1), 9 comparisons were significantly different (ANOVA: F = 18.3, df = 17, P less than 0.001). Discussion Container size plays a significant role within the mix of complex behaviors associated with mosquito oviposition site selection. Our finding that darker colors influence oviposition by Eastern Tree-hole Mosquitoes is similar to the findings of Collins and Blackwell (2000). Finding mosquito larvae in larger containers suggests that container size also plays a role in female mosquito oviposition choices. Successful oviposition in a range of container sizes and colors does not mean that all larvae would be successful. During this study, observation of late Figure 1. Mean (± 1 S.E.) percent occupancy of Aedes triseriatus (Eastern Tree-hole Mosquito) larvae by size and color of artificial containers over a three-year period. 2013 G.J. Torrisi and W.W. Hoback 367 instars and pupae were limited because of the short duration of each trial. Once the presence of larvae was observed, the trials were terminated within 3 weeks. However, we continued to rear late-stage larvae to adult stage in the laboratory Figure 2. Mean (± 1 S.E.) percent occupancy of Aedes triseriatus (Eastern Tree-hole Mosquito) larvae by color and size of artificial containers over a three-year period. S = small, L = large, w = white, g = green, and b = black. Table 1. Differences in percent of containers occupied between color-size combinations. P-values are based on Tukey-Kramer multiple comparison test. Container size: S = small, L = large; container color: w = white, g = green, b = black Comparison Difference of means P-value Lg - Sw 0.556 less than 0.001 Lg - Sg 0.519 less than 0.001 Lg - Lw 0.407 0.002 Lg - Sb 0.259 0.040 Lg - Lb 0.111 0.671 Lb - Sw 0.444 less than 0.001 Lb - Sg 0.407 0.002 Lb - Lw 0.296 0.017 Lb - Sb 0.148 0.396 Sb - Sw 0.296 0.017 Sb - Sg 0.259 0.040 Sb - Lw 0.148 0.396 Lw - Sw 0.148 0.396 Lw - Sg 0.111 0.671 Sg - Sw 0.037 0.995 368 Northeastern Naturalist Vol. 20, No. 2 for purposes of identification. In the field, on occasion, adult mosquitoes were observed visiting the containers. This observation suggests that female mosquitoes may have been appraising the available cues for oviposition selection (Lester and Pike 2003). Although initially these artificial habitats were sufficient to cause oviposition by gravid females, factors that contribute to larval development may have been lacking. It follows that longer trial durations might have led to all containers being selected after the more preferred large, dark containers were colonized. Further investigation over a longer time span would provide insight into the effects of congeners on ovipositing mosquitoes where ample containers are available for colonization. Past investigations suggest that deterrents to oviposition may include larval predators, nutrient quality, competition for nutrients, presence of conspecific eggs or larvae, competition from an alternative species having colonized the containers, or confinement of movement for existing larvae (Allan and Kline 1998, Braks et al. 2004, Etiam and Blaustein 2004, Jones and Schrieber 1994, Onyabe and Rottberg 1997, Yanoviak 1999, Zahiri and Rau 1998). Many of these factors may serve as barriers in the process of oviposition selection by gravid females. However, no predators or evidence of larval predation were observed during this investigation. As reported by Etiam and Blaustein (2004), there is a noticeable response of prey to risk of predation including behavioral changes such as reduced oviposition. This response would presume the existence of predators prior to selection and subsequent rejection of a given container because of detection of predator cues. However, low predator density during the onset of selection may not be detectable by the ovipositing mosquitoes (Eitam and Blaustein 2004). This scenario seems more likely because of the large number of containers selected for egg deposition in this investigation. In addition, samples from each trial indicated the same species was present in all containers and there was no evidence of an alternate mosquito species being present in any of the containers. In one sampling, we observed early instars along with late instars of Eastern Tree-hole Mosquitoes in the same container. Gravid female mosquitoes should select containers, artificial or natural, that provide for the success of their offspring. However, previous studies have shown that the size of the container does not necessarily correlate with successful development of mosquito larvae. For example, predators of mosquitoes are less likely to be found in small containers that serve as temporary habitats that are under the