<%BANNER%>

Aedes albopictus oviposition and larval density, development and interactions with Wyeomyia spp. in exotic bromeliads of...

University of Florida Institutional Repository
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1 Aedes albopictus OVIPOSITION AND LA RVAL DENSITY, DEVELOPMENT, AND INTERACTIONS WITH Wyeomyia spp. IN EXOTIC BROMELIADS OF SOUTHERN FLORIDA By ROBYN R. RABAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Robyn R. Raban

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3 To my parents, Bill and Judy Raba n and my grandfather, Bill Phill ips for their support for all my academic endeavors.

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4 ACKNOWLEDGMENTS I would like to thank L.P. Lounibos for his advi ce and support. I thank G. F. OMeara for his assistance in locating field sites, and his enth usiastic support for my research. I thank J.H. Frank for helpful suggestions on my research; N. Nishimura for help with my technical problems; and R. Escher for his mosquito rearin g guidance and for his provision of eggs for my research. Finally, I would like to thank the faculty, staff, and gr aduate students at FMEL, most of whom at one time or another, provid ed invaluable help to my research.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 Aedes albopictus AND Wyeomyia spp. IN THE EXOTIC BROMELIADS OF SOUTHERN FLORIDA.........................................................................................................13 Aedes albopictus in the United States.....................................................................................13 Wyeomyia mitchellii and Wyeomyia vanduzeei in the United States......................................14 Bromeliaceae as a Habitat......................................................................................................15 Interactions of A. albopictus and Wyeomyia spp. in Bromeliad Phytotelmata.......................17 2 INTERFERENCE COMPETITION AS A POTENTIAL MECHANISM FOR THE REDUCTION OF GROWTH AND SURVIVORSHIP OF A edes albopictus IN THE PRESENCE OF Wyeomyia spp. IN THE BROMELIADS OF SOUTHERN FLORIDA.....20 Introduction................................................................................................................... ..........20 Materials and Methods.......................................................................................................... .24 Chemical interference......................................................................................................24 Surface Area to Volume Ratio Encounter Competition Experiment..............................26 Habitat Complexity Experiment......................................................................................27 Resource Dependent Location of A. albopictus and Wyeomyia spp...............................28 Results........................................................................................................................ .............30 Chemical Interference.....................................................................................................30 Surface Area to Volume Ratio Experiment.....................................................................30 Habitat Complexity Experiment......................................................................................31 Resource-Dependent Foraging Location.........................................................................31 Discussion..................................................................................................................... ..........32 3 Wyeomyia spp. AS POTENTIAL PREDATORS OF A edes albopictus IN BROMELIADS IN SOUTHERN FLORIDA........................................................................53 Introduction................................................................................................................... ..........53 Materials and Methods.......................................................................................................... .54 Testing for Density Dependent Predation by Wyeomyia spp..........................................54 Testing for Resource Dependent Predation by Wyeomyia spp........................................54 Predation Observation.....................................................................................................55

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6 Results........................................................................................................................ .............55 Discussion..................................................................................................................... ..........56 4 DO W yeomyia spp.. LARVAE INHIBIT EGG HATCH OF A edes albopictus ?..................59 Introduction................................................................................................................... ..........59 Materials and Methods.......................................................................................................... .60 Results........................................................................................................................ .............61 Discussion..................................................................................................................... ..........62 5 FIELD STUDIES ON A edes albopictus AND Wyeomyia spp. IN EXOTIC BROMELIADS OF SOUTHERN FLORIDA........................................................................68 Introduction................................................................................................................... ..........68 Materials and Methods.......................................................................................................... .69 Differences in the Density of A. albopictus and Wyeomyia spp. within N. spectabilis and B. pyramidalis .....................................................................................69 Canopy Effects on Density..............................................................................................71 Results........................................................................................................................ .............72 Discussion..................................................................................................................... ..........73 6 OVIPOSITION AND LARVAL DEVELOPMENT OF A edes albopictus IN TWO EXOTIC SPECIES OF BROMELI AD IN SOUTHERN FLORIDA....................................91 Introduction................................................................................................................... ..........91 Materials and Methods.......................................................................................................... .94 Oviposition Experiment...................................................................................................94 Larval Development and Survivorship Experiment........................................................96 Results........................................................................................................................ .............96 Oviposition Experiment...................................................................................................96 Larval Development and Survivorship Experiment........................................................96 Discussion..................................................................................................................... ..........97 7 CONCLUSIONS..................................................................................................................100 LIST OF REFERENCES.............................................................................................................103 BIOGRAPHICAL SKETCH.......................................................................................................116

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7 LIST OF TABLES Table page 2-1 Means and standard errors for the concen trations of ammonia and pH preand postexperiment..................................................................................................................... .....36 2-2 Analysis of variance for A. albopictus average instar based on the presence or absence of Wyeomyia spp. and the surface:volume ratio of the artificial plant and the interaction of these two variables......................................................................................37 2-3 Analysis of variance for A. albopictus average survival based on the presence or absence of Wyeomyia spp., and the surface area:volume ra tio of the plant (size) and the interaction of these two variables.................................................................................38 2-4 Multivariate analysis of variance table for feeding location..............................................39 2-5 Analysis of variance for the effects of food level presence or absence of Wyeomyia spp. on the location of A. albopictus ..................................................................................40 2-6 Analysis of variance for the effects of food level and presence or absence of A. albopictus on the location of Wyeomyia spp......................................................................41 3-1 Number of Wyeomyia examined in predation experiments...............................................58 5-1 Analysis of variance for the density of mosquitoes per plant based on month (month of collection), site, and bromeliad species.........................................................................77 5-2 Analysis of variance for the density of mosquitoes per mL based on month (month of collection), site, and bromeliad species.............................................................................78 5-3 Analysis of variance for the density of each mosquito species per mL of water in each bromeliad species......................................................................................................79 5-4 Mean densities of mosquitoes (mos/plant) in B.pyramidalis and N. spectabilis based on location within the pl ant and plant species...................................................................80 5-5 Analysis of variance table for the eff ects of month and site on mos/mL and mos/plant...................................................................................................................... ......81 5-6 Analysis of variance results for the effect of month a nd sites on mosquito abundances (mos/plant) by bromeliad species...................................................................82 5-7 Average (SE) amount of water extracted from each plant species .................................83 5-8 A nested ANOVA table for effects of Oak vs. Palm canopy and bromeliad species ( B. pyramidalis vs. N. spectabilis ) on Wyeomyia spp. densities by mos/plant..................84

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8 5-9 Means and SE of densities of A. albopictus and Wyeomyia spp. by location within plants under oak and palm canopies..................................................................................85

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9 LIST OF FIGURES Figure page 2-1 Construction of the squares for the habitat complexi ty experiment..................................42 2-2 The folded shape of the high complexity and low complexity treatments and the orientation of the squares within the cones........................................................................43 2-3 Average instars of A. albopictus after 48 h exposure to Wyeomyia spp. through a dialysis membrane and without Wyeomyia in the control.................................................44 2-4 Mean survivorship of A. albopictus after 48 h exposure to Wyeomyia spp. through a dialysis membrane and without Wyeomyia spp. in the control. The error bars are standard error................................................................................................................. ....45 2-5 The average instar SE of A. albopictus in the absence or presence of Wyeomyia spp. in four plant sizes.......................................................................................................46 2-6 The average survivorship SE of A. albopictus in the absence or presence of Wyeomyia spp. in four plant sizes......................................................................................47 2-7 The average instar (+SE) of A. albopictus in two levels of habitat complexity and three levels of surface area to volume ratio.......................................................................48 2-8 Location of A. albopictus in the presence and absence of Wyeomyia ...............................49 2-9 Location of Wyeomyia spp. in the presence and absence of A. albopictus ........................50 2-10 Location of A. albopictus by food concentration and th e presence and absence of Wyeomyia spp....................................................................................................................51 2.11 Location of Wyeomyia spp. by food concentration and the presence and absence of A. albopictus ...........................................................................................................................52 4-1 Proportion hatch of viable e ggs SE in each treatment....................................................66 4-2 Proportion eggs damaged SE per treatment.....................................................................67 5-1 Map of collection sites.................................................................................................... ...86 5-2 Location of water samples.................................................................................................87 5-3 The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis and B. pyramidalis at each site..........................................................................................88 5-4 The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis at each site...................................................................................................................... ........89

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10 5-5 The monthly mean densities of Wyeomyia spp. and A. albopictus in B. pyramidalis at each site...................................................................................................................... ........90

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science Aedes albopictus OVIPOSITION AND LARVAL DE NSITY, DEVELOPMENT, AND INTERACTIONS WITH Wyeomyia spp. IN EXOTIC BROMELIADS OF SOUTHERN FLORIDA By Robyn R. Raban December 2006 Chair: L. P. Lounibos Major Department: Entomology and Nematology Wyeomyia mitchellii and Wyeomyia vanduzeei are indigenous mosquitoes that inhabit exotic and native bromeliads in so uthern Florida. In the mid-1980s Aedes albopictus invaded Florida where its immature stages occupied artificial and natural containers, including bromeliads. Previous studies have shown reduced abundance of A. albopictus in exotic bromeliads co-occupied by Wyeomyia spp. immatures, and reduced larval growth and survivorship in the presence of late instar Wyeomyia spp. Our study examined chemical interference, encounter competition, predation, and egg hatch inhibition as potenti al interactions between Wyeomyia spp. and A. albopictus in exotic bromeliads. Exposure to waste products of Wyeomyia spp. did not affect the growth or survivorship of A. albopictus Experimental alterations of su rface area to volume ratios and habitat complexity did not affect the decreased growth and survivorship of A. albopictus seen in the presence of Wyeomyia spp. Behavior experiments showed that A. albopictus larvae change their location within a cont ainer in the presence of Wyeomyia spp. This change in location may indicate a response of A. albopictus to encounters with Wyeomyia spp. Wyeomyia spp. showed no evidence of predation on A. albopictus but fourth instar Wyeomyia spp. were shown to inhibit the egg hatch of A. albopictus

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12 Field surveys and lab ex periments were conducted on A. albopictus and Wyeomyia spp. in Neoregelia spectabilis and Billbergia pyramidalis bromeliads. Higher densities of Wyeomyia spp. were found in B. pyramidalis while higher densities of A. albopictus were found in N. spectabilis Correlations between the number of mos quitoes per plant and the volume of water extracted were significant for Wyeomyia spp. and A. albopictus Overhead tree canopy type influenced Wyeomyia spp. larval densities in bromeliads. A. albopictus females oviposited preferentially in N. spectabilis and A. albopictus developed faster when exposed to Wyeomyia spp. within N. spectabilis as compared to the same exposure in B. pyramidalis Encounter competition with Wyeomyia spp. larvae is the most probable mechanism reducing the growth and survivorship of A. albopictus in exotic bromeliads in southern Florida, although other interactions may also influence the relative abundances of these mosquito species.

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13 CHAPTER 1 Aedes albopictus AND Wyeomyia spp. IN THE EXOTIC BR OMELIADS OF SOUTHERN FLORIDA With expanding global transport and commerce, humans have increased the distributions of many plant and animal species, often to novel lo cations. While some of these species fail to establish in these new locations (Mack et al. 200 0), some become successful biological invaders (Lounibos 2002). Aedes albopictus in the United States Since its establishment in North America in the 1980s (Spre nger and Wuithiranygool 1986), Aedes albopictus has invaded various habitats that have previously been occupied by other mosquito species. It has expanded its range throughout the south eastern and midwestern United States by its transport in used tires (Hawley 1988). In Florida, A. albopictus was first discovered in 1984, and is currently found throughout most of the state (O'Meara et al. 1995a, Juliano et al. 2004). Invasions by A. albopictus have been much discussed (Lounibos 2002), and this mosquito species continues to colonize new areas of western (A randa et al. 2006) and eastern Europe (Klobucar et al. 2006). Aedes albopictus is one of the commonest mosquitoes in natural and artifici al containers in the eastern United States and Brazil (Lounibos 2002). Aedes albopictus larvae are often found inhabiting man-made containers, but can also be found in natural phytotelmata such as treeholes (Novak et al. 1993) and bromeliads (Lounibos et al. 2003, O'Meara et al. 2003). Displacement by A. albopictus of mosquitoes that traditionally o ccurred in these habitats, such as Aedes aegypti (Juliano 1998), and Culex pipiens (Carrieri et al. 2003) has be en seen in some cases. To date, considerations of the interactions of A. albopictus with its competitiors have included mating interference (Nas ci et al. 1989), egg hatch in hibition (Edgerly et al. 1993, Edgerly et al. 1991), larval resource competiti on (Juliano 1998, Braks et al. 2004, Griswold and

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14 Lounibos 2005), predator-mediated competition (Gubler 1971, Lounibos et al. 2001, Griswold and Lounibos 2005), and intraguild predation (Edgerly et al. 1999). Yet, so far competitive exclusion of A. aegypti by A. albopictus has not occurred mainly due to suspected differences in the egg desiccation tolerances (Costanzo et al. 20 05b), or differences in macrohabitat preferences (Lounibos et al. 2001, Costanzo et al. 2005a). In some cases though, local extinction of A. aegypti occurred after the arrival of A. albopictus (OMeara et al. 1993). Aedes albopictus have been infected with over 22 species of arboviruses (Moore and Mitchell 1997). In North America, A. albopictus have been found infected in nature with LaCrosse (Gerhardt et al. 2001), Eastern Equine Encephalitis ( EEE) (Mitchell et al. 1992), and dengue viruses (Ibaez Bernal et al 1997). Therefore, there are poten tial health c oncerns related to A. albopictus populations. Wyeomyia mitchellii and Wyeomyia vanduzeei in the United States Wyeomyia mitchellii ranges throughout Mexico (Di az-Najera and Vargas 1973), the Caribbean region (Belkin and Heinemann 1975, Sh royer 1981), central a nd southern Florida (Darsie and Ward 2005), and Hawa ii (Shroyer 1981). This brom eliad specialist mosquito is commonly found co-occurring in Florida with another congeneric bromeliad specialist, Wyeomyia vanduzeei Dyar and Knab. Wyeomyia vanduzeei ranges through central and southern Florida (Darsie and Ward 2005), and much of the Caribbean region (Belkin and Heinemann 1975). Before the introduction of A. albopictus W. mitchellii and W. vanduzeei were the most common day-biting mosquito species in the mixed oak forests of southern Florida (Edman and Haeger 1977). Immatures of these Wyeomyia spp. are historically the most common mosquitoes in the epiphytic bromeliad phytotelmata of southern Florida (Fish 1976, Frank 1983, Frank and O' Meara 1985).

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15 Wyeomyia vanduzeei and W. mitchellii are diurnal species with peak diel biting activity occurring just prior to sunset (Edman and Haeger 1977) and oviposition occurring in the late daylight hours (Frank et al. 1985). In a study conducted by Edman a nd Haegar (1977), both W. vanduzeei and W. mitchellii adults appeared to be generalis t feeders, feeding on species of rabbits, deer, and birds. Wyeomyia vanduzeei are facultatively autogenous, while W. mitchelli are not (OMeara 1979), and their larv ae may be slightly segregated as W. vanduzeei lay more eggs in sunny versus shady locations, and thes e two mosquito species show preferences for different plants (Frank and O'Meara 1985). Although Wyeomyia spp. have not been found to vector arboviruses in Florida, Venezuelan Equine Encephalitis virus has been found in W. mitchellii (Scherer et al. 1971) and Ilheus and Wyeomyia viruses have been found in W. vanduzeei (Srihongse and Johnson 1965, 1967) Bromeliaceae as a Habitat Phytotelmata are water bodies that are held in plant structures su ch as flowers, leaf axils, or tree holes (Maguire 1971, Fish 1983, Clements 1999) The family Bromeliaceae contains many species which impound water in their leaf axils a nd are referred to as tank bromeliads (Frank 1983). There are over 2000 bromelia d species in the Americas (F rank 1983), with 16 species of bromeliads native to Florida, 7 of these being tank bromeliads that hold water throughout most of the year (Fish 1976). While bromeliads obtain ener gy from photosynthesis, the nu trients they sequester for photosynthesis are obtained in multiple ways. Ta nk bromeliads can absorb nutrients through scales on the shoot called trichomes (Pittendri gh 1948, Benzing et al. 1976, Benzing 2000), or by way of interfoliar roots (Pittendrigh 1948). Frank (1983) classifies nutrient acquisition by tank bromeliads into two categories, dendrophilous nutrition, which is rain throughfall from trees often collected by epiphytic bromeliads, and anemophilous nutrition, which are wind blown

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16 nutrients. Frank (1983) and Benzin g (2000) also describe the rele ase of nutrients provided by the wastes of faunal tank inhabitants which may contribu te to the nutrients available to the plant. In bromeliads, mosquitoes may encounter a wi de variety of other unrelated species. To date, bacteria, fungi, algae, protozoa, platyhe lminthes, rotifers, gast rotrichs, ostracods, nematodes, oligochaetes, crusta cea, acarids, Hemiptera, Coleopt era, Culicidae, Psychodidae, Syrphidae, Ceratopogonidae, Chironomidae, Sarcophaghidae, Tabanidae, Periscelidae, Tipulidae, Muscidae, Sciaridae, and odonates ha ve all been identified within bromeliad phytotelamata (Maguire 1971, Fish 1976, Frank 1983). Some chironomids, muscids, periscelids, and turbellarians are predatory and feed upon smalle r organisms and mosquito eggs, while some psychodids and sciarids feed on the submerged leaf litter in the bromeliad (Fish 1976). Bromeliads vary in their faunal compositions by the species of bromeliad and environmental conditions, such as sunlight and humidity (Laessl e 1961, Fish 1976). Since the popularization of exotic bromeliads as ornamental plants (Edman and Haeger 1977), there has been an increase in exotic bromeliad habitat for Wyeomyia spp. in Florida. Yet, with the arrival of the Mexican bromeliad weevil, Metamasius callizona there has been a decline of the native bromeliad Tillandsia utriculata (Frank and Thomas 1994), the common native phytotelmata of W. mitchellii and W. vanduzeei (Fish 1976). Exotic bromeliads, which have become another common habitat of these two spec ies, hold more water than the native bromeliad species and have a large centr al water-holding tank, uncommon in native bromeliads (OMeara et al. 2003). The two bromeliads which were the focus of most of this study are Billbergia pyramidalis the summer torch bromeliad, and Neoregelia spectabilis the painted fingernail plant. Billbergia pyramidalis and Neoregelia spectabilis are native to Brazil (Frank et al. 1988), and can both live

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17 as either an epiphyte or as a ground dwelling species. Neoregelia spectabilis is the larger of the two species and tends to be a dark green to da rk purple color with br ight purple tips. The inflorescence of N. spectabilis is nidulate, meaning it is nested into the central tank of the plant. Billbergia pyramidalis is a light green color and has a stalked inflorescence. Interactions of A. albopictus and Wyeomyia spp. in Bromeliad Phytotelmata There is evidence that Wyeomyia spp. larvae exhibit negative effects on A. albopictus within bromeliads. Possibly due to the fr eezing temperatures, or the absence of native bromeliads in northern Florida, W. mitchelliii and W. vanduzeei are infrequently found north of Orlando (O'Meara et al. 2003). In the absence of Wyeomyia spp. in northern Florida, A. albopictus has become a common mosquito in brom eliads, but in southern Florida where Wyeomyia spp. are abundant, A. albopictus individuals are scarce in bromeliads (O'Meara et al. 1995b, Lounibos et al. 2003). A study conducted in Hawaii determined that in the absence of Wyeomyia spp., A. albopictus dominated leaf axil phytotelmata (Shroyer 1981). This study attempts to further investigate th e results of Lounibos et al. (2003). In that study, 1st instar A. albopictus were exposed to varied quant ities of leaf litter with 1st or 4th instar Wyeomyia spp. in B. pyramidalis bromeliads. Aedes albopictus experienced a reduction in survivorship with reduced leaf litter and the presence of 4th instar Wyeomyia spp., but the presence of Wyeomyia spp. accounted for most of the reduction in survivorship. The mean growth stage of A. albopictus was only affected by the presence of 4th instar Wyeomyia spp. with no effect due to leaf litter. 1st instar Wyeomyia spp. had no effect on the growth or survivorship of A. albopictus Lounibos et al. (2003) hypothe sized that the reduction in growth and survivorship of A. albopictus in the presence of Wyeomyia spp. was attributable to interference competition. Lounibos et al. (2003) suggested that because the addition of leaf litter did not reduce the

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18 negative effects of Wyeomyia spp. on A. albopictus this interaction is inde pendent of resources. Interference competition is categorized into ove rgrowth, chemical, territorial, and encounter (Schoener 1983). Overgrowth interference comp etition occurs only in sessile organisms, and mosquito larvae are not known to e xhibit territorial interference be haviors, so these mechanisms are not considered in the possible competitive interactions between A. albopictus and Wyeomyia spp.. Chemical interference in mosquitoes results from the buil dup of larval waste products that lead to an environmental deterioration which can effect the growth and survivorship of individuals (Bedhomm e et al. 2005). Aedes albopictus has been found to be subject to chemical interference competition with the mosquito Tripteroides bambusa (Sunahara and Mogi 2002). Chemical intereference has also been demonstrated in Aedes sierrensis (Broadie and Bradshaw 1991), A. aegypti (Dye 1984), and Culex sitiens (Roberts 1998). Encounter competition usually occurs when physical contact between organisms causes a reduction in the feeding efficien cy of individuals (A nholt 1990). Encounter competition is known among A. sierrensis (Broadie and Bradshaw 1991), A. aegypti (Dye 1984) and Culex sitiens (Roberts 1998). Othe r mosquitoes such as Ochlerotatus cantans (Renshaw et al. 1993) are hypothesized to also exhibit en counter competition effects. Intraguild predation is anothe r important regulatory mechan ism in mosquito populations. Some mosquito species are facu ltative predators or cannibalis tic on smaller instars (Hinman 1934, Reisen and Emory 1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003, Edgerly et al. 1999, Koenraadt et al. 2004). Egg hatch inhibition occurs when mosquito larvae delay hatch of Aedes eggs. Egg hatch inhibition could increase the time to reproduction, which may reduce population growth and increase mortality in the do rmant egg stage of some species (Livdahl and

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19 Edgerly 1987). Some Aedes species exhibit varying degrees of egg hatch inhibition (Livdahl et al. 1984, Koenekoop and Livdahl 198 6, Livdahl and Edgerly 1987). Thus, the purpose of th e following experiments on A. albopictus and Wyeomyia spp. is to determine the mechanism of reduction of growth and survivorship of 1st instar A. albopictus in the presence of 4th instar Wyeomyia spp. in exotic bromeliads. As mentioned above, chemical interference competition, encounter competition, pr edation, and egg hatch inhibition are potential competitive mechanisms in mosquito populations. Previously none of these mechanisms have been studied between Wyeomyia spp. and A. albopictus within bromeliads, and thus these interactions are the fo cus of a set of experiments in this study. Furthermore, in attempt to add to th e current understanding of the occupancy by A. albopictus and Wyeomyia spp. of bromeliads in southe rn Florida (O'Meara et al. 1995b, Lounibos et al. 2003) multiple collections were made from two common ornamental bromeliad species B. pyramidalis and N. spectabilis Subsequent oviposition and larval competition experiments were conducted with these two br omeliad species to explain the patterns of mosquito abundance observed from field collections.

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20 CHAPTER 2 INTERFERENCE COMPETITION AS A POTENTIAL MECHANISM FOR THE REDUCTION OF GROWTH AND SURVIVORSHIP OF A edes albopictus IN THE PRESENCE OF Wyeomyia spp. IN THE BROMELIADS OF SOUTHERN FLORIDA. Introduction Competitive interactions can be a major fact or in determining community structure. Exploitative and interference competition are the tw o main mechanisms for interactions between individuals within a population (Schoener 1983). Exploitative competition has been the focus of many ecological studies, but interference competition often has a greater influence over species distribution and abundance (Case and Gilpin 1974). Schoener (1983) subcategorized exploitative competition into consumptive competition, which involves food resource competition, and preemptive competition for resource space. The subcategories of interference competition include (i) overgrowth, which invol ves individuals growing over one another and thus depriving their competitors of resources; (ii) chemical, whic h deals with toxins produced by some individuals harming other individuals; (iii) territorial, in which i ndividuals aggressively defend units of space to the detriment of the co mpetitors, and (iv) encounter competition in which interactions between mobile competitors cause harm by fighting, predation, or physical interference. Overlap is common in exploitativ e and interference competition (Schoener 1983). For example, interference competition affects competitors by changing the rate of resource exploitation, or in some cases resulting in in jury or death, which in turn affects population growth (Case and Gilpin 1974). Many plants, such as gra sses (Javaid et al. 2005, Singh et al. 2005) and pine trees (Nektarios et al. 2005), release al lelopathic chemicals that create an unfavorable environment for possible plant competitors. In some cases, the allelopathic chemicals released by plants are the main determinants of the plant community structure (Rasmussen and Rice 1971), and are

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21 therefore a major competitive infl uence in the community. Bacter ia also release allelopathic chemicals to limit the growth of th eir neighbors (Riley and Gordon 1999). Allelopathy has not been id entified in animals, but animals experience chemical interference caused by waste materials from othe r individuals. These waste products, such as ammonia and nitrates, cause a de terioration of the environment leading to a reduction in the growth and survivorship of othe r individuals (Bedhomme et al. 2005) For instance, intraspecific chemical interference competition has been shown among sea lamprey larvae (Rodrguez-Muoz et al. 2003), Paramecium (Gill 1972), tadpoles (Griffiths et al. 1991) and larvae of the mosquitoes A. aegypti (Bedhomme et al 2005, Dye 1984) and Culex pipiens molestus (Ikeshoji et al. 1976). Density dependent chemical inhi bition of larval growth has been demonstrated in other species of Aedes mosquitoes. Broadie and Bradshaw ( 1991) determined from a laboratory experiment that intraspecific chemical inte rference competition influenced pupation success, pupal weight and development time in Aedes sierrensis. Chemical interference has also been shown to affect the growth of A. albopictus Sunahara and Mogi (2002) determined that A. albopictus experienced a reduction in survivorship and pupation success from interspecific chemical interference competition with Tripteroides bambusa in bamboo stumps. Chemical inhibition of larval growth may be less common in other genera of mosquitoes. Wyeomyia smithii exhibit density dependent increases in development time and reductions in survivorship and pupal weight, but neither enco unter competition, chemical competition, nor cannibalism were found to contribute to these dens ity dependent changes in fitness (Broberg and Bradshaw 1995).

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22 While chemical inhibition of growth does occur within larval communities, encounter competition is often found to be the predominan t interference mechanism. Dye (1984) found that some strains of A. aegypti demonstrated intraspecific chemical interference, but chemical effects were small compared to those of encounter comp etition. Roberts (1998) al so found that chemical inhibition of growth occurred among Culex sitiens conspecifics, but that encounter competition was the stronger of the tw o competitive factors. Encounter competition was defined by Brian (1956) as the ability of an organism to harm another organism by directly attacking it, or indirectly by damaging its food resources or blocking its access to those resources. Direct attack encounter competition has been found to occur in mosquito larvae and often alters their survivorship. Among some filter-feeding mosquito species, 1st instar larvae may be attacked and often killed by fourth instar conspecifics in laboratory microcosms (e.g., Reisen and Emory 1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003). Mosquitoes may expe rience encounter competition when either high densities of individuals cause frequent encount ers with one another (B roadie and Bradshaw 1991, Roberts 1998) or larvae frequently encounter larger individuals of later instars (Dye 1984, Broadie and Bradshaw 1991). The combination of high densities a nd larger individuals can have the biggest effect on early instar mo squitoes (Broadie and Bradshaw 1991). In many cases, physical contact between organi sms can lead to a reduction in feeding efficiency or other metabolic costs (Anholt 1990). Physical contact was implicated as the cause of reduced feeding rates of A. sierrensis (Broadie and Bradshaw 1991) and Anopheles gambiae (Koenraadt et al. 2003) at high densities. Reductions in feeding efficiency can cause a lterations in life hist ory characteristics by increasing larval mortality and develo pment time and decreasing adult size. Aedes cantans

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23 experienced a reduction in adult size and an increase in larval mortality, which were hypothesized to be due to contact competition (Renshaw et al. 1993). Previous studies have tried to vary the e ffects of encounter competition by altering the water volume (Dye 1984), the surface to volum e ratio (Broadie and Bradshaw 1991, Roberts 1998), or habitat complexity (Broadie and Bradsh aw 1991). While experiments with alterations in the surface area to volume ra tio did not significantly allevi ate the effects of competition by changing the encounter rate be tween individuals (Broadie and Bradshaw 1991, Roberts 1998), there were successes with the alteration of wa ter volume (Dye 1984) and habitat complexity (Broadie and Bradshaw 1991). Habitat complexity affects th e levels of competition and predation among species (Hixon and Menge 1991, Hixon and Jones 2005). With increasi ng structural complexity of a habitat, the number of competitive refuges increases (M acArthur and Levins 1967, Finke and Denno 2002), and the number of physical enc ounters between predator and prey decreases (Murdoch and Oaten 1975). With more physical encounters in hab itats of decreased comp lexity, the occurrence of intraguild predation increas es (Marshall and Rypstra 1999, Roda et al. 2000, Finke and Denno 2002). In fact, most studies conducted on the effects of habitat complexity on competitive interactions have involved pr edator-prey interactions (e.g., Crowder and Cooper 1982, Schneider 1984, Diehl 1992, Babbitt and Tanner 1998, Alto et al 2005), although some studies have found benefits to competitors as well as prey populatio ns with increasing habitat complexity (Almany 2004). As with predator-prey in teractions, an increas e in habitat complex ity should decrease encounters between potential competitors. Therefore, structural complexity may provide refugia from physical encounters with non-predatory species.

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24 Behavioral changes in organisms that e xperience encounter competition are common. Anholt (1990) observed noticeable behavioral changes in feeding behavior of damselflies when exposed to different interspecific encounter ra tes. As mentioned before, physical encounters with other individuals can cause changes in feeding efficiency, a nd thus there should be a change in feeding behavior of individuals influenced by encounter competition. Lounibos et al. (2003) found that A. albopictus had a reduction in grow th and survivorship when exposed as 1st instars to 4th instars of Wyeomyia spp. in bromeliads and hypothesized interference competition as the mechanism for this e ffect. In this chapter, chemical interference competition and encounter competition are examined as potential types of interference to explain the negative effect of Wyeomyia spp. on larvae of A. albopictus. In this chapter, one experiment was conducte d to determine whether the waste products of Wyeomyia spp. reduce the growth or survivorship of A. albopictus Two experiments were conducted to determine whether physi cal contact with the larger 4th instar Wyeomyia spp. affected the growth and survivorship of A. albopictus The first of these experiments varied the surface area to volume ratio of arti ficial bromeliads in an attempt to change the frequency of encounters between the species. The second expe riment varied habitat complexity as an alternative method of changing th e frequency of encounters between species. Finally, the last experiment attempted to elucidat e changes in feeding location of A. albopictus in response to the presence of Wyeomyia spp.. Materials and Methods Chemical interference Twenty-five 1st instar colony-raised A. albopictus were added to 1000 Dalton, 31 mm diameter Spectra/Por Biotech Cellulose Ester di alysis tubes that cont ained 25 mL of sieved bromeliad water and 0.1 g of chopped live oak leaves ( Quercus virginiana) The leaves were

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25 dried at 68 C for 48 h before weighing. The bromeliad water was collected from approximately 60 plants, and sieved through a 130 m gauge mesh screen to remove detritus and macroscopic organisms. All sieved water was mixed into one large bucket, and then covered with a plastic bag for 2 days. The 1000 Dalton dialysis tubing was chosen fo r this experiment because its pore size allows waste products such as nitrates and ammonia to pass through, while excluding larger compounds such as food particles. Each tube was cut to 11.5 cm in length, which created approx. 8 X 3.1cm of aquatic habitat for the 1st instar larvae, and exce ss tubing of approximately 3.5 X 3.1cm was left over for folding the ends an d for suspending the tubing. A butterfly clip strung with wire was attached to the t ubing to suspend it in the center axil of a Billbergia pyramidalis bromeliad. The butterfly clip also kept th e tubing from sealing at the top, which would have prevented access to the air. The dialysis tubing had been preserved in 0.1% sodium azide solution, which was toxic to the larvae in preliminary tests. Therefore, a few days before use each dialysis tube was detoxified by soaking in distil led water 3 times for 30 minute intervals, after which it was placed in a 1.0 % sodium benzoate solution to prevent d ecay. On the day of the experiment, each tube was once again soaked 3 times for 30 min to remove the sodium benzoate, and then closed off at the bottom by folding and then sealing with a plastic-coated wire garbage tie. To the center axil of 10 of the 20 plants, 30 4th instar Wyeomyia spp. were added to 100 mL of sieved bromeliad water containing 0.4 g of chopped oak leaves. One hundred militers of sieved bromeliad water and 0.4 g of chopped oak leav es were added to the ce nter axil of the ten control plants. Then each plan t was randomly assigned to one of five cages where their bases

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26 were secured in a small plastic container and held for 48 h at 26C with a 12:12 h light dark cycle and approximately 80% relative humidity. The plants were checked every 12 h for leaki ng, which was observed in 10 of the twenty plants, a loss of around 30 mL by 24 h. At 24 h, more sieved bromeliad water was added to the leaky plants corresponding to th e amount of water loss detected in the plastic container holding the bromeliad. Ammonia levels and pH of the bromeliad wa ter were measured before and after the experiment. Ammonia concentration was recorded using the Fisher Scientific Accumet Portable AP63 pH/mV/ion meter using an ammonia probe, and pH was recorded using a Corning pH-20 meter. After 48 h, the water in each bromeliad was re moved by a pipette, and rinsed into a metal rearing pan with a spray bottle containing tap water. The number of surviving A. albopictus and the instars of each A. albopictus, determined by head capsule wi dths, were recorded for each replicate. An ANOVA was performed in SA S (2002) to determine whether the mean development stage or survivorship of A. albopictus differed between experimental (30 Wyeomyia ) and control (no Wyeomyia ) plants. Surface Area to Volume Ratio Encounter Competition Experiment Four different sized artificial bromeliads were built with circumferences of 4.5, 5.5, 7.0, and 9.0 cm at the 75 mL water level, represen ting surface area to volume ratios of 1.48, 1.82, 2.48, and 3.83 cm2/mL, respectively. The artificial brom eliads were constructed from 8.5 by 11 inch weatherproof map paper (IGage, Mapping Co rporation, Salt Lake City UT), and Perfect Glue No. 1 (Liquid Nails, Cleveland, OH) was used as an adhesive on the outer parts of the plant. The inner parts of the plant were water protect ed and sealed with non-toxic, waterproof, 100% silicone aquarium tank sealant (All-Glass Aquarium, Franklin, WI).

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27 Twenty-five 1st instar A. albopictus 0.5 g oak leaves, and 75 mL of sieved bromeliad water were added to 15 control bromeliads of each size. The other 15 of each size represented the experimental group and di ffered from the controls by the addition of 30 4th instar Wyeomyia spp.. Each replicate was placed into a plastic containe r of appropriate size a nd put onto one of three randomly chosen shelves in a climate controlled rearing room kept at 26C with a 12:12 light dark cycle and approximately 80% humidity. On the day of setting, each artificial bromeliad was marked with a pencil to indicate the 75 mL waterline. To compensate for evaporation, and the differences in evaporation based on the varied surface area to volume ratios among treatments, on the 4th and 8th day of the experiment each plant was refilled with sieved bromeliad water to its original 75 mL water level. After 10 days the experiment was terminated, and all of the A. albopictus in each replicate were counted, and thei r instars were recorded based on head capsule widths. Using PROC GLM in SAS (2002) an ANOVA was performed to detect significant variation among the bromeliad sizes and between the presen ce and absence of Wyeomyia spp. in the development rate (average instar) and survivorship (p ercent alive) of A. albopictus immatures. Habitat Complexity Experiment The habitat complexity experiment used the same artificial bromeliads described in the surface area to volume ratio experiment. In this experiment, the smallest artificial bromeliad with a circumference of 4.5 cm and a surface area to volume ratio of 1.48:1 was excluded because it was too narrow for the habitat comple xity manipulation and still allow for larval access to the air. In the other three sizes, 20 1st instar A. albopictus 20 4th instar Wyeomyia spp., 0.5 g oak leaves, and 75 mL of sieved bromeliad water were added to 30 bromeliads of each.

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28 Two different types of 3 cm2 squares were constructed from weatherproof paper to add habitat complexity to the experiment. The first type of paper square, which represents the low habitat complexity treatment, remained two dimens ional (Fig. 2-1). Three of these squares were added to half of each of the three bromeliad si zes. The second type of paper square, which represents high habitat complexity, was cut and folded to make a three dimensional structure (Fig. 2-2). When added to the cone, the low comp lexity square remained at the top or along the sides, while the high complexity square made a lattice-like structure within (Fig. 2-2). After 5 days, the experiment was terminat ed, and the numbers and developmental stage of the remaining 1st instar A. albopictus larvae were recorded as described in previous experiments. An ANOVA was performed in SA S (2002) with survivorship and development time of A. albopictus as dependent variables and surface to volume ratio of the artificial bromeliad (n=3) and the internal complexity (n=2) of the habitat as the i ndependent variables. Bonferroni-adjusted multivariate pairwise means comparisons followed detection of significant effects by ANOVA. Resource Dependent Location of A. albopictus and Wyeomyia spp. To determine whether foraging behavior of A. albopictus and Wyeomyia spp. changed in the presence of each other, or in different leve ls of food resources, an experiment was conducted in which three treatments within plastic cups received 0.1 g of dried chopped oak leaves ( Q. virgininia) as prepared in previous experiments, and the other three treatments received 0.5 g of the same leaves. The leaves were located at the bottom of all containers. As described in prior experiments, 75 mL sieved bromeliad water was al so added to each container. Each of the food levels had one treatment with 10 1st instar A. albopictus and 10 4th instar Wyeomyia spp., one treatment with only 10 1st instar A. albopictus and one treatment with only 4th instar Wyeomyia spp.. Each of these treatments was replicated two times. All Wyeomyia spp. were collected as

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29 larvae from bromeliads in Vero Beach and Fort Pierce, FL. All A. albopictus came from a colony described in the previous experiments. An instantaneous scan census (Martin and Bateson 1986) was implemented every hour for 7 h starting after a 3 h acclimation period. Treatments with only one species had fewer individuals, so behavioral obser vations were usually finished in approximately 15 seconds. The treatments with both species had more individual s to examine, so those observations often took as long as 45 seconds. The two replicates for ea ch treatment were sampled consecutively before moving onto the next treatment, and the treat ment observed first was changed at each observation. To make observations of 1st instar A. albopictus easier, and to prevent disturbing the larvae with light at every observation, treatmen ts were illuminated by a 60 watt desk lamp for the entirety of the experiment. Because the changes in location within the cont ainer were the focus of this experiment, the locations of each Wyeomyia spp. and each A. albopictus at each time were coded into one of four categories, predetermined from preliminary observa tions: 1) at the middle of the cup (1cm from top to 1cm from bottom); 2) on the bottom of cup ( bottom to 1 cm from bottom); 3) at the top of cup (water surface to 1 cm below the water surface with or without siphon extended for breathing) or 4) wandering (swi mming without noticeable fora ging behavior). Owing to the small size of 1st instar A. albopictus whether the larva was resti ng, filter feeding or browsing was not recorded because the differences in thes e behaviors often could not be determined. For instance, during preliminary examinations there were multiple occasions when A. albopictus was nearby a surface, so it appeared to be browsing on that surface, but after closer examination at a higher magnification, the larva was found to be fi lter feeding close to th e surface, not browsing upon it.

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30 At the end of the experiment, the raw data in each observation category were converted to a proportion based on the number of individuals of each species that showed the categorical behavior at the observation time. The proportional data were ar csine square root transformed and a multivariate analysis of variance (MANOVA) was performed with the effects of species, food, alone (whether with or wit hout the other species), time, replicate, and the interactions of species x treatment, species x food, and treatm ent and food. Further analysis of variance (ANOVA) analyses with subseque nt Tukey post-hoc tests differen tiated the effects found to be significant in the MANOVA. Results Chemical Interference There was no significant difference (F1,19=1.03, P =0.32) between the average instars (development rate) of A. albopictus in dialysis bags with or without Wyeomyia spp. outside the bag (Fig. 2-3). There was also no significant difference (F1,19=2.94, P =0.10) in the arcsine square root transformed survivor ship between the treatment and the control groups (Fig. 2-4). The mean values of ammonia, and pH show ed that there was little change in these concentrations between preand post-experiment (pH: t19=1.07, P =0.30; ammonia: t19=1.34, P =0.20) (Table 2-1). Surface Area to Volume Ratio Experiment The A. albopictus in the treatments with Wyeomyia spp. were found to have a significantly lower average instar (LS mean =2.72 0.07 SE) as compared to the LS mean (3.96 0.08 SE) with Wyeomyia spp. (Fig. 2-5). There was no signifi cant effect of bromeliad size on average instar, but there was a significant in teraction between the presence of Wyeomyia spp. and size (Table 2-2). From smallest to largest size, th e average instars (mean and standard error) at each surface area to volume ratio were 3.29 0.17, 3.54 0.16, 3.32 0.16, 3.22 0.12 respectively.