threat of drying out. Larger containers serve as a more permanent habitat and are more likely to contain mosquito predators that would reduce the abundance of the population within the container (Service 1977, Sunahara et al. 2002). Consequently, when selecting a suitable habitat, smaller containers are subject to habitat instability in terms of the loss of water, where large containers hold greater volumes of water and therefore provide a more stable habitat for female mosquitoes but may also be subject to greater predation risk (Minakawa et al. 2005). Lester and Pike (2003) concluded that container size alone may influence female mosquito oviposition site selection regardless of predator occupation of container habitats. 2013 G.J. Torrisi and W.W. Hoback 369 Elevation and container location might also influence which species selected the experimental containers. Placement of containers at 1 m above the forest floor may have attracted only the Eastern Tree-hole Mosquito. Future consideration must be given to ground-flying species and species that prefer tree-holes above 1 m from the ground. During our study, some containers had exposure to greater periods of sunlight because of their location closer to tree-fall gaps, while the remaining containers were located in areas with less light under the forest understory. This difference may have accounted for the lack of oviposition in some containers. In support of this possibility, Yanoviak (2001) reported that artificial tree-holes located in the understory attract more mosquitoes than artificial containers located in tree-fall gaps. During our experimentation, habitat stability was maintained by providing minimum water levels throughout the trials. Higher summer temperatures deplete water levels in containers that in turn lead to shorter development times for mosquitoes, especially when ample food is present. In this investigation, high summer temperatures may have resulted in fewer small containers being colonized because of the level of water falling below stable maintenance levels while the larger containers held adequate levels of water more consistently. In addition, higher water temperatures hinder container selection by female mosquitoes (Alto and Juliano 2001), perhaps forcing the female mosquitoes to continuing searching for a more suitable container. Container size alone does not dictate site selection. Other cues play a role in whether a gravid female will select a particular container. Factors include leaflitter quality, speed of decay, developmental time, water volume, lower water temperatures, leaf-fall diversity, and nutritional yield (Barrera et al. 2006, Bently and Day 1989, Reiskind et al. 2009, Yanoviak 1999). These factors become influential not only in site selection but also in larval maturation to adulthood and may actually limit oviposition even in the larger containers (Yanoviak 1999). Development may also be influenced by the effects of crowding, limiting the mobility of the larvae and reducing container selection by subsequent females (Yee et al. 2004). Investigating artificial containers in and around suburban homes and patchy forest surroundings revealed many large containers available to ovipositing mosquitoes. Large urns, flowerpots, birdbaths, fountains, and unattended swimming pools can all hold sufficient water and provide preferred habitats for mosquitoes. Once these containers are established as a breeding site, conspecific eggs then serve as additional attractant cues for gravid mosquitoes (Allan and Kline 1998, Onyabe and Roitberg 1998). Our results suggest that large container size may be more important than color for oviposition by gravid Eastern Tree-hole Mosquitoes in a temperate forest setting. Results of this investigation may add to the knowledge and applications of mosquito-control management and expand our understanding of mosquito behavior. 370 Northeastern Naturalist Vol. 20, No. 2 Acknowledgments The authors extend their gratitude to Apple Pools, Inc. for their initial financial support in providing the containers and paint used in this investigation. We thank P. Torrisi for her involvement in the proof reading and comments of the initial draft. Literature Cited Allan, S.A., and D.L. Kline. 1998. Larval-rearing water and preexisting eggs influence oviposition by Aedes aegypti and Aedes Albopictus (Diptera: Culicidae). Journal of Medical Entomology 35:943–947. Alto, B.W., and S.A. Juliano. 2001. Precipitation and temperature effects on populations of Aedes albopictus (Diptera: Culicidae): Implications for range expansion. Journal of Medical Entomology 38:646–656. Barrera, R., M. Amador, and G.C. Clark. 