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31 In treatments without Wyeomyia spp., A. albopictus were found to have a significantly lower survival (Table 2-3) (0.83 0.02 (mean SE)) compar ed to the control (0.89 0.02 (mean SE)). The largest three surface area to volume ratios contributed the most to the significant Wyeomyia spp. effect on survivorship (Fig 2-6) A Tukey post hoc test determined that the artificial bromeliad with the 6 cm diamet er, which was the second to smallest diameter, had a higher mortality rate than the other three sizes, but was onl y significantly different from the smallest size (Fig. 2-6). Habitat Complexity Experiment There was no difference in the survivorship of A. albopictus between high and low complexity treatments (F1,89=0.57, P =0.45) or surface area to volume ratios (F2,89=0.74, P =0.48). There was no significant difference in the development rate of A. albopictus among surface area to volume ratios (F2,89=1.29, P =0.28) but A. albopictus developed faster in high complexity treatments compared to the low complexity treatments (F1,89=30.02, P <0.01) (Fig 2-7) There was no significant interaction between surface area to volume ra tio and habitat complexity on developmental rate (F2,89=2.54, P =0.08), or on survivorship (F2,89=0.89, P =0.42). Resource-Dependent Foraging Location MANOVA indicated significant effects of sp ecies, presence of other species, and resource levels (Table 2-4). In general, Wyeomyia spp. foraged on the sides and on the bottom of containers more than A. albopictus (Figs. 2-8 and 2-9). Aedes albopictus were found at the top of the container more frequently and wandered more than Wyeomyia spp.. There were some significant differences between the locations of the mosquito species at the different food concentrations (Table 25 and 2-6). At high food concentrations Wyeomyia spp. allocated more time at th e water surface (Fig 2-11). Aedes. albopictus also spent more time at the water surface at high resour ce concentrations (Fig. 2-10). At low food concentrations,

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32 Wyeomyia spp. allocated more time on the bottom of the container, while A. albopictus occurred on the sides on the container more frequently (Figs 2-10 and 2-11). There was significant variation in the location of A. albopictus attributable to the presence of the other species and food level (Table 2-5). In the presence of Wyeomyia spp., A. albopictus spent more time on the top of the container and less at the bottom or on the side s of the containers (Fig. 2-8). There were significant interactions between the presence of the other species (alone effect) and species, and the presence of the other species and the level of food resources (Table 2-4). Thus, there was a difference in larval behavior of A. albopictus in the presence of Wyeomyia and the level of food resources also influenced the relati ve location of these two species. Discussion The chemical interference experiment provide d no evidence that waste products from Wyeomyia spp. larvae interfere with the growth or survivorship of A. albopictus. The buildup of waste products in experiments that exhibi ted chemical interference, such as among A. aegypti (Bedhomme et al. 2005, Dye 1984), may be less likely to occur in the bromeliad tank and axil habitat. Billbergia pyramidalis is both an epiphytic and grou nd dwelling plant (Frank et al. 1988). While there are no specific reports on the nitrogen abso rption abilities of B. pyramidalis in general, epiphytic bromeliads use dissolv ed nitrogen held in their tank water as their main nitrogen source (Benzing et al. 1976, Raven 1988). Epiphytic bromeliads also assimilate most of their nitrogen from amm onia (Endres and Mercier 2001), which is a common excretory product of mosquitoes (Clements 1999). By the end of the experiment, there was little change in the ammonium concentr ation or pH of the bromeliad water (Table 2-1 ), even though 30, 4th instar Wyeomyia spp., and 25 A. albopictus had been added to the tank for 48 hours. Because B. pyramidalis is sometimes epiphytic, it is possi ble that the plant absorbed the

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33 ammonia excreted by the mosquitoes, thus redu cing the probability of chemical interference from nitrogenous wastes in this phytotelm. On the other hand, at 24 hours as much as 30 mL of water was added to some of the plants that had le aked water. This addition of water could have reduced or eliminated the effect s of chemical interference of Wyeomyia spp. on A. albopictus in this experiment. The dialysis bag used in the chemical interference experiment physically separated A. albopictus from Wyeomyia spp. Thus, other potential forms of interspecific competition, such as encounter, resource, or space competition were eliminated. In some other experiments of this chapter, A. albopictus were in direct contact with Wyeomyia spp., which resulted in a significant reduction in growth of A. albopictus In the chemical interfer ence experiment, no significant effects were detected, suggesting that encount er, resource or space competition plays an important role in the reduction of growth and survivorship seen in th e other experiments. Previous experiments where surface area to volume ratios were manipulated in order to influence the impact of encounter competition have often been unsuccessful. Broadie and Bradshaw (1991) altered the su rface area to volume ratio of tr eehole microcosms, but only the density of A. sierrensis had an effect on pupation success, larval development time, or pupal weight. Anholt (1990) altered the surface area av ailable to damselfly larvae in cages, which had no effect on density dependent decreases in grow th and survival. Damselfly larvae exhibited a behavioral response to the increas e of structural complexity insi de cages, but habitat complexity did not change the decrease in growth, survival time, and size seen at high densities (Anholt 1990). In the current study, while the surface area to volume ratio of the plant had no effect on the development rate of A. albopictus there was a significant varia tion in survivorship of this

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34 species among the different surface area to volume ratios. Mean survivorship in size 2 differed significantly from size 1. The biological significance of this difference between these two sizes is unknown. Another unexpected result of th e surface area to volume ratio experiment was the reduced survivorship of A. albopictus in the absence of Wyeomyia spp.. The three largest surface area to volume ratios appeared to contribute to most of this reduction in survivorship, although the interaction between plant size and Wyeomyia spp. was not significan t (Table 2-3). While the results of the surface area to volum e ratio experiment do not explain the effects of Wyeomyia spp. on A. albopictus, increased habitat complexity in this experiment reduced the development time of A. albopictus in the presence of Wyeomyia spp.. In the habitat complexity experiment the addition of waterproof paper incr eased the foraging surface area This increase provided additional surface area to the developing A. albopictus a factor which was not included in other competition experiments in this thesis. In this experiment, while more foraging area was added to the treatments, the total foraging area between treatments was kept the same, because in both treatments the size of the paper added, and the number of pieces were kept constant, the paper was folded only to increase complexity. By the end of the experiment though, many of the low complexity treatment squares had become appr essed to the sides of th e artificial bromeliad, thus resulting in a decrease in the foraging surface area between the two habitat complexities. Therefore, habitat complexity may ha ve decreased the development time of A. albopictus in the presence of Wyeomyia spp., but the difference in foragi ng surface area between the habitat complexities may have contributed to this decrease in development time. If this effect is due to habitat complexity, its re levance under field conditions is uncertain. When Wyeomyia are present in bromeliads, which is the case in most of southern Florida (O Meara et al.1995b), then

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35 increased complexity could decrease the development time of A. albopictus Whether increased habitat complexity affects the developmental rate of A. albopictus in the absence of Wyeomyia spp. remains uncertain as no treatments without Wyeomyia were used in this experiment. The results of the behavior experiment indicated that Wyeomyia spp. spent most of the time foraging on the bottom of the container, especially during periods of low food concentrations. When in contact with Wyeomyia spp., A. albopictus spent significantly less time on the bottom of the container, and stayed the furthest away from Wyeomyia spp. by spending significantly more time at the t op of the container irrespective of food concentration. Such behavior of A. albopictus could indicate avoidance of Wyeomyia spp. These experiments do not support chemical interference as a likely competitive mechanism to explain negative effects of Wyeomyia spp. on A. albopictus From the habitat complexity experiment, encounter competition is the likely explanation for the increase in development time of A. albopictus in the presence of Wyeomyia spp., but, as mentioned before, increased foraging area may have al so contributed to this effect. Like other surface area to volume ratio habitat experiments performed with mosquitoes, this surface area to volume ratio experiment failed to explain the competitive interactions between A. albopictus and Wyeomyia spp.. The behavioral experiment provided some indication that A. albopictus larvae change location within the container in the presence of Wyeomyia spp. These changes in location could be a result of encounter competition causing A. albopictus to change their location to avoid encounters with Wyeomyia spp. This avoidance of Wyeomyia spp. may contribute to the decrease in development rate observed among A. albopictus in the presence of Wyeomyia spp..

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36 Table 2-1. Means and standard errors for the con centrations of ammonia and pH preand postexperiment. Paired t-tests showed no signifi cant differences (alpha= 0.05) betw een preand post-experiment means. pH (ppm) (mean SE) Ammonia (mean SE) Pre-experiment 6.21 0.02 0.22 0.01 Post experiment 6.19 0.02 0.21 0.01

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37 Table 2-2. Analysis of variance for A. albopictus average instar based on the presence or absence of Wyeomyia spp. and the surface:volume ratio of the artificial plant and the interaction of these two variables. F Df P -value Wyeomyia 162.38 1 <0.01 Bromeliad Size 2.01 3 0.11 Wyeomyia x Size 4.56 3 <0.01

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38 Table 2-3. Analysis of variance for A. albopictus average survival based on the presence or absence of Wyeomyia spp., and the surface area:volume ra tio of the plant (size) and the interaction of these two variables. F Df P -value Wyeomyia 6.48 1 0.01 Bromeliad Size 5.41 3 <0.01 Wyeomyia x Size 1.56 3 0.20

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39 Table 2-4. Multivariate analysis of variance table for feeding location. Effect Wilks Lambda Df P -value Species 65.20 6 <0.01 Alone 2.66 6 0.02 Food 8.30 6 <0.01 Time 0.88 42 0.68 Species x Alone 4.83 6 <0.01 Species x Food 4.68 6 <0.01 Alone x Food 3.84 6 0.02 The species effect refers to the location of either A. albopictus or Wyeomyia spp.. Alone refers to whether or not each species is alone or in the presence of the other species. The food category variables were either 0.1 g leaves (low treatment) or 0.5 g leaves (high treatment). Time is the hour of observation from the start of the experiment.

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40 Table 2-5. Analysis of variance for the e ffects of food level presence or absence of Wyeomyia spp. on the location of A. albopictus Combo indicates the trea tments that had both Wyeomyia spp. and A. albopictus. F Df P -value Combo High Food 15.40 3,63 <0.01 A. albopictus only High Food 3.49 3,63 0.02 Combo Low Food 12.24 3,63 <0.01 A. albopictus only Low Food 1.12 3,63 0.35

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41 Table 2-6. Analysis of variance for the effect s of food level and presence or absence of A. albopictus on the location of Wyeomyia spp. F Df P -value Combo High Food 14.55 3,63 <0.01 A. albopictus only High Food 33.87 3,63 <0.01 Combo Low Food 39.58 3,63 <0.01 A. albopictus only Low Food 15.58 3,63 <0.01 Combo indicates the tr eatments that had both Wyeomyia spp. and A. albopictus

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42 Figure 2-1. Construction of the squares for the habitat complexity experiment. A) the low complexity square is unaltered before in troduction to the bromeliad. B) the high complexity square was cut along the lin es indicated inside the square. A A B B 1.5 cm 3cm 3cm A B

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43 Figure 2-2. The folded shape of the high comple xity and low complexity treatments and the orientation of the squares within the cones. A) in the low complexity experiment the squares floated on the top, or laid on the side s of the cone. C) in the high complexity cone, the altered squares create a lattice-like formation on the inside of the cone. B) The diagram in the center shows the shape of the altered square after the cutting and folding described in Fig. 2-1. A C B

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44 Figure 2-3. Average instars of A. albopictus after 48 h exposure to Wyeomyia spp. through a dialysis membrane and without Wyeomyia in the control. The error bars are standard error.

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45 Figure 2-4. Mean survivorship of A. albopictus after 48 h exposure to Wyeomyia spp. through a dialysis membrane and without Wyeomyia spp. in the control. The error bars are standard error.

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46 Figure 2-5. The average instar SE of A. albopictus in the absence or presence of Wyeomyia spp. in four plant sizes. NoWy re fers to the treatments with no Wyeomyia spp., and Wy refers to treatments with Wyeomyia spp.. The size below these symbols refers to the surface area to volume ratio in the arti ficial bromeliad, proceeding from smallest (1) to largest (4), as qua ntified in Materials and Me thods. Means with common letters above the bars are not significantly different by ( P <0.05).

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47 Figure 2-6. The average survivorship SE of A. albopictus in the absence or presence of Wyeomyia spp. in four plant sizes. NoWy re fers to the treatments with no Wyeomyia spp., and Wy refers to treatments with Wyeomyia spp.. The size below these symbols refers to the surface area to volume ratio in the artificial bromeliad, proceeding from smallest (1) to largest (4), as quantified in Material s and Methods. Mean values without common letters above the ba rs are significantly different ( P <0.05).

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48 Figure 2-7. The average instar (+ SE) of A. albopictus in two levels of habitat complexity and three levels of surface ar ea to volume ratio. L and H denote the low and high complexity treatments. Numbers corres pond to increasing surface area to volume ratios, as indicated in Materials and Met hods. Mean values w ithout common letters above the bars are significantly different by a Bonferroni adjusted multivariate pairwise test.

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49 Figure 2-8. Location of A. albopictus in the presence and absence of Wyeomyia The bottom and top locations are significantly different between Wyeomyia and no Wyeomyia treatments (alpha<0.05) by a Tukey mean s comparison. The bars indicate SE.

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50 Figure 2-9. Location of Wyeomyia spp. in the presence and absence of A. albopictus No locations were signifi cantly different between A. albopictus and no A. albopictus treatments (alpha<0.05) by a Tukey mean s comparison. The bars indicate SE.

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51 Figure 2-10. Location of A. albopictus by food concentration and the presence and absence of Wyeomyia spp.. The bars indicate SE. Different letters over the same bar category indicate significantly different means am ong the food concentrations (high and low) and treatments ( Wyeomyia spp. and No Wyeomyia spp.) for each location (side, bottom, top and wander) (alpha=0.05).

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52 Figure 2.11: Location of Wyeomyia spp. by food concentration and the presence and absence of A. albopictus The bars indicate SE. Different letters over the same bar category indicate significantly different means am ong the food concentrations (high and low) and treatments ( A. albopictus spp. and No A. albopictus spp.) for each location (side, bottom, top and wander) (alpha=0.05)

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53 CHAPTER 3 Wyeomyia spp. AS POTENTIAL PREDATORS OF A edes albopictus IN BROMELIADS IN SOUTHERN FLORIDA Introduction In mosquito populations predation can effect the survivorship (G riswold and Lounibos 2006, Blaustein et al. 1995, Fincke et al. 1997), and development time (Alto et al. 2005, Grill and Juliano 1996) of the prey as predators consum e prey or the prey become less active in the presence of predators. These alterations in survivorship and development time can effect the population and community structur e of both the predator and prey species (Sih 1985). Intraguild predation is the killing and cons umption of competitors that consume similar resources, which has an immediat e energetic benefit and a benefi cial reduction in exploitative competition for the predator (Polis et al. 1989). Age and size dependent intraguild predation is common throughout all ecological systems, and many predators are cannibalistic on smaller sized or younger conspecifics (Po lis et al. 1989). Often intra guild predation occurs between competitors with the greatest resource overlap, and may be a result of the increased encounter rates due to niche overlap (revi ewed by Polis et al. 1989). Mosquitoes that are predominantly br owsers and filter feeders, such as Aedes and Anopheles species (Clements 1999), may become canni bals (Hinman 1934, Reisen and Emory 1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003) or facultative predators (Edgerly et al. 1999, Koenraadt et al. 2003) under certain co nditions. Koenekoop and Livdahl (1986) and Edgerly et al. (1999) found that cannibalism in Aedes triseriatus was resource dependent with lower resource availability lead ing to higher cannibalism. Koenraadt et al. (2003, 2004) found no increase in cannibalism or predation by Anopheles gambiae complex mosquitoes related to resource availability. Increased habitat complexity also reduced predation by A. triseriatus on congeners, but the opposite effect was seen with A. aegypti (Edgerly et al. 1999). Koenraadt et

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54 al. (2004) also showed that higher densities of anopheline mosquitoes in small spaces increased cannibalistic and predatory behavior. Although facultative predation may infl uence the population dynamics of many Aedes and Anopheles mosquitoes, W. mitchellii and W. vanduzeei have yet to be examined as potential predators of A. albopictus in bromeliads. Because predation is an important mechanism shown to effect the survivorship and development ti me of mosquito species in other studies, Wyeomyia spp. larvae from Florida bromeliads were dissected after exposure to A. albopictus to determine whether these Wyeomyia spp. may be facultative predators of A. albopictus Materials and Methods Testing for Density Dependent Predation by Wyeomyia spp. In this experiment, three densities (0, 25, 50) of 1st instar A. albopictus were placed into plastic containers containing 75 mL sieved br omeliad water and 0.5 g dried (68C for 48 h), chopped Quercus virginiana leaves. Thirty-six containers we re used, allowing 12 replicates of each density. To each container thirty 4th instar Wyeomyia spp. were added after previously being kept in tap water without food for 36 h prior to the expe riment. The lack of food for Wyeomyia spp. prior to the experiment was to ensure that all gut contents examined after the experimentation resulted from the food obtained during the experiment, a nd not prior feedings. After 12 h, 6 containers were removed from each density, and all live 4th instar Wyeomyia spp. larvae were dissected. At 24 h, the remaining containers were removed and again all of the live 4th instar Wyeomyia spp. larvae were dissect ed. Dead larvae of Wyeomyia spp., and pupae were not dissected. Testing for Resource Dependent Predation by Wyeomyia spp. In a second experiment, two de nsities of food resources (0.1 g and 0.5 g) of dried (68C for 48 h), chopped Q. virginiana leaves were placed into 24 plas tic containers with 75 mL sieved

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55 bromeliad water and 25 1st instar A. albopictus Twenty-five 4th instar Wyeomyia spp. were added to each container. Twelve hours and 96 h afte r the start of the experi ment, 6 containers at each food level were re moved and their live 4th instar Wyeomyia spp. were dissected. As before, dead Wyeomyia spp., and pupae were not dissected. Predation Observation Visual examinations of the behavior of A. albopictus and Wyeomyia spp. were conducted in plastic cont ainers. Ten 1st instar A. albopictus were added with either (1) 0.1 g food and no Wyeomyia spp.; (2) 0.1 g food and 10 4th instar Wyeomyia spp.; (3) 0.5 g food and no Wyeomyia spp.; or (3) 0.5 g food and 10 4th instar Wyeomyia spp.. There were two replicates of each combination. The food was dried, chopped Q. virginiana leaves as used in the two previously described predation experiments. Two cups at each food level were selected, every hour for 10 h, to be examined for signs of predation by Wyeomyia spp. on A. albopictus Predatory behavior was classified as chewing, biting, grabbing or actually consuming another organism. Each examination occurred for 1 min, and since light was necessary for visualization of the 1st instar A. albopictus a desk light kept the cups illuminated for the entire 10 h duration of the experiment. Results No A. albopictus body parts were found in the guts of any of the 941 Wyeomyia spp. dissected (Table 3-1). All guts of Wyeomyia spp., including the treatment without A. albopictus contained brown organic material but no signs of head capsules, or any other body parts, of A. albopictus There were also no signs of aggression upon A. albopictus by Wyeomyia spp. throughout the entire duration of the predation observation experiment.

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56 Discussion From these experiments, it seems very unlikely that Wyeomyia spp. prey upon A. albopictus When field-collected larvae of W. vanduzeei were examined by Fish (1976), no remains from any invertebrates were found; onl y organic particulate matter, protozoans and pollen grains were found in the gut contents. In a study by Broberg and Bradshaw (1995), the pitcher plant mosquito, Wyeomyia smithii was found to not exhibit density dependent cannibalism. Therefore, although othe r detritivores, such as some Aedes and Anopheles species, can be cannibalistic or facultative predators, it seems that Wyeomyia spp. of North America do not exhibit these characteristics, at least under the density and re source conditions examined in the lab. As an adaptation to the variable wate r and resource levels in a bromeliad, W. vanduzeei can develop slowly on limited food resources (Frank and Curtis 1977), so the Wyeomyia spp. of Florida bromeliads may not have adopted a pred ation strategy, because th ey have adapted to a food limited environment. Most Aedes and Anopheles species that are cannibalistic or facultative predators would starve at food levels that Wyeomyia spp. can subsist upon (Barrera and Medialdea 1996). As with any lab experiment, the applicability of these results to nature comes into question. As mentioned in other chapters bromeliad phytotelmata vary in size, structure, complexity, water holding capacity, and faunal composition. All of these factors could influence the expression of predation in Wyeomyia spp.. Variations of brome liad phytotelm size, structure, and complexity have been examined to some extent in this study. In this study, whether conducted within an actual bromeliad or an artif icial one, there was no si gnificant reduction in survivorship of A. albopictus that would indicate pred ation. In conclusion, if there is predation

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57 by Wyeomyia spp. on A. albopictus it occurs so rarely as to be unimportant in overall survivorship of A. albopictus

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58 Table 3-1. Number of Wyeomyia examined in predation experiments. Density Dependent Resource Dependent Time 0 Albo. 25 Albo. 50 Albo. Low food high food 12 h 111 123 128 74 111 24 h 117 126 151 n/a n/a 96 h n/a n/a n/a 87 158 This table compiles the number of dissected Wy eomyia spp. from two different experiments. The time box indicates the number of hours after A. albopictus were added that Wyeomyia were examined. The next three categories, 0 Al bo., 25 Albo., and 50 Albo., correspond to the first experiment in which the Wyeomyia were examined in the presence of 0, 25 and 50 1st instar A. albopictus. The next two categories, low food and high food, indicate th e treatments in the second experiment in which Wyeomyia spp. were examined after exposure to 0.1 g or 0.5 g of food in the presence of 25 1st instar A. albopictus.

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59 CHAPTER 4 DO W yeomyia spp.. LARVAE INHIBIT EGG HATCH OF A edes albopictus ? Introduction Eggs of Aedes spp. hatch after submersion in wate r, and low oxygen levels caused by nearby microbial growth stimulate hatching (G jullin et al. 1941, Judson 1960, Fallis and Snow 1983). Most eggs will hatch in response to th e initial stimulus, but some hatching is delayed, awaiting later stimuli (Gille tt et al. 1977, Livdahl and Ko enekoop 1985, Andreadis 1990). Suppression of egg hatch by larvae occurs wh en the larvae consume mi crobes on and near the eggs, thus causing a reduction in the local mi crobial population, and a su bsequent increase in oxygen resulting in inhibition (Gillett et al. 1977, E dgerly and Marvier 1992) There are also abiotic secondary determinants of hatch inhibi tion, such as temperature (Mallack et al. 1964), photoperiod (Horsfall 1956, McHaffey and Ha rwood 1970, McHaffey 1972, Shroyer and Craig 1980), and variations in wet a nd dry periods or humidity (A ndreadis 1990, Clements 1999), which will also delay hatch by putting eggs in to quiescence or diapause until conditions are favorable for hatching Egg hatch inhibition is hypothesized to benefit larvae by delaying hatch when competition for resources is high (Livdahl et al. 1984, Livdahl and E dgerly 1987), risk of predation is high (Koenekoop and Li vdahl 1986), or when abiotic c onditions are unf avorable for development (Shroyer and Craig 1980, Clements 1999). For many Aedes species, these benefits are thought to outweigh the costs of e gg hatch inhibition, which increases the time until reproduction. Increasing the time to reproduc tion may lead to a reduc tion in population growth and may increase mortality in the dormant egg st age of aedine mosquitoes (Livdahl and Edgerly 1987).

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60 Because many Aedes mosquitoes exhibit some level of hatch inhibition (Edgerly et al. 1993), it is important to examine this role of competition on Aedes hatch. Within the exotic bromeliads of southern Florida, larval competition between Wyeomyia spp. and A. albopictus is thought to influence the mosquito community stru cture in this system (Lounibos et al. 2003), although the mechanism for this competition has ye t to be determined. Field collections from exotic bromeliads in southern Flor ida show that high densities of 4th instar Wyeomyia spp. are common in exotic bromeliads (Raban unpublished data). Additionally, Wyeomyia larvae develop slowly, as an adaptation to the variable water and resour ces levels in a bromeliad (Frank and Curtis 1977), so it is feasib le that these large, later inst ar larvae are present for long durations. Also, W. mitchellii and W. vanduzeei are found in the larval stage throughout the year, while some A. albopictus overwinter as eggs. Thus even an initial spring A. albopictus cohort could experience egg hatch inhibition from the presence of Wyeomyia spp. Therefore, a resource dependent egg inhibiti on experiment was conducted to determine whether older instar Wyeomyia spp. inhibit the hatch of A. albopictus eggs and whether inhib ition varies with larval food level. Materials and Methods Fifty A. albopictus eggs on each of 56 papers inserted individually in plastic cups were submerged in 75 mL of sieved bromeliad water. All A. albopictus eggs used in the study were from one oviposition paper, re sulting from approximately 3 da ys of oviposition from a Florida colony of this species supplemente d irregularly with wild mosqu itoes. All eggs were counted under a dissecting microscope to ensure no hatch or damage before the start of the experiment. Twenty-eight of these cups were randomly a ssigned to a low food level of 0.1 g of dried, chopped, live oak leaves ( Q. virginiana ), and the remaining 28 were assigned a high food level of 0.5 g of dried, chopped, live oa k leaves. All leaves were dr ied at 41C for 72 h before the

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61 experiment. At each food level, 20 field collected, 4th instar Wyeomyia spp. were added to 14 of the cups. The other fourteen cups contained only the 50 A. albopictus eggs and served as controls. After 48 h the experiment was terminat ed, and the egg hatch ra tes were recorded for each cup. The number of eggs hatched was determined by counting the number of A. albopictus larvae present and the number of eggs that ha tched on the paper. Whichever hatch number was higher, number of larvae or the number of eggs hatched on the paper, was considered the hatch number for that cup. In almost all cases, the number of eggs hatched on the paper exceeded or equaled the number of larvae found in the cup. The bleaching technique of Trpis (1970) was used to determine the viability of the unhatched eggs. Eggs were considered viable if they had a fully developed embryo. Empty eggs and partial or shriveled embryos were considered inviable and were omitted in the data analysis. On the other hand, if eggs were not wholly enca sed and appeared to be crushed or damaged, these eggs were classified as damaged. Due to the high number of inviable eggs, an ANOVA was conducted on their incidence to ensure their uniform spread across treatments. To ensure homogeneity of variances, hatch rates were arcsine square root transformed before an ANOVA was performed in SAS (2002) with a subsequent Tukeys post-hoc test A Kruskal-Wallis test and a Dunnets test was performed on the damaged egg data. Two separate treatments had one replicate each that was omitted from the analysis because they both had a 0.0% hatch rate with only 2 to 4 viable eggs per replicate. Results Overall, 29.0% 2.4% (mean SE) of all e ggs were inviable, with no difference among treatments (F3,50= 1.88, P = 0.14). There was a significant difference between the hatch rates of the treatments (F3,50=6.28, P =0.02), which was attributed to the high food with Wyeomyia spp. treatment being significantly different than both food tr eatments without Wyeomyia spp. (Fig. 4-

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62 1). There was a lower hatch rate for the treatments with Wyeomyia spp. as compared to the treatments without Wyeomyia spp., although this difference was only significant for the higher food concentration (Fig. 4-1). On the othe r hand, there was a significant variation ( 2=8.06, df=3, P =0.04) among treatments in the in cidence of damaged eggs (Fi g. 4-2). Significantly more damaged eggs were observed in the low food treatment with Wyeomyia spp. compared to both the food treatments without Wyeomyia spp. (Fig. 4-2). Discussion In this experiment, there was a significantly lower hatch rate of A. albopictus in the presence of fourth instar Wyeomyia spp at higher food concentrati ons. These findings are not overly surprising as previous studies by Edgerly et al. (1993) showed reduced hatch of A. albopictus in the presence of high de nsities of larger instar Aedes larvae at 24 h. While the hatch rate of A. albopictus in the lower resource treatment with Wyeomyia spp. was lower than the lower resource control, this difference was not significant, indicating that resource level may influence the egg hatch inhibition in A. albopictus Even though in this experiment egg hatch inhibition by Wyeomyia on A. albopictus was found to influence the hatch rate at high food con centrations, the effects on hatch rate were only moderate. In this experiment, treatments with Wyeomyia experienced only an approximately 9% decrease in hatch rate at 48 h, a nd 80% to 90% of viable eggs st ill hatched in all treatments. Therefore, while there may be egg hatch inhibition, if the hatch rate s seen in this experiment are indicative of natural field hatch rates, th en the influence of egg hatch inhibition on A. albopictus development is not pronounced and does not ex plain the decrease in development rate of A. albopictus in the presence of Wyeomyia spp. seen in the surface area to volume ratio experiment, and the larval experiment in Lounibos et al. (2003).

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63 The reduced effects of egg hatch inhibition on A. albopictus are not surprising, as Edgerly et al. (1993) found that at higher larval densities A. albopictus egg hatch was inhibited, but at reduced rates as compared to other Aedes spp. mosquitoes tested. So, in general it is possible that A. albopictus does not experience pronounced egg hatc h inhibition. Also, this experiment was allowed to run for 48 h, while the Edgerly et al. (1993) experiment was terminated at 24 h, so it is possible that most pronounced egg hatch inhibition occurs in the first 24 h, which was missed in a 48 h experiment. Livdahl et al. (1984) and Livda hl and Edgerly (1987) found that Aedes triseriatus hatch rates are reduced by large densities of late instar conspeci fics. These authors hypothesized that large 4th instar larvae inhibit hatc h through their elevated grazi ng intensity, which reduces oxygen-depleting microbes. The si gnificantly larger amount of e gg damage seen in the presence of Wyeomyia spp. in this experiment may be a re sult of browsing by the late instar Wyeomyia spp. on the eggs of A. albopictus Because the percentage of e ggs damaged was highest in the low resource group (but not significantly different from high resources) with Wyeomyia spp., it suggests that with fewer l eaf resources to graze upon, Wyeomyia spp. may have increased their grazing upon A. albopictus eggs. The majority of egg damage probably occurred after hatch or did not effect hatch rate, b ecause the high resource with Wyeomyia spp. treatment had lower hatch rate than the lower resource with Wyeomyia treatment, but the lower resource treatment with Wyeomyia spp. had higher egg damage. Like wise, the lower resource with Wyeomyia spp. treatment did not have a significan tly different hatch rate from e ither of the controls at each resource level, but it did have significantly more egg damage. Thus, there seems to be no strong relationship between hatch rate and egg damage.

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64 In nature, egg hatch inhibition of A. albopictus within bromeliads could differ from the laboratory results. In the t ypical bromeliad habitat many ot her organisms could also be influencing the bacterial conten t of the area around the eggs and, thus, the hatch rate of A. albopictus eggs. Many other bacteriaand detritusconsuming macroinvertebrates are known to inhabit bromeliads (Fish 1976, Frank 1983). Additionally, the bromeliad itself can influen ce the chemical composition of its contents based on its physiological needs (see bromeliad as a ha bitat section in Chapter 1 of this thesis). For instance, Fallis and Snow (1983) determined that an increase in water nitrogen induced hatch in Aedes punctor and nitrogen is one of the main chem icals that is absorbed by bromeliads (Benzing 2000), and thus may influence the hatch of A. albopictus Fallis and Snow (1983) also found that the change in oxygen content, and not the concentration of oxygen was the hatching trigger for A. punctor which may also be regulated by the bromeliad. So, although Wyeomyia spp. may inhibit the hatch of A. albopictus eggs at high resource levels it is possible that other organisms or processes within the bromeliad can alter egg hatch inhibition in A. albopictus With more macroinvertebrates in the aqua tic community, there co uld be more grazing and an increase in localized oxygen levels near th e eggs, and thus more i nhibition. In contrast, Edgerly and Marvier (1992) hypothe sized that at a certain dens ity, the number of organisms surrounding eggs can be great enoug h to deplete the overall oxygen levels in the water, and thus stimulate egg hatch. Although as mentioned befo re, Edgerly et al. ( 1993) found that at high densities A. albopictus had a low level of hatch inhibiti on compared to congeners, so higher densities of mosquitoes may have little effect on the overall hatch of A. albopictus In this experiment the eggs of A. albopict us were submerged below the water line in a cluster on the side of the container. In th e location study from Chapter 2 of this thesis

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65 Wyeomyia spp. larvae were located more often at the bottom and at the to p of the container and less on the sides. If A. albopictus does not oviposit its eggs on the inner ax il walls of bromeliads, then this experiment may misrepresent the in fluence of Wyeomyia s pp. on egg hatch inhibition of A. albopictus. There is no ev idence that A. albopictus lays its eggs on the sides of the bromeliads, nor is it probable that their eggs are laid in one cluste r as in this experiment. Further investigation into the location and distribution of oviposite d eggs of A. albopictus within bromeliads is needed to determine the relevance of this experiment to natural conditions.

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66 Figure 4-1. Proportion hatch of viable eggs SE in each treatment. High Food refers to high food treatments, and Low food refers to low food treatments. No Wy indicates treatments with no Wyeomyia spp. and Wy indicates treatments with Wyeomyia spp.. Treatments with the same letter above the SE bar are not significantly different based on a Tukey test.

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67 Figure 4-2. Proportion eggs damaged SE per treatment. High Food refers to high food treatments, and Low food refers to low food treatments. No Wy indicates treatments with no Wyeomyia spp. and Wy indicates treatments with Wyeomyia spp.. Treatments with the same letter above the SE bar are not significantly different based on a Dunnetts Test.

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68 CHAPTER 5 FIELD STUDIES ON A edes albopictus AND Wyeomyia spp. IN EXOTIC BROMELIADS OF SOUTHERN FLORIDA Introduction In a study conducted by OMeara et al. (1995b), Wyeomyia spp. were the most abundant mosquitoes in bromeliads throughout southern Florida, but in northern Florida where W. vanduzeei and W. mitchelii are absent, A. albopictus was the most common mosquito species in bromeliads. In the southern sites with Wyeomyia spp., A. albopictus was often found in great numbers in nearby artificial contai ners. For example, when vases were placed near bromeliads in Vero Beach, Florida, A. albopictus larvae were subsequently collected from all the vases but from only 40 percent of the bromeliads. In the study of Lounibos et al. (2003), A. albopictus was also found in bromeliads in grea ter numbers in the absence of Wyeomyia spp.. Wyeomyia spp. vary in larval abundance and ovipos itional preference based on the species of bromeliad. In collections by OMeara et al. (1995b), Wyeomyia spp. were less abundant in Neoregelia spectabilis than in Aechmea fasciata Frank and OMeara (1985) also found that W. vanduzeei showed an oviposition prefer ence for the native bromeliad Tillandsia utriculata over another native bromeliad, Catopsis berteroniana The purpose of the current study was to furt her explore the differences in abundance and distribution of Wyeomyia spp. and A. albopictus within bromeliads based on the location within the plant (axil or central tank), by bromeliad species, and by macrohabitat. Neoregelia spectabilis and B. pyramidalis are two exotic bromeliad speci es commonly featured in the residential landscaping of many homes in the citie s of Vero Beach and Fort Pierce, Florida. These two plants were chosen for further st udy because they were common and distinctly different in size, color, and shape, but are ofte n grown in the same location. In the current study,

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69 N. spectabilis and B. pyramidalis were sampled to compare the abundances of Wyeomyia spp. and A. albopictus in these two bromeliad species. Due to the differences in their physical characteristics these bromeliads may have different abundances of mosquitoes. These samples were further divided by location wi thin the plant, i.e. lateral ax il or central axil, to determine whether the abundance of Wyeomyia spp. or A. albopictus varied by location within the plant. It is also hypothesized that the abundance of mosquitoes will no t vary by location within the bromeliad, because bromeliad studies by Frank and Curtis (1977) have indicated that during rainfall eggs may be washed to other locations within the plant. Thus, if rainfall occurs frequently enough there should be a rather homog eneous distribution of larvae throughout the plant. To further describe habitat preferences of bromeliad-inhabiting mosquitoes, mosquitoes were collected from the field to determine whether mosquito abundance varied between two canopy types, mainly oak tree and palm tree. Frank and OMeara (1985) determined that W. mitchellii preferred shady habitats an d, thus, different canopy types could also provide shade variations that influence the a bundance of bromeliad mosquitoes. Materials and Methods Differences in the Density of A. albopictus and Wyeomyia spp. within N. spectabilis and B. pyramidalis The aquatic contents of B. pyramidalis and N. spectabilis bromeliads were collected with a meat baster monthly from September 2005 to July 2006 at eight sites in Vero Beach and Fort Pierce, Florida, and from two s ites in Orlando, Florida (Fig 5-1). On a few occasions plants in Tampa Bay and Washington Oaks Park were also sampled (Fig 5-1). At each site, five to twenty-five plants of eac h species were sampled every 4 to 8 weeks. To avoid taking repeated samples from the same plants, at each sampling the approximate locations of the samples were recorded, although repeated sampling may have occu rred at two of the smaller Vero Beach, FL

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70 sites due to a reduced number of plants. The sites where re-sampling of plants was suspected were omitted from all analyses. From each plant two samples were taken by removing all extractable fluid contents with the baster. One sample consisted of the contents of the central axil of the plant, while a separate sample was taken of the lateral axil contents. The central axil sample was extracted from the area of water held in th e center of the plant (photo A in Fig. 2.2), and the lateral axil sample was extracted from the spaces created by the lateral axils of the plant (photo B in Fig. 2.2). Due to the fact that B. pyramidalis with inflorescences held very little, if any, water, only B. pyramidalis that were not flowering were samp led in this study. Conversely, since N. spectabilis holds very little if any water in its central tank when it is not flowering, only flowering N. spectabilis were sampled in this study. The sp ecies and development stage of each mosquito immature collected were record ed. To aid in the identification of 1st instar larvae, all samples were initially examined in trays under a dissecting scope. Studies by Frank et al. (1977) have shown a correlation between br omeliad size and the number of mosquito larvae and pupae. After February 2006, in the current study the amount of water in each bromeliad was also measured to determine whether a correlation exists between the amount of water in a bromeliad and the abundance of either species of mosquito and their total. Thus, the analysis of the data from these collections is br oken into two categories 1) all collections, with analyses being based on total mo squitoes per plant, a nd 2) collections after February 2006, which were analyzed by numbers of mosquitoes per volume of water. ANOVA calculated from type III sums of squares was app lied to detect significant variations in the dependent variables (densitiy of mosquitoes per mL and per plant) in relation to the independent variables (bromeliad species). Means comparisons were conducted with Tukeys tests. The

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71 density means (mos/mL and mos/plant) were also compared by a paired t-test in relation to the location within the plant (lateral vs. central axil). A repeated measures ANOVA, with subsequent means comparisons by Tukeys tests, wa s applied to detect significant variations in the dependent density variables by the month of collection. Correlations were performed using Spearmans between the volume of water in bromeliads and the numbers of A. albopictus and Wyeomyia spp. found within. Canopy Effects on Density A study was also conducted on the differences in density of Wyeomyia spp. and A. albopictus in bromeliads based on the surrounding ha bitat. Preliminary observations during collections showed that there may be fewer Wyeomyia spp. and A. albopictus in bromeliads with palm trees overhead versus in bromeliads shaded by oak trees. These habitats differ in amount of sun exposure, leaf litter, and throughfall input into the bromeliads below. The largest sample site in Vero Beach, FL (VB-5) contained four clumps of interspersed N. spectabilis and B. pyramidalis under palm trees and seve n clumps of interspersed N. spectabilis and B. pyramidalis under oak trees. In both habitats B. pyramidalis was the more common of the two bromeliad species. Once in April 2006 and once in June 2006 two samples, one from the central axil, and one from th e lateral axil, of fifteen B. pyramidalis and nine N. spectabilis were taken from under either an oak tree or a palm tr ee. In order to avoid sampling the same plants twice, the location of the first collection was different from the location of the second collection. The mosquitoes collected were counted and reco rded as described in the previous section. Significant effects of canopy type on the numbers of Wyeomyia spp. in bromeliads were tested by nested ANOVA with bromeliad species ( B. pyramidalis vs. N. spectabilis ) nested within canopy type (Oak vs. Palm). Aedes albopictus was omitted from ANOVA analysis as no larvae of this species were found in the oak location.

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72 Results Both mosquitoes per plant (mos/plant) and per unit volume (mos/mL) varied significantly between plant species (Table 5-1 and Table 5-2). Aedes albopictus had a higher density (mos/mL and mos/plant) in N. spectabilis, but Wyeomyia spp. was found in significantly higher densities in B. pyramidalis (Tables 5-3 and 5-4). Aedes albopictus had higher densities (mos/plant) in the central axils of N. spectabilis (t217= 2.22, P =0.03), but showed no difference in density between the central and lateral axils in B. pyramidalis (t242= 0.86, P= 0.39) (Table 5-4). Aedes albopictus also showed no difference in density (mos/mL) between the central and lateral axils in either N. spectabilis (t172= -0.70, P = 0.49) or B. pyramidalis (t193= 1.00, P = 0.32). Wyeomyia spp. showed no differences in densities (m os/plant) between the lateral and central axils of B. pyramidalis (t242= 1.38, P = 0.17) or N. spectabilis (t217= 1.60, P = 0.11). However, Wyeomyia spp. did have higher mean densities (m os/mL) in the lateral axils of both N. spectabilis (t172= 2.27, P = 0.02) and B. pyramidalis (t193= 2.52, P = 0.01) (Table 5-4). The densities (mos/plant) of Wyeomyia spp. and A. albopictus varied by the month of collection ( W .F6= 8.74, P <0.01 and A. albo .F6= 12.36, P <0.01) and by the site of collection ( W .F8= 10.18, P <0.01and A. albo .F8= 2.15, P =0.03). The density of A. albopictus in B. pyramidalis was relatively constant throughout the year, but in N. spectabilis the density at each site of A. albopictus increased in September and in April through July (Fig. 5-3). In B. pyramidalis the density of Wyeomyia spp. increased in September, decreased in November through March, and increased in April and in July. Aedes albopictus also experienced similar changes in density (mos/mL) over time, but Wyeomyia spp. density (mos/mL) did not differ significantly among months (Tab les 5-5, 5-6, Figs. 5-4, 5-5,). There were also distinct differences in the water holding capacity between the plants (Table 5-7). More water was extracted on average for N. spectabilis than B. pyramidalis (Table

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73 5-7). When the data were analyzed separately by the location within the plant there was significantly more water in N. spectabilis than B. pyramidalis in both lateral and central axils (Table 5-7). There were positive correlations between the number of Wyeomyia spp. ( 400= 0.12; P =0.01), the number of A. albopictus ( 400= 0.26; P <0.01), and the total mosquitoes ( 400= 0.18 P <0.01) and the amount of water extracted from the bromeliad. Wyeomyia spp. differed in densities be tween the palm and oak sites (Table 5-8), and by plant species in the palm and oak sites (Table 5-8). Wyeomyia spp. were less abundant in the palm sites, and in both the oak and palm sites Wyeomyia spp. was found in a significantly greater densities in B. pyramidalis plants (Table 5-9). Discussion In the current surveillance Wyeomyia spp. immatures were denser in B. pyramidalis than in N. spectabilis. OMeara et al. (1995b) showed that Wyeomyia spp. were more abundant in Aechmea. fasciata than in N. spectabilis On the other hand, A. albopictus had much higher mean densities in N. spectabilis than in B. pyramidalis The differences in abundances in each plant could indicate that A. albopictus is avoiding contact with Wyeomyia spp. by ovipositing in bromeliads not occupied by Wyeomyia spp., but in an experiment conducted by Lounibos et al. (2003), A. albopictus showed no difference in ovipositional preference between B. pyramidalis with Wyeomyia spp. present or absent. The Lounibos et al. (2003) experiment indicates that ovipositing A. albopictus are not responding to cues from Wyeomyia spp. larvae. The Washington Oaks site had the greatest abundance of A. albopictus and the lowest abundance of Wyeomyia spp. The studies by OMeara et al. (1995b) and Lounibos et al. (2003) indicate that more northern sites like Wa shington Oaks have more A. albopictus in their bromeliads than in the southern sites. These two studies also indicated that lower abundances of Wyeomyia spp. occurred in northern Florida, which was likely due to the intolerance of Wyeomyia spp. to

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74 temperatures below freezing, or to the rarity of native bromeliad phytotlemata in northern Florida. There were differences in the densities of Wyeomyia spp. and A. albopictus by location within the plant. Wyeomyia spp. had higher densities (mos/m L) in the lateral axils of B. pyramidalis and N. spectabilis so either Wyeomyia spp. oviposits more frequently in lateral axils, or if Wyeomyia spp. oviposit more eggs into the centra l axil, then some eggs or larvae are washed by rainfall into the lateral axils. Frank et al. (1976) ob served that female W. vandueezi oviposit more frequently in to the central axils of T. utriculata but were still f ound in the lateral axils of that bromeliad species. In a Frank and Curtis (1977) study, W. vandueezi were found to be easily washed out of the central tank of the bromeliad during rainfall. Therefore, it is unknown whether the lateral axils an d central axils have the same number of mosquitoes due to oviposition preference or due to egg a nd larval movement during rainfall. The Frank et al. (1977) study determined that there was a correlati on between the size of the bromeliad and the number of mosquitoes wi thin the bromeliad. The Frank et al. (1977) experiment used the total water ho lding capacity of the bromeliad as the indicator of the size of the bromeliad. In the current research, the actual am ount of water within the bromeliad, not the total holding capacity, was used to measure the de nsities of mosquitoes per milliliter of extracted fluid. While the quantity of wate r actually present is probably not as good an indicator of size as the total water holding capacity of the brome liad, it gives an approximation of the habitat available to the mosquito at the time of sampling. There was positive correlation between the amount of water present in the bromeliad and the number of mosquitoes extr acted, although its strength vari ed between mosquito species. Aedes albopictus had a stronger positive correlation with water volume than Wyeomyia spp.,

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75 which is possibly due to the fact that Wyeomyia spp. are bromeliad specialists, while A. albopictus is a container generalist. Wyeomyia spp. are probably better adapted to the variable bromeliad environment, and thus can thrive under a variety of bromeliad conditions, including reduced water. For instance, Frank and Cur tis (1977) hypothesized that the prolonged larval development of W. vanduzeei under reduced food conditions was an adaptation to the variable habitat in bromeliads. Aedes albopictus on the other hand, can also have prolonged larval development with reduced res ources (Barrera and Medialdea 1996), but probably cannot prolong its life as long as Wyeomyia spp.. Thus, under variable conditions A. albopictus females have to either be more selective for environments w ith more water for their oviposition, or risk a reduction in survivorship due to desiccati on or decreased space. Conversely, ovipositing Wyeomyia spp. females do not have to be as selectiv e for the amount of wa ter in the bromeliad habitat, as their larvae can tolerate alterations in wa ter conditions. In the habitat study Wyeomyia spp., but not A. albopictus, differed in mean densities under different canopy types. It is pos sible that with more samples, A. albopictus would have also differed in mean densities, because the total number of A. albopictus recovered from all plants was small. Since the palm trees offered less protection from the sun, the bromeliads under palms were exposed to more insolation. In a study by Frank and OMeara (1985), W. mitchellii showed a preference for shaded habitats. In the curren t study, on two occasions the larvae collected from most sites were identified to species. During both examin ations about 80-85% of the Wyeomyia spp. collected were W. mitchellii with the remaining being W. vanduzeei Thus, the decrease in density of Wyeomyia spp. in the palm habitat was most likely due to the preference of Wyeomyia mitchellii for shaded areas.