2006. Ecological factors influencing Aedes aegypti (Diptera: Culicidae) productivity in artificial containers in Salinas, Puerto Rico. Journal of Medical Entomology 43:484–492. Bentley, M.D., and J.F. Day. 1989. Chemical ecology and behavioral aspects of mosquito oviposition. Annual Review of Entomology 34:401–421. Braks, M.A.H., N.A. Honorio, L.P. Lounibos, R. Lourenco-de-Oliviera, and S.A. Juliano. 2004. Interspecific competition between two invasive species of container mosquitoes, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), in Brazil. Annals of the Entomological Society of America 97:130–139. Center for Disease Control (CDC). 2010a. Dengue. Available online at http://www.cdc. gov/dengue/epidemiology/index.html. Accessed on 10 April 2012. CDC. 2010b. La Crosse Encephalitis. Available online at http://www.cdc.gov/lac/tech/ epi.html. Accessed on 10 April 2012. Collins, L.E., and A. Blackwell. 2000. Colour cues for oviposition behaviour in Toxorhynchitis moctezuma and Toxorhynchitis amboinensis mosquitoes. Journal of Vector Ecology 25:127–135. Etiam, A., and L. Blaustein. 2004. Oviposition habitat selection by mosquitoes in response to predator (Notonecta maculate) density. Physiological Entomology 29:188–191. Frank, J.H. 1985. Use of an artificial bromeliad to show the importance of color value in restricting colonization of bromeliads by Aedes aegypti and Culex quinquefasciatus. Journal of the American Mosquito Control Association 1:28–32. Frank, J.H. 1986. Bromelaids as ovipositional sites for Wyeomyia mosquitoes: Form and color influence behaviour. Florida Entomologist 69:728–742. Hayes, E.B., and D.J. Gubler. 2006. West Nile virus: Epidemiology and clinical features of an emerging epidemic in the United States. Annual Review of Medicine 57:181–194. Jones, C.J., and E.T. Schrieber. 1994. Color and height affects oviposition site preferences of Toxorhynchitis splendens and Toxorhynchitis rutilus rutilus (Diptera: Culicidae) in the laboratory. Environmental Entomology 28:130–135. Kitching, R.L. 1971. An ecological study of water-filled tree holes and their position in the woodland ecosystem. Journal of Animal Ecology 40:281–302. Lester, P.J., and A.J. Pike. 2003. Container surface area and water depth influence the population dynamics of the mosquito Culex pervigilians (Diptera: Culicidae) and its associated predators in New Zealand. Journal of Vector Ecology 28:267–274. 2013 G.J. Torrisi and W.W. Hoback 371 Minakawa, N., G. Sonye, and G.Yan. 2005. Relationships between the occurrences of Anopheles gambriae s. l. (Diptera: Culicidae) and size and stability of larval habitats. Journal of Medical Entomology 42:295–300. New York State Department of Health (NYSDH). 2012. Mosquitoes and West Nile virus: Fight the bite. Available online at http://www.nyhealth.gov/publications/2731/. Accessed on 15 April 2012. Onyabe, D.Y., and B.D. Rottberg. 1997. The effect of conspecifics on oviposition site selection and oviposition behavior in Aedes togoi (Theobold) (Diptera: Culicidae). The Canadian Entomologist 129:1173–1176. Reiskind, M.H., K.L. Greene, and L.P. Lounibos. 2009. Leaf species identity and combination affect performance and oviposition choice of two container mosquito species. Ecological Entomology 34:447–456. Available online at http://www.ncbi.nlm.nih. gov/pmc/articles/PMC2712298. Rodriguez-Tovar, M.L., M.H. Badii, J.K. Olson, and A. Flores-Suarez. 2000. Oviposition preference of Aedes aegypti (L.) in artificial containers in Nuevo Leon, Mexico. Southwestern Entomologist 25:55–58. Service, M.W. 1977. Mortalities of the immature stages of species B of Anopheles gambiae complex in Kenya: Identification of predators, and effects of insecticidal spraying. Journal of Medical Entomology 13:535–545. Sunahara, T., K. Ishizaka, and M. Mogi. 2002. Habitat size: A factor determining the opportunity for encounters between mosquito larvae and aquatic predators. Journal of Vector Ecology 27:8–20. Yanoviak, S.P. 1999. Effects of leaf-litter species on macroinvertebrate community properties and mosquito yield in neotropical tree-hole microcosms. Oecologia 120:147–155. Yanoviak, S.P. 2001. Container color and location affect macroinvertebrate community structure in artificial tree holes in Panama. Florida Entomologi st 84:265–271. Yee, D.A., B. Kesavaraju, B., and S.A. Juliano. 2004. Larval feeding behavior of three co-occurring species of container mosquitoes. Journal of Vector Ecology 29:315–322. Zahiri, N., and M.E. Rau. 1998. Oviposition attraction and repellency of Aedes aegypti (Diptera: Culicidae) to waters from conspecific larvae subjected to crowding, confinement, starvation, or infection. Journal of Medical Entomology 3 5:782–787.