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76 Even though Wyeomyia spp. had the higher mean density in all plants, Wyeomyia spp. had a higher mean density in B. pyramidalis while A. albopictus had a higher mean density in N. spectabilis In the following chapter, oviposition and larval competition experiments were conducted to explain the causes of th e differing relative abundances of Wyeomyia spp. and A. albopictus in these two bromeliad species.

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77 Table 5-1. Analysis of variance for the densit y of mosquitoes per plant based on month (month of collection), site, and bromeliad speci es (N. spectabilis vs.B. pyramidalis). A repeated measures ANOVA was appplied to the month variable. df F-statistic P -value Month 6,472 0.27 0.53 Site 7,472 12.36 <0.01 Bromeliad species 1,472 7.92 <0.01

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78 Table 5-2. Analysis of variance for the density of mosquitoes per mL based on month (month of collection), site, and bromeliad species (N. spectabilis vs. B. pyramidalis). df F-statistic P -value Month 4,378 4.50 <0.01 Site 7,378 3.33 <0.01 Bromeliad species 1,378 42.92 <0.01 A repeated measures ANOVA was appplied to the month variable.

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79 Table 5-3. Analysis of variance for the density of each mosquito species per mL of water in each bromeliad species. Mosquito Bromeliad Mean SE (mos/mL) F-Statistic df P -value N. spectabilis 0.03.11 A. albopictus B. pyramidalis 0.0003.0003 7.16 1,399 <0.01 N. spectabilis 0.09.01 Wyeomyia spp. B. pyramidalis 0.26.02 42.53 1,399 <0.01 These means are representative of the data taken after February 2006. All samples including those with no mosquitoes were included in this analysis.

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80 Table 5-4. Mean densities of mosquitoes (mos/plant) in B.pyrami dalis and N. spectabilis based on location within the plant and plant species. 1: F1,472=42.53, P <0.01; 2: F1,472= 18.07, P <0.01; 3: F1,472= 18.51, P <0.01. Species Location albopictus abundance (mos/plant) Mean SE Wy. spp. abundance (mos/plant) Mean SE Total A. albopictus Abundance (mos/plant) Mean SE Total Wy spp. abundance (mos/plant) Mean SE Total mosquito abundance (mos/plant) Mean SE N. spectabilis central axil 1.67 0.32 2.53 0.44 2.58 0.39 5.67 0.77 8.30 0.89 N. spectabilis lateral axil 0.92 0.16 3.13 0.41 B. pyramidalis central axil 0.06 0.03 7.87 0.54 0.11 0.04 16.48 1.01 16.60 1.02 B. pyramidalis lateral axil 0.05 0.02 8.62 0.60

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81 Table 5-5. Analysis of variance table for th e effects of month and site on mos/mL and mos/plant. Mosquito Effect F-statistic df P -value mos/mL A. albopictus Month 9.17 4,378 <0.01 mos/mL Wyeomyia spp. Month 2.07 4,378 0.07 mos/plant A. albopictus Month 12.36 6,472 <0.01 mos/plant Wyeomyia spp. Month 8.74 6,472 <0.01 mos/plant A. albopictus Site 2.15 8,472 0.03 mos/plant Wyeomyia spp. Site 10.18 8,472 <0.01

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82 Table 5-6. Analysis of variance results for the effect of month and sites on mosquito abundances (mos/plant) by bromeliad species. Bromeliad Mosquito Effect F-statistic df P -value B. pyramidalis Total Mosquitoes Month 25.58 6,252 <0.01 Site 7.53 7,252 <0.01 N. spectabilis Total Mosquitoes Month 7.14 6,225 <0.01 Site 13.47 8,225 <0.01 B. pyramidalis Wyeomyia spp. Month 24.13 6,252 <0.01 Site 7.30 7,252 <0.01 N. spectabilis Wyeomyia spp. Month 3.86 6,225 <0.01 Site 16.86 8,225 <0.01 B. pyramidalis A. albopictus Month 6.37 6,252 <0.01 Site 0.64 7,252 0.73 N. spectabilis A. albopictus Month 12.99 6,225 <0.01 Site 2.02 8,225 0.05

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83 Table 5-7. Average (SE) amount of wa ter extracted from each plant species N. spectabilis lateral axil B. pyramidalis lateral axil N. spectabilis central axil B. pyramidalis central axil Total water N. spectabilis Total water B. pyramidalis Mean water (mL) SE 56.04 2.96 39.03 2.36 53.08 3.81 33.28 1.45 109.12 5.80 79.09 2.90 ANOVA results F1,198= 3.02; P <0.01by plant species F1,198= 47.59; P <0.01by plant species

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84 Table 5-8. A nested ANOVA table for effects of Oak vs. Palm canopy and bromeliad species ( B. pyramidalis vs. N. spectabilis ) on Wyeomyia spp. densities by mos/plant. F df P -value Wyeomyia spp. Oak vs. Palm 26.61 1,32 <0.01 B. pyramidalis vs. N. spectabilis 8.77 1,32 <0.01 Oak vs. Palm ( B. pyramidalis vs. N. spectabilis ) 6.73 1,28 0.01

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85 Table 5-9. Means and SE of densities of A. albopictus and Wyeomyia spp. by location within plants under oak and palm canopies. Mean SE Wyeomyia spp. Oak B. pyramidalis central axil 15.73 3.20 B. pyramidalis lateral axil 7.80 1.16 N. spectabilis central axil 3.85 0.92 N. spectabilis lateral axil 4.08 0.73 Wyeomyia spp.Palm B. pyramidalis central axil 1.00 0.50 B. pyramidalis lateral axil 0.75 0.41 N. spectabilis central axil 0.57 0.43 N. spectabilis lateral axil 0.14 0.14 A. albopictus Oak B. pyramidalis central axil 0 B. pyramidalis lateral axil 0 N. spectabilis central axil 0 N. spectabilis lateral axil 0 A. albopictus Palm B. pyramidalis central axil 0.13 0.13 B. pyramidalis lateral axil 0.38 .26

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86 Figure 5-1. Map of collection s ites. Figure adapted from OM eara et al. 1995b, Fig. 1, Pg. 218.

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87 Photo A Photo B Figure 5-2. Location of water samples. The wh ite arrow indicates the location where wate r samples were taken from the central a xil (Photo A) and the latera l axils (Photo B) of a N. spectabilis

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88 Figure 5-3. The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis and B. pyramidalis at each site. Error bars represent SE of the means at each site. For B. pyramidalis n= 243 plants (12.88 1.58 plants sampled per month at each site (mean SE)), and for N. spectabilis n= 218 plants (9.53 1.00 plants sampled per month at each site (mean SE)).

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89 Month Sept/Oct Nov/Dec Feb Mar Apr May Jun J ul y Mean number of mosquitoes per N. spectabilis at each site 0 5 10 15 20 25 30 35 A. albopictus Wyeomyia spp. a bd b bd bdc cd c c b cd b b b bd a e Figure 5-4: The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis at each site. Error bars represent SE of the means at each site. Means without a common letter written above indi cate significant differences for Wyeomyia spp.. Means without a common letter written be low indicate significant differences for A. albopictus

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90 Month Sep t / O ct Nov/D e c Feb Mar Apr Ma y Jun J u l y Mean number of mosquitoes per B, pyramidalis at each site 0 10 20 30 40 50 A. albopictus Wyeomyia spp. a b c c b c b bc a b b b b b b b Figure 5-5. The monthly mean densities of Wyeomyia spp. and A. albopictus in B. pyramidalis at each site. Error bars represent SE of the means at each site. Means without a common letter written above indi cate significant differences for Wyeomyia spp.. Means without a common letter written be low indicate significant differences for A. albopictus

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91 CHAPTER 6 OVIPOSITION AND LARVAL DEVELOPMENT OF A edes albopictus IN TWO EXOTIC SPECIES OF BROMELIAD IN SOUTHERN FLORIDA Introduction Oviposition behavior by the female determines th e aquatic habitat of th e mosquitos larval stages (Clements 1999). Thus, it is not surprising that most eco logical theories on oviposition selection are generally based on the assumpti on that adults choose oviposition sites that maximize the survivorship and reproductive ou tput of their offspring and minimize their development time (Srivastava and Lawton 1998). At least for some herbivor ous insects, there is some indication that fitness considerations of ovi positing females are also important in choice of oviposition sites (Mayhew 2001). Whatever the ecological significance of oviposition preference, numerous experiments have explored the cues that in sects use to select oviposition sites. Culicids select oviposition sites based on visual olfactory and tactile cues (Bentley and Day 1989). The most common visual ovipositional st imuli of studied culici ds are color cues. Members of the genus Toxorhynchites are often attracted to black colored containers (Hilburn et al. 1983, Jones and Schreiber 1994, Collins and Blackwell 2000), as are many species of the genus Aedes (Beckel 1955, Wilton 1968). Culex mosquitoes are most often found to be attracted to black and red (Dhileepan 1997), although colo r preference cannot be generalized by genus. Aedes triseriatus are attracted to darker colors in the blue spectrum (Williams 1962, McDaniel et al. 1976), and A. aegypti responded most strongly to black ar tificial bromeliads (Frank 1985). There are also many chemical ovipositional cues originating from the habitat and from conspecifics, predators, parasite s and other species. There ar e numerous studies which indicate that mosquitoes orient to cues from the or ganic material within the habitat (reviewed by Clements 1999). For instance, in a laboratory study conducted by Wilton (1968), A. triseriatus

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92 oviposited significantly more eggs in water collect ed from a treehole than in distilled water of similar color. Culex spp. have also been found to respond to habitat specific chemical cues derived from bacteria or the or ganic matter of the habitat (Ike shoji et al. 1967, Beehler et al. 1994). Species of other mosquito genera also or ient to chemical oviposition cues derived from the habitat (Ikeshoji and Mulla 1970b, Bentley et al. 1979, Millar et al. 1992, Beehler et al. 1994). Habitat humidity (Kennedy 1942), salinity (Nav arro et al. 2003), or algal content (Bond et al. 2005) also influenced the ovipositi on site selection of mosquitoes. Mosquitoes often respond to the presence of conspecifics and other species when choosing oviposition sites. Some of the first olf actory studies conducted on Culicidae involved the discovery of chemical oviposition cues in the egg rafts Culex spp. (Hazard et al. 1967, Ikeshoji et al. 1967, Osgood 1971, Bruno and Laurence 1979, La urence and Pickett 1982). In the Culex egg raft experiments, female mosquitoes were attrac ted to oviposition sites that contained conspecific eggs. Other mosquito species have shown a pr eference for ovipositing in the presence of conspecific larvae (Soman and Reuben 1970, Ben tley et al. 1976, Allan and Kline 1998, Mokany and Shine 2003), or larvae of other species (Bentley et al 1976, Allan and Kline 1998). Mosquitoes are hypothesized to be attracted to ov iposit in habitats with conspecifics, or other species, because the presence of other mosquitoes is a possible indication of a successful larval habitat (Clements 1999). Some culicids have ovipositional preferences related to the presence or absence of predators. In a study conduc ted by Munga et al. (2006), Anopheles gambiae avoided ovipositing in containers that had been prec onditioned with the waste of either of two predators. Avoidance of oviposition in the presence of predators has also been seen for Ochlerotatus australis

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93 (Mokany and Shine 2003), Culiseta longiareolata (Blaustein et al. 2004), and Aedes taeniorhynchus (Ritchie and Laidlawbell 1994). Once eggs are laid, the conditions within the habitat influence larval development. Since A. albopictus is a container-inhabiting mo squito, variations in natura l and artificial container types occupied by this species influence larval development and survivorship. In treehole communities, resource quantity within the ha bitat can influence larval development and survivorship (Fish and Carpenter 1982, Leonard and Juliano 1995, Walker et. al 1997) and also influence the community structure (Srivastava a nd Lawton 1998). The permanence of the habitat also influences the development of larvae (Blaustein and Chase 2007), with drying of the habitat often decreasing larv al survivorship. In southern Florida, A. albopictus is common in artificial or natural containers (O'Meara et al. 1995a) and is occasionally found inhabiting bromeliads (O'Meara et al. 1995b). Within these bromeliad habitats A. albopictus often co-occurs with other species such as W. mitchellii and W. vanduzeei and occasionally with A. aegypti Aedes bahamensis, Culex quinquefasciatus and ulex biscaynesis (Frank 1985, OMeara et al. 1995b, OMeara et al. 2003) Past studies on oviposition by A. albopictus have demonstrated a preference for darker colors (Gubler 1971, Yap 1975), and a lack of response to the pr esence of the bromeliad specialists W. vanduzeei and W. mitchellii (Lounibos et al. 2003). Wyeomyia vanduzeei showed a preference for oviposition into flowering Tillandsia utriculata (Frank and OMeara 1985). During the field survey discussed in Chapte r 5 of this thesis, there was a significant difference between the densities of A. albopictus larvae in different spec ies of bromeliads. Of the two species of bromeliads that were the focus of the field survey, N. spectabilis had significantly highe r densities of A. albopictus than B. pyramidalis In order to understand the

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94 differences in mosquito densities in these tw o plant species, an oviposition experiment was implemented to determine whether oviposition wa s a factor that contributed to the higher densities of A. albopictus in N. spectabilis and a larval developm ent and survivorship experiment was conducted to see whethe r the growth and survivorship of A. albopictus differed between N. spectabilis and B. pyramidalis Materials and Methods Oviposition Experiment To determine whether A. albopictus preferentially oviposits into N. spectabilis in comparison to B. pyramidalis, an experiment was conducted in which 15 gravid A. albopictus females were added by mouth aspirator to each of ten cages containing both one N. spectabilis and one B. pyramidalis bromeliad. In accordance with approved animal-care protoc ol (VB-17 project of the University of Florida), each female had been bloodfed on a chicke n five days before the start of the experiment and held without an oviposition site. Only mo squitoes that appeared fully engorged were removed from the main colony cage, and then tran sferred to a cage kept at 26C in a climatecontrolled insectary, with access to a 20% sucrosewater solution for the five days prior to the start of the experiment. The colony from which the females were selected originated from collections from southern Florida. The N. spectabilis plants in the experiment were colle cted from one residence in the city of Gotha in central Florida, and the B. pyramidalis plants were collected from multiple residences in Vero Beach a nd Fort Pierce, Florida. The N. spectablis plants chosen for the experiment all contained partially submerge d, nidulate inflorescences in the central tank (Benzing 2000) and, thus, held larger amounts of water than non-flowering individuals. Because N. spectabilis with inflorescences were the plants sa mpled in the field survey conducted in

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95 Chapter 5, using flowering individuals for experime nts may help to explain some of the findings in that chapter. On the other hand, the B. pyramidalis in this experiment did not have inflorescences, as their formation often destroys the water holding capacity of this species of bromeliad. Non-flowering B. pyramidalis were also the only B. pyramidalis sampled in the field surveys of Chapter 5. Each plant was thoroughly washed by first rinsing in a bucket of water and then by spraying with a high powered hose. Due to the fact that it is very difficult to remove all of the leaves and organisms from the bromeliads, all pl ants were allowed to dry in an air-conditioned laboratory from 8 to 10 days to ensure the death of all organisms due to desiccation. The plants were then rewashed with a hose to rem ove any remains resulting from the drying. Each replicate was enclosed in a 1.0 m wide and 0.76 m high pyramidal cage located within a outdoor screened encl osure constructed for studies of mosquito flight behavior (Bidlingmayer 1977). Within each cage, specime ns of each bromeliad species were paired, albeit the N. spectabilis were always slightly bigger. 1.0 g of chopped, dried (48 h at 75C) Q. virginiana oak leaves, and 150 mL of sieved bromeliad water collected from both B. pyramidalis and N. spectabilis bromeliads from the Vero Beach and Fo rt Pierce, Florida areas were added to the central tank of each bromeliad. The water of each species was pooled, so the water added to each plant was a mixture from both bromelia d species. The water was sieved through a 130 m mesh to remove macroinvertebrates and detritu s. The number of wa ter holding leaves was counted as an estimate of plant water holding capacity. The experiment was run for 7 days, after which the remaining water in each bromeliad was removed by a pipette, and each leaf containing water was rinsed with tap water into a metal rearing pan. Each pan was kept for 10 days at 26C in a climate-cont rolled rearing room and

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96 covered with a sheet of glass to prevent possible oviposition from stray mosquitoes in the rearing room. On each day, the pans were checked for newly hatched A. albopictus and the sums of hatched larvae in the two bromeliad species were regarded as products of recent oviposition and compared by a paired t-test. Larval Development and Survivorship Experiment Fourteen plants, 7 B. pyramidalis and 7 N. spectabilis from the previous experiment were washed in the manner as described above, and 1.0 g of chopped oak leaves ( Q. virginiana ), and 150 mL of sieved bromeliad water were added to the central tank of each bromeliad. Then after 20 4th instar Wyeomyia spp. and 20 1st instar A. albopictus had been added into the center tank, the plants were placed into metal cages in a 26C climate controlled rearing room. After 3 days, 50 mL of water was added to the plants to compensate for evaporation. After 6 days, all of the contents of the bromeliads were rinsed into a pan in the manner as the oviposition experiment. Variations between plant specie s in the number and developmental stage of surviving A. albopictus were analyzed by ANOVA in SAS ( 2002). The survivorship data was arcsine square root transforme d to meet the homogeneity of variances assumption of ANOVA. Results Oviposition Experiment Aedes albopictus oviposited significantly more (t9= 2.95, P <0.01) eggs into N. spectabilis (mean SE=33.50.38) than B. pyramidalis (mean SE=15.80 5.29). Neoregelia spectabilis had significantly more (t9= 3.33, P <0.01) (mean SE= 9.60 0.70) water-holding axils than B. pyramidalis (mean SE= 7.1 0.46). Larval Development and Survivorship Experiment A. albopictus had a significantly higher (F1,12= 67.41, P <0.01) average instar number within N. spectabilis (mean SE=3.96 0.22) than in B. pyramidalis (mean

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97 SE=2.31.08).The mean survivorships of A. albopictus in N. spectabilis (0.56 0.08 (SE) )and B. pyramidalis (0.67 0.04 (SE)) were not significantly different (F1,12= 1.43, P =0.25). Discussion While there was significantly more oviposition by A. albopictus in N. spectabilis this experiment did not consider the cause of this ovipositional preference The difference could be based on coloration differences between N. spectabilis and B. pyramidalis N. spectablis is dark green with dark purple tips as comp ared to the entirely light-green B. pyramidalis In an experiment conducted by Frank (1985), other species of mosquitoes occasionally found in bromeliads, C. quinquefasciatus A. aegypti and Toxorhynchites rutilus preferentially oviposited in darkly colored artificial bromeliads, as opposed to lighter green artificial bromeliads. In this same study, Wyeomyia spp. preferred to oviposit in the lighter green ar tificial bromeliads. Therefore, it seems possible that A. albopictus like other container-gene ralist mosquito species, has a preference for the darker colored bromeliad N. spectabilis Another visual cue besides color that may have affected the experiment was the size of the plants. Generally in the field, a nd in this experiment as well, N. spectabilis is a horizontally larger plant with mu ch longer leaves. B. pyramidalis tends to be smaller overall with shorter leaves and a more vertically dominant stature than horizontally dominant. The paired t-test demonstrated that the N. spectablis also has more water holding axils per plant than the B. pyramidalis plants in this experiment. Bentley and Day (1989) stated that speci alist mosquitoes, those which have more restricted ovipositional s ites, such as crab holes, bromeliads and natural or artificial containers, tend to rely strongly on visual cues to aid in the identification of ovipositional sites. While this statement may also favor the color differe nce hypothesis for the observed ovipositional preference of A. albopictus for N. spectablis the fact that there was a difference in the size of the

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98 two plants could also be a factor. Neoregelia spectabilis the larger of the two bromeliad species, may be visually more attractive to A. albopictus regardless of color. This larger size is not an artifact of the experiment, because in nature N. spectabilis are commonly larger than B. pyramidalis. Therefore, although the difference may be due to size, there are still important ecological implications to this difference. On the other hand, visual cues may be of litt le importance to the ovipositional decision of A. albopictus in bromeliads. Aedes albopictus could be responding to habitat-based chemical cues unique to each plant species. For instan ce, the submerged nidulate inflorescence of N. spectaibilis may provide additional nutr ients or chemical compounds to the central tank water, while B. pyramidalis does not receive this input, because it creates tall, stalky inflorescenses. Since the water from both species was pooled, each species received water originating from both B. pyramidalis and N. spectabilis Therefore, it is unlikely th at chemical compounds within the water contributed to the oviposition preference of A. albopictus for N. spectabilis in this experiment. The experiment ran for 7 days, so only chemical cues that a ccumulated in this short period of time could have affected the preference of A. albopictus for N. spectabilis. The larval competition experiment within N. spectabilis and B. pyramidalis showed that A. albopictus can develop at different rates based on the species of bromeliad. The multitude of structural differences in size and shape between the two plant species could contribute to the developmental differences of A. albopictus within the two plants. In Ch apter 2 of this thesis, and in treehole microcosms study by Broadie and Bradshaw 1991, surface area to volume ratio was not a factor influencing the development rate of Aedes mosquitoes. So, it is possible that other structural differences account for the increased development rate of A. albopictus in N. spectabilis

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99 Neoregelia spectabilis has a sunken inflorescence which was decaying during this experiment, while B. pyramidalis did not have this decaying inflorescence. Therefore, it is possible that this sunken inflorescence provide d additional nutrients that increased the food resources available to A. albopictus. Further experiments are necessa ry to elucidate the causes of the developmental differences of A. albopictus seen in these two bromeliad species. This experiment shows the importance of bromeliad species in the distribution of A. albopictus So far in North America, A. albopictus has been found to be infected with LaCrosse (Gerhardt et al. 2001), Eastern Equine Encephalitis (EEE) (Mit chell et al. 1992), and dengue viruses (Ibaez Bernal et al. 1997), and transm its Dengue (DEN) in other areas of the world (Hawley 1988). As this mosquito species is a health concern, then the understanding of its ovipositional preferences within bromeliads can be benefici al to determining the risk of transmission of these viruses to human populations. In Brazil, bromeliad eradication projects have been implemented to eliminate these supposed development sites of dengue vectors (Benzing 2000). With th e higher populations of A. albopictus in northern Florida where Wyeomyia spp. is absent (OMeara et al. 1995b), furt her studies on the ovipositional preferences of A. albopictus in bromeliad species could help reduce risk by applying control measures only to bromeliad species that are of highest concern.

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100 CHAPTER 7 CONCLUSIONS In aquatic container habitats, high densities of mosquito larvae can increase development time and reduce survivorship and pupal weight (e.g. Frank and Curtis 1977, Mogi 1984, Livdahl 1982, Broadie and Bradshaw 1991). In the surface area to volume ratio experiment of Chapter 2, and a larval competition experiment conducted by Lounibos et al. (2003), A. albopictus suffered reduced growth and survivorsh ip in the presence of larger 4th instar Wyeomyia spp.. Resource competition is common among mosquito larvae, and interference competition in the forms of chemical interference (Dye 1984, Sunhara and Mogi 2002, Be dhomme et al. 2005), encounter competition (Dye 1984, Roberts 1998, Broadie and Bradshaw 1991), and facultative or intraguild predation (e.g. Reisen and Em ory 1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003) also influence larval mosquito communi ties. The results of this study suggest that encounter competition, not chemical interference or predation, causes the reduced growth and survivorship of early instar A. albopictus in the presence of 4th instar Wyeomyia spp. in bromeliads. Increasing the habitat complexity in an artificial bromeliad increased the developmental rate of A. albopictus in the presence of Wyeomyia spp.. However, because the effect of habitat complexity on the development time of A. albopictus in the absence of Wyeomyia spp. was not explored, the relatio nship of habitat complexity to A. albopictus development may be unrelated to interactions with Wyeomyia spp. Even though large numbers of mosquitoes were used in the chemical interference experiments, excretory products of Wyeomyia spp. did not affect the growth or survivorship of A. albopictus in bromeliads. Neither the pH nor a mmonia concentration of bromeliad water differed before and after the experiment. Because bromeliads assimilate ammonia from their

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101 central tank and axils (Benzing 2000), the likelihood of chemical interference caused by mosquito waste products may be reduced in this phytotelmata. The dissections of over 1200 Wyeomyia spp. larvae exposed to multiple prey density and resource dependent conditions, and observations on the behavior of A. albopictus and Wyeomyia spp., provided no evidence of predation by Wyeomyia spp. upon A. albopictus If Wyeomyia spp. larvae are facultative predators of A. albopictus then the occurrence of predation is probably very rare and does not greatly effect the dynamics of these species. Egg hatch inhibition is a competitive mechanis m demonstrated in laboratory studies of Aedes mosquitoes (Edgerly et al. 1993). Egg hatch inhibition could change the population dynamics of affected species by ch anging the rate at which larvae enter a habitat and, thus, might cause changes in larval species composition (Livdahl and Edgerly 1987). In this study, Wyeomyia spp. larvae inhibited the egg hatch of A. albopictus at high resource levels, but only moderately reduced the overall hatch rate. Thus in bromeliad habitats the rate at which A. albopictus larvae hatch may be altered by high densities of Wyeomyia spp., but this effect is probably minor among competitive intera ctions between these two species. In field collections the densities of Wyeomyia spp. and A. albopictus differed between two species of exotic bromeliad. Aedes albopictus larvae were more common in N. spectabilis, and Wyeomyia spp. were more common in B. pyramidalis Experiments determined that A. albopictus deposited more eggs and developed more quickly in N. spectabilis the bromeliad species with the higher densities of larvae of this species in fi eld collections. The choice of oviposition in N. spectabilis over B. pyramidalis is probably not due to the ability of the A. albopictus females to detect Wyeomyia spp. within the plant, becau se Lounibos et al. (2003) determined that A. albopictus did discriminate between B. pyramidalis without or without

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102 Wyeomyia spp. Therefore, the oviposition preference of A. albopictus is likely due to chemical or visual differences between the two bromeliad species. While A. albopictus larvae developed faster in N. spectabilis when exposed to Wyeomyia spp. than under the same conditions in B. pyramidalis, the cause of this difference is unknown. There are many structural differences between the two host plant species, one being differing surface area to volume ratios, but in the encount er competition experiments conducted in this study, there was no effect of vary ing surface area to volume ratio of the plant on survivorship or growth of A. albopictus in the presence of Wyeomyia spp.. However, this encounter competition experiment was conducted in an artificial bromeliad, so the outcome of competition between A. albopictus and Wyeomyia spp. based on the surface area to volume ratio may be different under field conditions with actual bromeliads. As there are many differences between the two plant species, further investigations are needed to determine the causes of the more rapid developmental rate of A. albopictus within N. spectabilis

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106 Dye. C. 1984. Competition amongst larval Aedes aegypti : role of interference. Ecological Entomology 16 : 335-357. Edgerly, J. S., and M. A. Marvier. 1992. To hatch or not to hatch egg hatch response to larval density and to larval contact in a treehole mosquito. Ecological Entomology 17 : 28-32. Edgerly, J. S., M. S. Willey, and T. P. Livdahl. 1993. The community ecology of Aedes egg hatching implications for a mosqu ito invasion. Ecological Entomology 18 : 123-128. Edgerly, J. S., M. S. Willey, and T. Livdahl. 1999. Intraguild predati on among larval treehole mosquitoes, Aedes albopictus Ae. aegypti and Ae. triseriatus (Diptera: Culicidae), in laboratory microcosms. Jour nal of Medical Entomology 36 : 392-399. Edman, J. D., and J. S. Haeger. 1977. Host-f eeding patterns of Florida mosquitoes. V. Wyeomyia Journal of Medical Entomology 14 : 477-479. Endres, L., and H. Mercier. 2001. Ammonium and urea as nitrogen sources for bromeliads. Journal of Plant Physiology 158 : 205-212. Fallis, S. P., and K. R. Snow. 1983. The hatching stimulus for eggs of Aedes punctor (Diptera, Culicidae). Ecological Entomology 8 : 23-28. Finke, D.L. and R.F. Denno. 2002. Intraguild pred ation diminished in complex-structured vegetation: implications fo r prey suppression. Ecology 83 : 643-652. Fincke, O.M., S.P. Yanoviak, and D. Hansc hu. 1997. Predation by odonates depresses mosquito abundance in water-filled tree holes in Panama. Oecologia 112 : 244-253. Fish, D. 1976. Structure and composition of the aquatic invertebrate community inhabiting epiphytic bromeliads in sout h Florida and the discovery of an insectivorous bromeliad. PhD dissertation. University of Fl orida, Gainesville, Florida. 78 pp. Fish, D. 1983. Phytotelmata: flora and fauna. Pages 1-27 i n J. H. Frank and L. P. Lounibos, editors. Phytotelmata: Terrestrial Plants as Hosts for Aquatic Insect Communities. Plexus Publishing, Medford, New Jersey. Fish, D., and S. R. Carpenter. 1982. Leaf litter and larval mosquito dynamics in tree-hole ecosystems. Ecology 63 : 283-288. Frank, J. H. 1983. Bromeliad phytotelmata and thei r biota, especially mosquitoes. Pages 101-128 in J. H. Frank and L. P. Lounibos, editors. Ph ytotelmata: Terrestrial Plants as Hosts for Aquatic Insect Communities. Plexus Publishing, Medford, New Jersey.

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107 Frank, J. H. 1985. Use of an artificial bromelia d to show the importance of color value in restricting colonizati on of bromeliads by Aedes aegypti and Culex quinquefasciatus Journal of The American Mosquito Control Association 1 : 28-32. Frank, J.H. and G.A. Curtis. 1977. Bionomics of bromeliad-inhabiting mosquitoes. IV. Egg mortality of Wyeomyia vanduzeei caused by rainfall. Mosquito News 37 : 239-245. Frank, J.H., G.A Curtis, and H.T. Evans. 1976. On the dynamics of bromeliad-inhabiting mosquitoes. I. Some factors influencing oviposition by Wyeomyia vanduzeei Mosquito News 36 : 25. Frank, J. H., G. A. Curtis and H. T. Evans. 1977. On the bionomics of bromeliad-inhabiting mosquitoes. II. The relationship of brom eliad size to the number of immature Wyeomyia vanduzeei and Wy. medioalbipes Mosquito News 37 : 180-192. Frank, J.H., Lynn, H.C., and J.M. Goff. 1985. Diurnal oviposition by W. mitchellii and W. vanduzeei. Florida Entomologist 68 : 493-496. Frank, J. H., and G. F. O'Meara. 1985. Infl uence of microhabitat and macrohabitat on distribution of some bromelia d-inhabiting mosquitoes. Ento mologia Experimentalis et Applicata 37 : 169-174. Frank, J. H., J. P. Stewart, and D. A. Wats on. 1988. Mosquito larvae in axils of the imported bromeliad Billbergia pyramidalis in southern Florida. Florida Entomologist 71 : 33-43. Frank, J. H., and M. C. Thomas. 1994. Metamasius callizona (Chevrolat) (Coleoptera, Curculionidae), an immigrant pest, destr oys bromeliads in Florida. Canadian Entomologist 126 : 673-682. Gerhardt, R. R., K. L. Gottfried, C. S. Apperson, B. S. Davis, P. C. Erwin, A. B. Smith, N. A. Panella, E. E. Powell, and R. S. Nasci. 2001. First isolation of La Crosse virus from naturally infected Aedes albopictus Emerging Infectious Diseases 7 : 807-811. Gill, D.E. 1972. Intrinsic rates of increase, satura tion densities, and competitive ability. I. An experiment with Paramecium American Naturalist 106 : 461-471. Gillett, J. D., E. A. Roman, and V. Phillips. 1977. Erratic hatching in Aedes eggs: new interpretation. Proceedings of the Royal So ciety of London Series B-Biological Sciences 196 : 223-232. Gjullin, C. N., C.P.Hegarty, and W.B.Bollen, 1941. The necessity of low oxygen concentration for the hatching of Aedes mosquito eggs. Journal of Cellu lar and Comparative Physiology 17 : 193-202.

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114 Schoener, T. W. 1983. Field experiments on inte rspecific competition. American Naturalist 122 : 240-285. Sih, A. 1985. Evolution, predator avoidance, and unsuccessful predation. American Naturalist. 125 : 153-157. Singh, H. P., D. R. Batish, J. K. Pandher, and R. K. Kohli. 2005. Allelopa thic studies of common wheat ( Triticum aestivum L.). Weed Biology and Management 5 : 105-109. Shroyer, D. A. 1981. Establishment of Wyeomyia mitchellii on the island of Oahu, Hawaii. Mosquito News 41 : 805-806. Shroyer, D. A., and G. B. Craig. 198 0. Egg hatchability and diapause in Aedes triseriatus (Diptera, Culicidae) : temperature induced a nd photoperiod induced latencies. Annals of the Entomological Society of America 73 : 39-43. Soman, R. S., and R. Reuben. 1970. Studies on the preference shown by ovipositing females of Aedes aegypti for water containing immature stages of the same species. Journal of Medical Entomology 7 : 485-489. Sprenger, D., and T. Wuithiranygool. 1986. The discovery and distribution of Aedes albopicuts in Harris County, Texas. Journal of the American Mosquito Control Association 2 : 217219. Srihongse, S.C., and C.M. Johnson. 1965. Wyeomy ia sub-group of arbovirus: isolation from man. Science. 149 : 863-864. Srihongse, S.C., and C.M. Johnson. 1967. The isolat ion of Ilheus virus from a man in Panama. American Journal of Tropi cal Medicine and Hygiene. 16 : 516-518. Srivastava, D.S., and J.H. Lawton. 1998. Why mo re productive sites have more species: an experimental test of theory using tree -hole communities. The American Naturalist 152 : 510-529. Sunahara, T., and M. Mogi. 2002. Priority effect s of bamboo-stump mosquito larvae: influences of water exchange and leaf litter input. Ecological Entomology. 3 :346-354. Trpis, M. 1970. A new bleaching and decalcifyin g method for general use in zoology. Canadian Journal of Zoology 48 : 892-893. Walker, E. D., M. G. Kaufman, M. P. Ayres, M. H. Riedel, and R. W. Merritt. 1997. Effects of variation in quality of leaf detritus on growth of the eastern tree-hole mosquito, Aedes triseriatus (Diptera: Culicidae). Canadian Journal of Zoology 75 : 706-718 Williams, R. E. 1962. Effect of coloring ovi position media with regard to mosquito Aedes triseriatus (Say). Journal of Parasitology 48: 919925.

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PAGE 116

116 BIOGRAPHICAL SKETCH Robyn Raban was born in Lewiston, Maine, and then at the age of twelve moved to Phoenix, Arizona. After graduating from Mount ain Pointe High School, she went on to the University of California, Berkeley where she ear ned a degree in environmental science. During her experiences at UC Berkeley she was able to study tropical ecology in Costa Rica, and work with the Smithsonian Institute on mangrove ecology research in Panama. Through her experiences in central America, she became in terested in mosquito biology, and medical entomology. She will continue to pursue her intere sts in mosquito biology with her next degree in mosquito genetics and arbovirology.


Permanent Link: http://ufdc.ufl.edu/UFE0017921/00001

Material Information

Title: Aedes albopictus oviposition and larval density, development and interactions with Wyeomyia spp. in exotic bromeliads of southern Florida
Physical Description: Mixed Material
Language: English
Creator: Raban, Robyn R. ( Dissertant )
Lounibos, Leon P. ( Thesis advisor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006
Copyright Date: 2006

Subjects

Subjects / Keywords: Entomology and Nematology thesis, M.S
Dissertations, Academic -- UF -- Entomology and Nematology

Notes

Abstract: Wyeomyia mitchellii and Wyeomyia vanduzeei, are indigenous mosquitoes that inhabit exotic and native bromeliads in southern Florida. In the mid-1980s Aedes albopictus invaded Florida where its immature stages occupied artificial and natural containers, including bromeliads. Previous studies have shown reduced abundance of A. albopictus in exotic bromeliads co-occupied by Wyeomyia spp. immatures, and reduced larval growth and survivorship in the presence of late instar Wyeomyia spp. Our study examined chemical interference, encounter competition, predation, and egg hatch inhibition as potential interactions between Wyeomyia spp. and A. albopictus in exotic bromeliads. Exposure to waste products of Wyeomyia spp. did not affect the growth or survivorship of A. albopictus. Experimental alterations of surface area to volume ratios and habitat complexity did not affect the decreased growth and survivorship of A. albopictus seen in the presence of Wyeomyia spp. Behavior experiments showed that A. albopictus larvae change their location within a container in the presence of Wyeomyia spp. This change in location may indicate a response of A. albopictus to encounters with Wyeomyia spp. Wyeomyia spp. showed no evidence of predation on A. albopictus, but fourth instar Wyeomyia spp. were shown to inhibit the egg hatch of A. albopictus. Field surveys and lab experiments were conducted on A. albopictus and Wyeomyia spp. in Neoregelia spectabilis and Billbergia pyramidalis bromeliads. Higher densities of Wyeomyia spp. were found in B. pyramidalis, while higher densities of A. albopictus were found in N. spectabilis. Correlations between the number of mosquitoes per plant and the volume of water extracted were significant for Wyeomyia spp. and A. albopictus. Overhead tree canopy type influenced Wyeomyia spp. larval densities in bromeliads. A. albopictus females oviposited preferentially in N. spectabilis, and A. albopictus developed faster when exposed to Wyeomyia spp. within N. spectabilis as compared to the same exposure in B. pyramidalis. Encounter competition with Wyeomyia spp. larvae is the most probable mechanism reducing the growth and survivorship of A. albopictus in exotic bromeliads in southern Florida, although other interactions may also influence the relative abundances of these mosquito species.
Abstract: Aedes, bromeliad, competition, mosquito, wyeomyia
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 116 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

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Permanent Link: http://ufdc.ufl.edu/UFE0017921/00001

Material Information

Title: Aedes albopictus oviposition and larval density, development and interactions with Wyeomyia spp. in exotic bromeliads of southern Florida
Physical Description: Mixed Material
Language: English
Creator: Raban, Robyn R. ( Dissertant )
Lounibos, Leon P. ( Thesis advisor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006
Copyright Date: 2006

Subjects

Subjects / Keywords: Entomology and Nematology thesis, M.S
Dissertations, Academic -- UF -- Entomology and Nematology

Notes

Abstract: Wyeomyia mitchellii and Wyeomyia vanduzeei, are indigenous mosquitoes that inhabit exotic and native bromeliads in southern Florida. In the mid-1980s Aedes albopictus invaded Florida where its immature stages occupied artificial and natural containers, including bromeliads. Previous studies have shown reduced abundance of A. albopictus in exotic bromeliads co-occupied by Wyeomyia spp. immatures, and reduced larval growth and survivorship in the presence of late instar Wyeomyia spp. Our study examined chemical interference, encounter competition, predation, and egg hatch inhibition as potential interactions between Wyeomyia spp. and A. albopictus in exotic bromeliads. Exposure to waste products of Wyeomyia spp. did not affect the growth or survivorship of A. albopictus. Experimental alterations of surface area to volume ratios and habitat complexity did not affect the decreased growth and survivorship of A. albopictus seen in the presence of Wyeomyia spp. Behavior experiments showed that A. albopictus larvae change their location within a container in the presence of Wyeomyia spp. This change in location may indicate a response of A. albopictus to encounters with Wyeomyia spp. Wyeomyia spp. showed no evidence of predation on A. albopictus, but fourth instar Wyeomyia spp. were shown to inhibit the egg hatch of A. albopictus. Field surveys and lab experiments were conducted on A. albopictus and Wyeomyia spp. in Neoregelia spectabilis and Billbergia pyramidalis bromeliads. Higher densities of Wyeomyia spp. were found in B. pyramidalis, while higher densities of A. albopictus were found in N. spectabilis. Correlations between the number of mosquitoes per plant and the volume of water extracted were significant for Wyeomyia spp. and A. albopictus. Overhead tree canopy type influenced Wyeomyia spp. larval densities in bromeliads. A. albopictus females oviposited preferentially in N. spectabilis, and A. albopictus developed faster when exposed to Wyeomyia spp. within N. spectabilis as compared to the same exposure in B. pyramidalis. Encounter competition with Wyeomyia spp. larvae is the most probable mechanism reducing the growth and survivorship of A. albopictus in exotic bromeliads in southern Florida, although other interactions may also influence the relative abundances of these mosquito species.
Abstract: Aedes, bromeliad, competition, mosquito, wyeomyia
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 116 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 003757687
System ID: UFE0017921:00001


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Aedes albopictus OVIPOSITION AND LARVAL DENSITY, DEVELOPMENT, AND
INTERACTIONS WITH Wyeomyia spp. IN EXOTIC BROMELIADS OF SOUTHERN
FLORIDA



















By

ROBYN R. RABAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2006
































Copyright 2006

by

Robyn R. Raban





































To my parents, Bill and Judy Raban and my grandfather, Bill Phillips for their support for all my
academic endeavors.









ACKNOWLEDGMENTS

I would like to thank L.P. Lounibos for his advice and support. I thank G. F. O'Meara for

his assistance in locating field sites, and his enthusiastic support for my research. I thank J.H.

Frank for helpful suggestions on my research; N. Nishimura for help with my technical

problems; and R. Escher for his mosquito rearing guidance and for his provision of eggs for my

research. Finally, I would like to thank the faculty, staff, and graduate students at FMEL, most

of whom at one time or another, provided invaluable help to my research.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............7................

LI ST OF FIGURE S .............. ...............9.....


AB S TRAC T ........._. ............ ..............._ 1 1...

CHAPTER


1 Aedes albopictus AND Wyeomyia spp. IN THE EXOTIC BROMELIADS OF
SOUTHERN FLORIDA. .............. ...............13___ .......


Aedes albopictus in the United States............... ...... ........... ...........1
Wyeomyia mitchellii and Wyeomyia vanduzeei in the United State s ................ .................1 4
Bromeliaceae as a Habitat ................ ......... .. ........... .......1
Interactions of A. albopictus and Wyeomyia spp. in Bromeliad Phytotelmata ................... ....1 7

2 INTERFERENCE COMPETITION AS A POTENTIAL MECHANISM FOR THE
REDUCTION OF GROWTH AND SURVIVORSHIP OF Aedes albopictus IN THE
PRESENCE OF Wyeomyia spp. IN THE BROMELIADS OF SOUTHERN FLORIDA. ....20

Introducti on .................. ...............20._ ___......
M materials and M ethods .............. ...............24....
Chemical interference................. .. .. .. ..............2
Surface Area to Volume Ratio Encounter Competition Experiment .............. ................26
Habitat Complexity Experiment.................. .... ................2
Resource Dependent Location of A. albopictus and Wyeomyia spp. ............. ................28
Re sults................... ..... ...............30.......
Chemical Interference ................ ....... .............3
Surface Area to Volume Ratio Experiment ................. ........._. ...... 30__. ...
Habitat Complexity Experiment ......................... ......... .... ........
Resource-Dependent Foraging Location ...._. ................. ........___.........3
Discussion ................. ...............32....... ......


3 Wyeomyia spp. AS POTENTIAL PREDATORS OF Aedes albopictus IN
BROMELIADS IN SOUTHERN FLORIDA .............. ...............53....

Introducti on ........._ ....... __. ...............53....
M materials and M ethods ................... ........... .. .. ..............5
Testing for Density Dependent Predation by Wyeomyia spp. ............. .....................5
Testing for Resource Dependent Predation by Wyeomyia spp............... ..................5
Predation Observation .............. ...............55....












Re sults ................ ...............55.................
Discussion ................. ...............56.................


4 DO Wyeomyia spp.. LARVAE INHIBIT EGG HATCH OF Aedes albopictus? ..................59


Introducti on ................. ...............59.................
M materials and M ethods .............. ...............60....
Re sults ................ ...............61.................
Discussion ................. ...............62.................


5 FIELD STUDIES ON Aedes albopictus AND Wyeomyia spp. IN EXOTIC
BROMELIADS OF SOUTHERN FLORIDA. ......___. ...... .___ .... _. ...........6


Introducti on ........._ ....... __. ...............68....
M materials and M ethods .............. .. .. ..... ..... ... ...............6

Differences in the Density ofA. albopictus and Wyeomyia spp. within N.

spectabilis and B. pyramnidalis ............_. ...._.. ...._... ...........6
Canopy Effects on Density ............._. ...._... ...............71...
Re sults............._. ...._... ...............72....
Discussion ............._. ...._... ...............73....


6 OVIPOSITION AND LARVAL DEVELOPMENT OF Aedes albopictus IN TWO
EXOTIC SPECIES OF BROMELIAD IN SOUTHERN FLORIDA .................. ...............91


Introducti on ................. ...............91.................
M materials and M ethods .............. ...............94....

Oviposition Experiment. ............. ... .... ...............94..
Larval Development and Survivor ship Experiment ............. ..... ...............96
Re sults............. ..... .. ...............96..

Oviposition Experiment. ............. ... .... ...............96..
Larval Development and Survivor ship Experiment ............. ..... ...............96
Discussion ............. ..... ...............97...


7 CONCLUSIONS .............. ...............100....


LI ST OF REFERENCE S ............. ..... ._ ...............103..


BIOGRAPHICAL SKETCH ............. .....__ ...............116..










LIST OF TABLES


Table page

2-1 Means and standard errors for the concentrations of ammonia and pH pre- and post-
experiment. ........._.._.. ...._... ...............36....

2-2 Analysis of variance for A. albopictus average instar based on the presence or
absence of Wyeomyia spp. and the surface:volume ratio of the artificial plant and the
interaction of these two variables. ........._ ....__ ......__ ......_ ............37

2-3 Analysis of variance for A. albopictus average survival based on the presence or
absence of Wyeomyia spp., and the surface area:volume ratio of the plant (size) and
the interaction of these two variables. ..........._... ...............38.....__.._....

2-4 Multivariate analysis s of variance table for feeding location ................. ............. .......3 9

2-5 Analysis of variance for the effects of food level presence or absence of Wyeomyia
spp. on the location of A. albopictus ................. ...............40......__. ..

2-6 Analysis of variance for the effects of food level and presence or absence ofA.
albopictus on the location of Wyeomyia spp............... ...............41..

3-1 Number of Wyeomyia examined in predation experiments. ............... ...................5

5-1 Analysis of variance for the density of mosquitoes per plant based on month (month
of collection), site, and bromeliad species ................. ........._. ......77.........

5-2 Analysis of variance for the density of mosquitoes per mL based on month (month of
collection), site, and bromeliad species .............. ...............78....

5-3 Analysis of variance for the density of each mosquito species per mL of water in
each bromeliad species. ............. ...............79.....

5-4 Mean densities of mosquitoes (mos/plant) in B.pyramidalis and N. spectabilis based
on location within the plant and plant species. ............. ...............80.....

5-5 Analysis of variance table for the effects of month and site on mos/mL and
mos/pl ant. ........._.._.. ...._... ...............8 1....

5-6 Analysis of variance results for the effect of month and sites on mosquito
abundances (mos/plant) by bromeliad species ................. ...............82........... ...

5-7 Average (+SE) amount of water extracted from each plant species. ........._.._... ........._....83

5-8 A nested ANOVA table for effects of Oak vs. Palm canopy and bromeliad species
(B. pyramnidalis vs. N. spectabilis) on Wyeomyia spp. densities by mos/plant. ........._......84










5-9 Means and SE of densities ofA. albopictus and Wyeomyia spp. by location within
plants under oak and palm canopies. ............. ...............85.....










LIST OF FIGURES


Figure page

2-1 Construction of the squares for the habitat complexity experiment. ............. .... ........._...42

2-2 The folded shape of the high complexity and low complexity treatments and the
orientation of the squares within the cones ................. ...............43........... ..

2-3 Average instars ofA. albopictus after 48 h exposure to Wyeomyia spp. through a
dialysis membrane and without Wyeomyia in the control. ............. .....................4

2-4 Mean survivorship of A. albopictus after 48 h exposure to Wyeomyia spp. through a
dialysis membrane and without Wyeomyia spp. in the control. The error bars are
standard error. ............. ...............45.....

2-5 The average instar & SE ofA. albopictus in the absence or presence of Wyeomyia
spp. in four plant sizes.. ............ ...............46.....

2-6 The average survivorship a SE of A. albopictus in the absence or presence of
Wyeomyia spp. in four plant sizes. ................. ...............47....____....

2-7 The average instar (+SE) of A. albopictus in two levels of habitat complexity and
three levels of surface area to volume ratio. ............. ...............48.....

2-8 Location ofA. albopictus in the presence and absence of Wyeomyia .............. ................49

2-9 Location of Wyeomyia spp. in the presence and absence ofA. albopictus. .......................50

2-10 Location ofA. albopictus by food concentration and the presence and absence of
Wyeomyia spp... ........... ...............51......

2.11 Location of Wyeomyia spp. by food concentration and the presence and absence ofA.
albopictus. ................. ...............52....... ......

4-1 Proportion hatch of viable eggs & SE in each treatment..........._.._.. ......._.._........._..66

4-2 Proportion eggs damaged +SE per treatment. ........._..._.._ ...._._ ....._._. .........6

5-1 M ap of collection sites. .............. ...............86....

5-2 Location of water samples. ........._..._._ ...............87.._.._._ ....

5-3 The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis
and B. pyramnidalis at each site. ............. ...............88.....

5-4 The monthly mean densities of Wyeomyia spp. and A. albopictus in N. spectabilis at
each site............... ...............89..











5-5 The monthly mean densities of Wyeomyia spp. and A. albopictus in B. pyramnidalis at
each site............... ...............90..









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

Aedes albopictus OVIPOSITION AND LARVAL DENSITY, DEVELOPMENT, AND
INTERACTIONS WITH Wyeomyia spp. INT EXOTIC BROMELIADS OF SOUTHERN
FLORIDA

By Robyn R. Raban
December 2006

Chair: L. P. Lounibos
Maj or Department: Entomology and Nematology

Wyeomyia mitchellii and Wyeomyia vanduzeei, are indigenous mosquitoes that inhabit

exotic and native bromeliads in southern Florida. In the mid-1980s Aedes albopictus invaded

Florida where its immature stages occupied artificial and natural containers, including

bromeliads. Previous studies have shown reduced abundance of A. albopictus in exotic

bromeliads co-occupied by Wyeomyia spp. immatures, and reduced larval growth and

survivorship in the presence of late instar Wyeomyia spp.

Our study examined chemical interference, encounter competition, predation, and egg

hatch inhibition as potential interactions between Wyeomyia spp. and A. albopictus in exotic

bromeliads. Exposure to waste products of Wyeomyia spp. did not affect the growth or

survivorship ofA. albopictus. Experimental alterations of surface area to volume ratios and

habitat complexity did not affect the decreased growth and survivorship ofA. albopictus seen in

the presence of Wyeomyia spp. Behavior experiments showed that A. albopictus larvae change

their location within a container in the presence of Wyeomyia spp. This change in location may

indicate a response ofA. albopictus to encounters with Wyeomyia spp. Wyeomyia spp. showed

no evidence of predation on A. albopictus, but fourth instar Wyeomyia spp. were shown to inhibit

the egg hatch of A. albopictus.









Field surveys and lab experiments were conducted on A. albopictus and Wyeomyia spp.

in Neoregelia spectabilis and Billbergia pyramida~lis bromeliads. Higher densities of Wyeomyia

spp. were found in B. pyramnidalis, while higher densities ofA. albopictus were found in N.

spectabilis. Correlations between the number of mosquitoes per plant and the volume of water

extracted were significant for Wyeomyia spp. and A. albopictus. Overhead tree canopy type

influenced Wyeomyia spp. larval densities in bromeliads. A. albopictus females oviposited

preferentially in N. spectabilis, and A. albopictus developed faster when exposed to Wyeomyia

spp. within N. spectabilis as compared to the same exposure in B. pyramnidalis.

Encounter competition with Wyeomyia spp. larvae is the most probable mechanism

reducing the growth and survivorship of A. albopictus in exotic bromeliads in southern Florida,

although other interactions may also influence the relative abundances of these mosquito species.









CHAPTER 1
Aedes albopictus AND Wyeomyia spp. INT THE EXOTIC BROMELIADS OF SOUTHERN
FLORIDA

With expanding global transport and commerce, humans have increased the distributions

of many plant and animal species, often to novel locations. While some of these species fail to

establish in these new locations (Mack et al. 2000), some become successful biological invaders

(Lounibos 2002).

Aedes albopictus in the United States

Since its establishment in North America in the 1980s (Sprenger and Wuithiranygool

1986), Aedes albopictus has invaded various habitats that have previously been occupied by

other mosquito species. It has expanded its range throughout the southeastern and midwestern

United States by its transport in used tires (Hawley 1988). In Florida, A. albopictus was first

discovered in 1984, and is currently found throughout most of the state (O'Meara et al. 1995a,

Juliano et al. 2004). Invasions by A. albopictus have been much discussed (Lounibos 2002), and

this mosquito species continues to colonize new areas of western (Aranda et al. 2006) and

eastern Europe (Klobucar et al. 2006).

Aedes albopictus is one of the commonest mosquitoes in natural and artificial containers in

the eastern United States and Brazil (Lounibos 2002). Aedes albopictus larvae are often found

inhabiting man-made containers, but can also be found in natural phytotelmata such as treeholes

(Novak et al. 1993) and bromeliads (Lounibos et al. 2003, O'Meara et al. 2003). Displacement

by A. albopictus of mosquitoes that traditionally occurred in these habitats, such as Aedes aegypti

(Juliano 1998), and Culex pipiens (Carrieri et al. 2003) has been seen in some cases.

To date, considerations of the interactions of A. albopictus with its competitors have

included mating interference (Nasci et al. 1989), egg hatch inhibition (Edgerly et al. 1993,

Edgerly et al. 1991), larval resource competition (Juliano 1998, Braks et al. 2004, Griswold and









Lounibos 2005), predator-mediated competition (Gubler 1971, Lounibos et al. 2001, Griswold

and Lounibos 2005), and intraguild predation (Edgerly et al. 1999). Yet, so far competitive

exclusion ofA. aegypti by A. albopictus has not occurred mainly due to suspected differences in

the egg desiccation tolerances (Costanzo et al. 2005b), or differences in macrohabitat preferences

(Lounibos et al. 2001, Costanzo et al. 2005a). In some cases though, local extinction of A.

aegypti occurred after the arrival ofA. albopictus (O'Meara et al. 1993).

Aedes albopictus have been infected with over 22 species of arboviruses (Moore and

Mitchell 1997). In North America, A. albopictus have been found infected in nature with

LaCrosse (Gerhardt et al. 2001), Eastern Equine Encephalitis (EEE) (Mitchell et al. 1992), and

dengue viruses (Ibafiez Bernal et al. 1997). Therefore, there are potential health concerns related

to A. albopictus populations.

Wyeomyia mitchellii and Wyeomyia vanduzeei in the United States

Wyeomyia mitchellii ranges throughout Mexico (Diaz-Naj era and Vargas 1973), the

Caribbean region (Belkin and Heinemann 1975, Shroyer 1981), central and southern Florida

(Darsie and Ward 2005), and Hawaii (Shroyer 1981). This bromeliad specialist mosquito is

commonly found co-occurring in Florida with another congeneric bromeliad specialist,

Wyeomyia vanduzeei Dyar and Knab. Wyeomyia vanduzeei ranges through central and southern

Florida (Darsie and Ward 2005), and much of the Caribbean region (Belkin and Heinemann

1975).

Before the introduction ofA. albopictus, W. mitchellii and W. vanduzeei were the most

common day-biting mosquito species in the mixed oak forests of southern Florida (Edman and

Haeger 1977). Immatures of these Wyeomyia spp. are historically the most common mosquitoes

in the epiphytic bromeliad phytotelmata of southern Florida (Fish 1976, Frank 1983, Frank and

O' Meara 1985).









Wyeomyia vanduzeei and W. mitchellii are diurnal species with peak diel biting activity

occurring just prior to sunset (Edman and Haeger 1977) and oviposition occurring in the late

daylight hours (Frank et al. 1985). In a study conducted by Edman and Haegar (1977), both W.

vanduzeei and W. mitchellii adults appeared to be generalist feeders, feeding on species of

rabbits, deer, and birds. Wyeomyia vanduzeei are facultatively autogenous, while W. mitchelli

are not (O'Meara 1979), and their larvae may be slightly segregated as W. vanduzeei lay more

eggs in sunny versus shady locations, and these two mosquito species show preferences for

different plants (Frank and O'Meara 1985).

Although Wyeomyia spp. have not been found to vector arboviruses in Florida, Venezuelan

Equine Encephalitis virus has been found in W. mitchellii (Scherer et al. 1971) and Ilheus and

Wyeomyia viruses have been found in W. vanduzeei (Srihongse and Johnson 1965, 1967)

Bromeliaceae as a Habitat

Phytotelmata are water bodies that are held in plant structures such as flowers, leaf axils, or

tree holes (Maguire 1971, Fish 1983, Clements 1999). The family Bromeliaceae contains many

species which impound water in their leaf axils and are referred to as tank bromeliads (Frank

1983). There are over 2000 bromeliad species in the Americas (Frank 1983), with 16 species of

bromeliads native to Florida, 7 of these being tank bromeliads that hold water throughout most of

the year (Fish 1976).

While bromeliads obtain energy from photosynthesis, the nutrients they sequester for

photosynthesis are obtained in multiple ways. Tank bromeliads can absorb nutrients through

scales on the shoot called trichomes (Pittendrigh 1948, Benzing et al. 1976, Benzing 2000), or by

way of interfoliar roots (Pittendrigh 1948). Frank (1983) classifies nutrient acquisition by tank

bromeliads into two categories, dendrophilous nutrition, which is rain throughfall from trees

often collected by epiphytic bromeliads, and anemophilous nutrition, which are wind blown









nutrients. Frank (1983) and Benzing (2000) also describe the release of nutrients provided by the

wastes of faunal tank inhabitants which may contribute to the nutrients available to the plant.

In bromeliads, mosquitoes may encounter a wide variety of other unrelated species. To

date, bacteria, fungi, algae, protozoa, platyhelminthes, rotifers, gastrotrichs, ostracods,

nematodes, oligochaetes, crustacea, acarids, Hemiptera, Coleoptera, Culicidae, Psychodidae,

Syrphidae, Ceratopogonidae, Chironomidae, Sarcophaghidae, Tabanidae, Periscelidae,

Tipulidae, Muscidae, Sciaridae, and odonates have all been identified within bromeliad

phytotelamata (Maguire 1971, Fish 1976, Frank 1983). Some chironomids, muscids,

periscelids, and turbellarians are predatory and feed upon smaller organisms and mosquito eggs,

while some psychodids and sciarids feed on the submerged leaf litter in the bromeliad (Fish

1976). Bromeliads vary in their faunal compositions by the species of bromeliad and

environmental conditions, such as sunlight and humidity (Laessle 1961, Fish 1976).

Since the popularization of exotic bromeliads as ornamental plants (Edman and Haeger

1977), there has been an increase in exotic bromeliad habitat for Wyeomyia spp. in Florida. Yet,

with the arrival of the Mexican bromeliad weevil, M\~etamasiust~~~ttt~~ttt~~ callizona, there has been a decline

of the native bromeliad Tillandsia~~ll1~~~~111~~~ utriculata (Frank and Thomas 1994), the common native

phytotelmata of W. mitchellii and W. vanduzeei (Fish 1976). Exotic bromeliads, which have

become another common habitat of these two species, hold more water than the native bromeliad

species and have a large central water-holding tank, uncommon in native bromeliads (O'Meara

et al. 2003).

The two bromeliads which were the focus of most of this study are Billbergia pyramnidalis,

the summer torch bromeliad, and Neoregelia spectabilis, the painted fingernail plant. Billbergia

pyramnidalis and Neoregelia spectabilis are native to Brazil (Frank et al. 1988), and can both live









as either an epiphyte or as a ground dwelling species. Neoregelia spectabilis is the larger of the

two species and tends to be a dark green to dark purple color with bright purple tips. The

inflorescence of N. spectabilis is nidulate, meaning it is nested into the central tank of the plant.

Billbergia pyranzida~lis is a light green color and has a stalked inflorescence.

Interactions of A. albopictus and Wyeomyia spp. in Bromeliad Phytotelmata

There is evidence that Wyeonzyia spp. larvae exhibit negative effects on A. albopictus

within bromeliads. Possibly due to the freezing temperatures, or the absence of native

bromeliads in northern Florida, W. nitchelliii and W. vanduzeei are infrequently found north of

Orlando (O'Meara et al. 2003). In the absence of Wyeonzyia spp. in northern Florida, A.

albopictus has become a common mosquito in bromeliads, but in southern Florida where

Wyeonyia spp. are abundant, A. albopictus individuals are scarce in bromeliads (O'Meara et al.

1995b, Lounibos et al. 2003). A study conducted in Hawaii determined that in the absence of

Wyeonzyia spp., A. albopictus dominated leaf axil phytotelmata (Shroyer 1981).

This study attempts to further investigate the results of Lounibos et al. (2003). In that

study, 1st instar A. albopictus were exposed to varied quantities of leaf litter with 1st or 4th instar

Wyeonzyia spp. in B. pyra~nidalis bromeliads. Aedes albopictus experienced a reduction in

survivorship with reduced leaf litter and the presence of 4th instar Wyeonzyia spp., but the

presence of Wyeonzyia spp. accounted for most of the reduction in survivorship. The mean

growth stage of A. albopictus was only affected by the presence of 4th instar Wyeonzyia spp. with

no effect due to leaf litter. 1st instar Wyeonzyia spp. had no effect on the growth or survivorship

of A. albopictus.

Lounibos et al. (2003) hypothesized that the reduction in growth and survivorship of A.

albopictus in the presence of Wyeonzyia spp. was attributable to interference competition.

Lounibos et al. (2003) suggested that because the addition of leaf litter did not reduce the










negative effects of Wyeomyia spp. on A. albopictus, this interaction is independent of resources.

Interference competition is categorized into overgrowth, chemical, territorial, and encounter

(Schoener 1983). Overgrowth interference competition occurs only in sessile organisms, and

mosquito larvae are not known to exhibit territorial interference behaviors, so these mechanisms

are not considered in the possible competitive interactions between A. albopictus and Wyeomyia

spp..

Chemical interference in mosquitoes results from the buildup of larval waste products that

lead to an environmental deterioration which can effect the growth and survivorship of

individuals (Bedhomme et al. 2005). Aedes albopictus has been found to be subj ect to chemical

interference competition with the mosquito Tripteroides bamnbusa (Sunahara and Mogi 2002).

Chemical interference has also been demonstrated in Aedes sierrensis (Broadie and Bradshaw

1991), A. aegypti (Dye 1984), and Culex sitiens (Roberts 1998).

Encounter competition usually occurs when physical contact between organisms causes a

reduction in the feeding efficiency of individuals (Anholt 1990). Encounter competition is

known among A. sierrensis (Broadie and Bradshaw 1991), A. aegypti (Dye 1984) and Culex

sitiens (Roberts 1998). Other mosquitoes such as Ochlerotatus cantans (Renshaw et al. 1993)

are hypothesized to also exhibit encounter competition effects.

Intraguild predation is another important regulatory mechanism in mosquito populations.

Some mosquito species are facultative predators or cannibalistic on smaller instars (Hinman

1934, Reisen and Emory 1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003, Edgerly et

al. 1999, Koenraadt et al. 2004). Egg hatch inhibition occurs when mosquito larvae delay hatch

of Aedes eggs. Egg hatch inhibition could increase the time to reproduction, which may reduce

population growth and increase mortality in the dormant egg stage of some species (Livdahl and










Edgerly 1987). Some Aedes species exhibit varying degrees of egg hatch inhibition (Livdahl et

al. 1984, Koenekoop and Livdahl 1986, Livdahl and Edgerly 1987).

Thus, the purpose of the following experiments on A. albopictus and Wyeomyia spp. is to

determine the mechanism of reduction of growth and survivorship of 1st instar A. albopictus in

the presence of 4th instar Wyeomyia spp. in exotic bromeliads. As mentioned above, chemical

interference competition, encounter competition, predation, and egg hatch inhibition are potential

competitive mechanisms in mosquito populations. Previously none of these mechanisms have

been studied between Wyeomyia spp. and A. albopictus within bromeliads, and thus these

interactions are the focus of a set of experiments in this study.

Furthermore, in attempt to add to the current understanding of the occupancy by A.

albopictus and Wyeomyia spp. of bromeliads in southern Florida (O'Meara et al. 1995b,

Lounibos et al. 2003) multiple collections were made from two common ornamental bromeliad

species B. pyramnidalis and N. spectabilis. Subsequent oviposition and larval competition

experiments were conducted with these two bromeliad species to explain the patterns of

mosquito abundance observed from field collections.









CHAPTER 2
INTERFERENCE COMPETITION AS A POTENTIAL MECHANISM FOR THE
REDUCTION OF GROWTH AND SURVIVORSHIP OF Aedes albopictus IN THE
PRESENCE OF Wyeomyia spp. IN THE BROMELIADS OF SOUTHERN FLORIDA.

Introduction

Competitive interactions can be a maj or factor in determining community structure.

Exploitative and interference competition are the two main mechanisms for interactions between

individuals within a population (Schoener 1983). Exploitative competition has been the focus of

many ecological studies, but interference competition often has a greater influence over species

distribution and abundance (Case and Gilpin 1974). Schoener (1983) subcategorized

exploitative competition into consumptive competition, which involves food resource

competition, and preemptive competition for resource space. The subcategories of interference

competition include (i) overgrowth, which involves individuals growing over one another and

thus depriving their competitors of resources; (ii) chemical, which deals with toxins produced by

some individuals harming other individuals; (iii) territorial, in which individuals aggressively

defend units of space to the detriment of the competitors, and (iv) encounter competition in

which interactions between mobile competitors cause harm by fighting, predation, or physical

interference. Overlap is common in exploitative and interference competition (Schoener 1983).

For example, interference competition affects competitors by changing the rate of resource

exploitation, or in some cases resulting in injury or death, which in turn affects population

growth (Case and Gilpin 1974).

Many plants, such as grasses (Javaid et al. 2005, Singh et al. 2005) and pine trees

(Nektarios et al. 2005), release allelopathic chemicals that create an unfavorable environment for

possible plant competitors. In some cases, the allelopathic chemicals released by plants are the

main determinants of the plant community structure (Rasmussen and Rice 1971), and are









therefore a maj or competitive influence in the community. Bacteria also release allelopathic

chemicals to limit the growth of their neighbors (Riley and Gordon 1999).

Allelopathy has not been identified in animals, but animals experience chemical

interference caused by waste materials from other individuals. These waste products, such as

ammonia and nitrates, cause a deterioration of the environment leading to a reduction in the

growth and survivorship of other individuals (Bedhomme et al. 2005). For instance, intraspecific

chemical interference competition has been shown among sea lamprey larvae (Rodriguez-Mufioz

et al. 2003), Parantecium (Gill 1972), tadpoles (Griffiths et al. 1991) and larvae of the

mosquitoes A. aegypti (Bedhomme et al. 2005, Dye 1984) and Culex pipiens nzolestus (Ikeshoji

et al. 1976).

Density dependent chemical inhibition of larval growth has been demonstrated in other

species of Aedes mosquitoes. Broadie and Bradshaw (1991) determined from a laboratory

experiment that intraspecific chemical interference competition influenced pupation success,

pupal weight and development time in Aedes sierrensis. Chemical interference has also been

shown to affect the growth of A. albopictus. Sunahara and Mogi (2002) determined that A.

albopictus experienced a reduction in survivorship and pupation success from interspecific

chemical interference competition with Tripteroides ba~nbusa in bamboo stumps.

Chemical inhibition of larval growth may be less common in other genera of mosquitoes.

Wyeonzyia snzithii exhibit density dependent increases in development time and reductions in

survivorship and pupal weight, but neither encounter competition, chemical competition, nor

cannibalism were found to contribute to these density dependent changes in fitness (Broberg and

Bradshaw 1995).









While chemical inhibition of growth does occur within larval communities, encounter

competition is often found to be the predominant interference mechanism. Dye (1984) found that

some strains ofA. aegypti demonstrated intraspecific chemical interference, but chemical effects

were small compared to those of encounter competition. Roberts (1998) also found that chemical

inhibition of growth occurred among Culex sitiens conspecifics, but that encounter competition

was the stronger of the two competitive factors.

Encounter competition was defined by Brian (1956) as the ability of an organism to harm

another organism by directly attacking it, or indirectly by damaging its food resources or

blocking its access to those resources. Direct attack encounter competition has been found to

occur in mosquito larvae and often alters their survivorship. Among some fi1ter-feeding

mosquito species, 1st instar larvae may be attacked and often killed by fourth instar conspecifies

in laboratory microcosms (e.g., Reisen and Emory 1976, Koenekoop and Livdahl 1986,

Koenraadt et al. 2003). Mosquitoes may experience encounter competition when either high

densities of individuals cause frequent encounters with one another (Broadie and Bradshaw

1991, Roberts 1998) or larvae frequently encounter larger individuals of later instars (Dye 1984,

Broadie and Bradshaw 1991). The combination of high densities and larger individuals can have

the biggest effect on early instar mosquitoes (Broadie and Bradshaw 1991).

In many cases, physical contact between organisms can lead to a reduction in feeding

efficiency or other metabolic costs (Anholt 1990). Physical contact was implicated as the cause

of reduced feeding rates of A. sierrensis (Broadie and Bradshaw 1991) and Anopheles gamnbiae

(Koenraadt et al. 2003) at high densities.

Reductions in feeding efficiency can cause alterations in life history characteristics by

increasing larval mortality and development time and decreasing adult size. Aedes cantans









experienced a reduction in adult size and an increase in larval mortality, which were

hypothesized to be due to contact competition (Renshaw et al. 1993).

Previous studies have tried to vary the effects of encounter competition by altering the

water volume (Dye 1984), the surface to volume ratio (Broadie and Bradshaw 1991, Roberts

1998), or habitat complexity (Broadie and Bradshaw 1991). While experiments with alterations

in the surface area to volume ratio did not significantly alleviate the effects of competition by

changing the encounter rate between individuals (Broadie and Bradshaw 1991, Roberts 1998),

there were successes with the alteration of water volume (Dye 1984) and habitat complexity

(Broadie and Bradshaw 1991).

Habitat complexity affects the levels of competition and predation among species (Hixon

and Menge 1991, Hixon and Jones 2005). With increasing structural complexity of a habitat, the

number of competitive refuges increases (MacArthur and Levins 1967, Finke and Denno 2002),

and the number of physical encounters between predator and prey decreases (Murdoch and

Oaten 1975). With more physical encounters in habitats of decreased complexity, the occurrence

of intraguild predation increases (Marshall and Rypstra 1999, Roda et al. 2000, Finke and Denno

2002). In fact, most studies conducted on the effects of habitat complexity on competitive

interactions have involved predator-prey interactions (e.g., Crowder and Cooper 1982, Schneider

1984, Diehl 1992, Babbitt and Tanner 1998, Alto et al. 2005), although some studies have found

benefits to competitors as well as prey populations with increasing habitat complexity (Almany

2004). As with predator-prey interactions, an increase in habitat complexity should decrease

encounters between potential competitors. Therefore, structural complexity may provide refugia

from physical encounters with non-predatory species.









Behavioral changes in organisms that experience encounter competition are common.

Anholt (1990) observed noticeable behavioral changes in feeding behavior of damselflies when

exposed to different interspecific encounter rates. As mentioned before, physical encounters

with other individuals can cause changes in feeding efficiency, and thus there should be a change

in feeding behavior of individuals influenced by encounter competition.

Lounibos et al. (2003) found that A. albopictus had a reduction in growth and survivorship

when exposed as 1st instars to 4th instars of Wyeomyia spp. in bromeliads and hypothesized

interference competition as the mechanism for this effect. In this chapter, chemical interference

competition and encounter competition are examined as potential types of interference to explain

the negative effect of Wyeomyia spp. on larvae ofA. albopictus.

In this chapter, one experiment was conducted to determine whether the waste products of

Wyeomyia spp. reduce the growth or survivorship of A. albopictus. Two experiments were

conducted to determine whether physical contact with the larger 4th instar Wyeomyia spp.

affected the growth and survivorship ofA. albopictus. The first of these experiments varied the

surface area to volume ratio of artificial bromeliads in an attempt to change the frequency of

encounters between the species. The second experiment varied habitat complexity as an

alternative method of changing the frequency of encounters between species. Finally, the last

experiment attempted to elucidate changes in feeding location ofA. albopictus in response to the

presence of Wyeomyia spp..

Materials and Methods

Chemical interference

Twenty-five 1st instar colony-raised A. albopictus were added to 1000 Dalton, 31 mm

diameter Spectra/Por Biotech Cellulose Ester dialysis tubes that contained 25 mL of sieved

bromeliad water and 0. 1 g of chopped live oak leaves (Quercus virginiana). The leaves were









dried at 68 OC for 48 h before weighing. The bromeliad water was collected from approximately

60 plants, and sieved through a 130 Clm gauge mesh screen to remove detritus and macroscopic

organisms. All sieved water was mixed into one large bucket, and then covered with a plastic

bag for 2 days.

The 1000 Dalton dialysis tubing was chosen for this experiment because its pore size

allows waste products such as nitrates and ammonia to pass through, while excluding larger

compounds such as food particles. Each tube was cut to 11.5 cm in length, which created

approx. 8 X 3.1cm of aquatic habitat for the l't instar larvae, and excess tubing of approximately

3.5 X 3.1cm was left over for folding the ends and for suspending the tubing. A butterfly clip

strung with wire was attached to the tubing to suspend it in the center axil of a Billbergia

pyramnidalis bromeliad. The butterfly clip also kept the tubing from sealing at the top, which

would have prevented access to the air.

The dialysis tubing had been preserved in 0.1% sodium azide solution, which was toxic to

the larvae in preliminary tests. Therefore, a few days before use each dialysis tube was

detoxified by soaking in distilled water 3 times for 30 minute intervals, after which it was placed

in a 1.0 % sodium benzoate solution to prevent decay. On the day of the experiment, each tube

was once again soaked 3 times for 30 min to remove the sodium benzoate, and then closed off at

the bottom by folding and then sealing with a plastic-coated wire garbage tie.

To the center axil of 10 of the 20 plants, 30 4th instar Wyeomyia spp. were added to 100

mL of sieved bromeliad water containing 0.4 g of chopped oak leaves. One hundred militers of

sieved bromeliad water and 0.4 g of chopped oak leaves were added to the center axil of the ten

control plants. Then each plant was randomly assigned to one of five cages where their bases









were secured in a small plastic container and held for 48 h at 260C with a 12:12 h light dark

cycle and approximately 80% relative humidity.

The plants were checked every 12 h for leaking, which was observed in 10 of the twenty

plants, a loss of around 30 mL by 24 h. At 24 h, more sieved bromeliad water was added to the

leaky plants corresponding to the amount of water loss detected in the plastic container holding

the bromeliad.

Ammonia levels and pH of the bromeliad water were measured before and after the

experiment. Ammonia concentration was recorded using the Fisher Scientific Accumet Portable

AP63 pH/mV/ion meter using an ammonia probe, and pH was recorded using a Corning pH-20

meter.

After 48 h, the water in each bromeliad was removed by a pipette, and rinsed into a metal

rearing pan with a spray bottle containing tap water. The number of surviving A. albopictus and

the instars of each A. albopictus, determined by head capsule widths, were recorded for each

replicate. An ANOVA was performed in SAS (2002) to determine whether the mean

development stage or survivorship ofA. albopictus differed between experimental (30

Wyeomyia) and control (no Wyeomyia) plants.

Surface Area to Volume Ratio Encounter Competition Experiment

Four different sized artificial bromeliads were built with circumferences of 4.5, 5.5, 7.0,

and 9.0 cm at the 75 mL water level, representing surface area to volume ratios of 1.48, 1.82,

2.48, and 3.83 cm2/mL, respectively. The artificial bromeliads were constructed from 8.5 by 11

inch weatherproof map paper (IGage, Mapping Corporation, Salt Lake City UT), and Perfect

Glue No. 1 (Liquid Nails, Cleveland, OH) was used as an adhesive on the outer parts of the

plant. The inner parts of the plant were water protected and sealed with non-toxic, waterproof,

100% silicone aquarium tank sealant (All-Glass Aquarium, Franklin, WI).









Twenty-five 1st instar A. albopictus, 0.5 g oak leaves, and 75 mL of sieved bromeliad water

were added to 15 control bromeliads of each size. The other 15 of each size represented the

experimental group and differed from the controls by the addition of 30 4th instar Wyeomyia spp..

Each replicate was placed into a plastic container of appropriate size and put onto one of three

randomly chosen shelves in a climate controlled rearing room kept at 260C with a 12:12 light

dark cycle and approximately 80% humidity. On the day of setting, each artificial bromeliad

was marked with a pencil to indicate the 75 mL waterline. To compensate for evaporation, and

the differences in evaporation based on the varied surface area to volume ratios among

treatments, on the 4th and 8th day of the experiment each plant was refilled with sieved bromeliad

water to its original 75 mL water level. After 10 days the experiment was terminated, and all of

the A. albopictus in each replicate were counted, and their instars were recorded based on head

capsule widths.

Using PROC GLM in SAS (2002) an ANOVA was performed to detect significant

variation among the bromeliad sizes and between the presence and absence of Wyeomyia spp. in

the development rate (average instar) and survivorship (percent alive) of A. albopictus

immatures.

Habitat Complexity Experiment

The habitat complexity experiment used the same artificial bromeliads described in the

surface area to volume ratio experiment. In this experiment, the smallest artificial bromeliad

with a circumference of 4.5 cm and a surface area to volume ratio of 1.48:1 was excluded

because it was too narrow for the habitat complexity manipulation and still allow for larval

access to the air. In the other three sizes, 20 1st instar A. albopictus, 20 4th instar Wyeomyia spp.,

0.5 g oak leaves, and 75 mL of sieved bromeliad water were added to 30 bromeliads of each.









Two different types of 3 cm2 Squares were constructed from weatherproof paper to add

habitat complexity to the experiment. The first type of paper square, which represents the low

habitat complexity treatment, remained two dimensional (Fig. 2-1). Three of these squares were

added to half of each of the three bromeliad sizes. The second type of paper square, which

represents high habitat complexity, was cut and folded to make a three dimensional structure

(Fig. 2-2). When added to the cone, the low complexity square remained at the top or along the

sides, while the high complexity square made a lattice-like structure within (Fig. 2-2).

After 5 days, the experiment was terminated, and the numbers and developmental stage

of the remaining 1st instar A. albopictus larvae were recorded as described in previous

experiments. An ANOVA was performed in SAS (2002) with survivorship and development

time ofA. albopictus as dependent variables and surface to volume ratio of the artificial

bromeliad (n=3) and the internal complexity (n=2) of the habitat as the independent variables.

Bonferroni-adjusted multivariate pairwise means comparisons followed detection of significant

effects by ANOVA.

Resource Dependent Location of A. albopictus and Wyeomyia spp.

To determine whether foraging behavior ofA. albopictus and Wyeomyia spp. changed in

the presence of each other, or in different levels of food resources, an experiment was conducted

in which three treatments within plastic cups received 0. 1 g of dried chopped oak leaves (Q.

virgininia) as prepared in previous experiments, and the other three treatments received 0.5 g of

the same leaves. The leaves were located at the bottom of all containers. As described in prior

experiments, 75 mL sieved bromeliad water was also added to each container. Each of the food

levels had one treatment with 10 1st instar A. albopictus and 10 4th instar Wyeomyia spp., one

treatment with only 10 1st instar A. albopictus, and one treatment with only 4th instar Wyeomyia

spp.. Each of these treatments was replicated two times. All Wyeomyia spp. were collected as










larvae from bromeliads in Vero Beach and Fort Pierce, FL. All A. albopictus came from a

colony described in the previous experiments.

An instantaneous scan census (Martin and Bateson 1986) was implemented every hour for

7 h starting after a 3 h acclimation period. Treatments with only one species had fewer

individuals, so behavioral observations were usually finished in approximately 15 seconds. The

treatments with both species had more individuals to examine, so those observations often took

as long as 45 seconds. The two replicates for each treatment were sampled consecutively before

moving onto the next treatment, and the treatment observed first was changed at each

ob servati on. To make observations of 1st instar A. albopictus easier, and to prevent disturbing

the larvae with light at every observation, treatments were illuminated by a 60 watt desk lamp for

the entirety of the experiment.

Because the changes in location within the container were the focus of this experiment, the

locations of each Wyeomyia spp. and each A. albopictus at each time were coded into one of four

categories, predetermined from preliminary observations: 1) at the middle of the cup (1cm from

top to 1cm from bottom); 2) on the bottom of cup (bottom to 1 cm from bottom); 3) at the top of

cup (water surface to 1 cm below the water surface with or without siphon extended for

breathing) or 4) wandering (swimming without noticeable foraging behavior). Owing to the

small size of 1st instar A. albopictus, whether the larva was resting, filter feeding or browsing

was not recorded because the differences in these behaviors often could not be determined. For

instance, during preliminary examinations there were multiple occasions when A. albopictus was

nearby a surface, so it appeared to be browsing on that surface, but after closer examination at a

higher magnification, the larva was found to be filter feeding close to the surface, not browsing

upon it.










At the end of the experiment, the raw data in each observation category were converted to

a proportion based on the number of individuals of each species that showed the categorical

behavior at the observation time. The proportional data were arcsine square root transformed

and a multivariate analysis of variance (MANOVA) was performed with the effects of species,

food, alone (whether with or without the other species), time, replicate, and the interactions of

species x treatment, species x food, and treatment and food. Further analysis of variance

(ANOVA) analyses with subsequent Tukey post-hoc tests differentiated the effects found to be

significant in the MANOVA.

Results

Chemical Interference

There was no significant difference (F1,19=1.03, P=0.32) between the average instars

(development rate) of A. albopictus in dialysis bags with or without Wyeongia spp. outside the

bag (Fig. 2-3). There was also no significant difference (F1,19=2.94, P=0.10) in the arcsine

square root transformed survivorship between the treatment and the control groups (Fig. 2-4).

The mean values of ammonia, and pH showed that there was little change in these

concentrations between pre- and post-experiment (pH: tl9=1.07, P=0.30; ammonia: tl9=1.34,

P=0.20) (Table 2-1).

Surface Area to Volume Ratio Experiment

The A. albopictus in the treatments with Wyeongia spp. were found to have a significantly

lower average in star (LS mean =2.72 & 0.07 SE) as compared to the LS mean (3.96 & 0.08 SE)

with Wyeongia spp. (Fig. 2-5). There was no significant effect of bromeliad size on average

instar, but there was a significant interaction between the presence of Wyeongia spp. and size

(Table 2-2). From smallest to largest size, the average instars (mean and standard error) at each

surface area to volume ratio were 3.29 & 0.17, 3.54 & 0.16, 3.32 & 0.16, 3.22 & 0.12 respectively.









In treatments without Wyeomyia spp., A. albopictus were found to have a significantly

lower survival (Table 2-3) (0.83 & 0.02 (mean & SE)) compared to the control (0.89 & 0.02

(mean & SE)). The largest three surface area to volume ratios contributed the most to the

significant Wyeomyia spp. effect on survivorship (Fig 2-6). A Tukey post hoc test determined

that the artificial bromeliad with the 6 cm diameter, which was the second to smallest diameter,

had a higher mortality rate than the other three sizes, but was only significantly different from

the smallest size (Fig. 2-6).

Habitat Complexity Experiment

There was no difference in the survivorship of A. albopictus between high and low

complexity treatments (F1,89=0.57, P=0.45) or surface area to volume ratios (F2,89=0.74, P=0.48).

There was no significant difference in the development rate ofA. albopictus among surface area

to volume ratios (F2,89=1.29, P=0.28) but A. albopictus developed faster in high complexity

treatments compared to the low complexity treatments (F1,89=30.02, P<0.01) (Fig 2-7) There

was no significant interaction between surface area to volume ratio and habitat complexity on

developmental rate (F2,89=2.54, P=0.08), or on survivorship (F2,89=0.89, P=0.42).

Resource-Dependent Foraging Location

MANOVA indicated significant effects of species, presence of other species, and

resource levels (Table 2-4). In general, Wyeomyia spp. foraged on the sides and on the bottom of

containers more than A. albopictus (Figs. 2-8 and 2-9). Aedes albopictus were found at the top

of the container more frequently and wandered more than Wyeomyia spp..

There were some significant differences between the locations of the mosquito species at

the different food concentrations (Table 2-5 and 2-6). At high food concentrations Wyeomyia

spp. allocated more time at the water surface (Fig 2-11). Aedes. albopictus also spent more time

at the water surface at high resource concentrations (Fig. 2-10). At low food concentrations,










Wyeonyia spp. allocated more time on the bottom of the container, while A. albopictus occurred

on the sides on the container more frequently (Figs 2-10 and 2-11). There was significant

variation in the location of A. albopictus attributable to the presence of the other species and

food level (Table 2-5).

In the presence of Wyeonzyia spp., A. albopictus spent more time on the top of the

container and less at the bottom or on the sides of the containers (Fig. 2-8). There were

significant interactions between the presence of the other species (alone effect) and species, and

the presence of the other species, and the level of food resources (Table 2-4). Thus, there was a

difference in larval behavior of A. albopictus in the presence of Wyeonzyia, and the level of food

resources also influenced the relative location of these two species.

Discussion

The chemical interference experiment provided no evidence that waste products from

Wyeonyia spp. larvae interfere with the growth or survivorship of A. albopictus. The buildup of

waste products in experiments that exhibited chemical interference, such as among A. aegypti

(Bedhomme et al. 2005, Dye 1984), may be less likely to occur in the bromeliad tank and axil

habitat. Billbergia pyranzida~lis is both an epiphytic and ground dwelling plant (Frank et al.

1988). While there are no specific reports on the nitrogen absorption abilities of B.

pyra~nidalis, in general, epiphytic bromeliads use dissolved nitrogen held in their tank water as

their main nitrogen source (Benzing et al. 1976, Raven 1988). Epiphytic bromeliads also

assimilate most of their nitrogen from ammonia (Endres and Mercier 2001), which is a common

excretory product of mosquitoes (Clements 1999). By the end of the experiment, there was little

change in the ammonium concentration or pH of the bromeliad water (Table 2-1 ), even though

30, 4th instar Wyeonyia spp., and 25 A. albopictus had been added to the tank for 48 hours.

Because B. pyra~nidalis is sometimes epiphytic, it is possible that the plant absorbed the









ammonia excreted by the mosquitoes, thus reducing the probability of chemical interference

from nitrogenous wastes in this phytotelm. On the other hand, at 24 hours as much as 30 mL of

water was added to some of the plants that had leaked water. This addition of water could have

reduced or eliminated the effects of chemical interference of Wyeomyia spp. on A. albopictus in

this experiment.

The dialysis bag used in the chemical interference experiment physically separated A.

albopictus from Wyeomyia spp. Thus, other potential forms of interspecific competition, such as

encounter, resource, or space competition were eliminated. In some other experiments of this

chapter, A. albopictus were in direct contact with Wyeomyia spp., which resulted in a significant

reduction in growth ofA. albopictus. In the chemical interference experiment, no significant

effects were detected, suggesting that encounter, resource or space competition plays an

important role in the reduction of growth and survivorship seen in the other experiments.

Previous experiments where surface area to volume ratios were manipulated in order to

influence the impact of encounter competition have often been unsuccessful. Broadie and

Bradshaw (1991) altered the surface area to volume ratio of treehole microcosms, but only the

density ofA. sierrensis had an effect on pupation success, larval development time, or pupal

weight. Anholt (1990) altered the surface area available to damselfly larvae in cages, which had

no effect on density dependent decreases in growth and survival. Damselfly larvae exhibited a

behavioral response to the increase of structural complexity inside cages, but habitat complexity

did not change the decrease in growth, survival time, and size seen at high densities (Anholt

1990).

In the current study, while the surface area to volume ratio of the plant had no effect on

the development rate of A. albopictus, there was a significant variation in survivorship of this










species among the different surface area to volume ratios. Mean survivorship in size 2 differed

significantly from size 1. The biological significance of this difference between these two sizes is

unknown.

Another unexpected result of the surface area to volume ratio experiment was the reduced

survivorship ofA. albopictus in the absence of Wyeomyia spp.. The three largest surface area to

volume ratios appeared to contribute to most of this reduction in survivorship, although the

interaction between plant size and Wyeomyia spp. was not significant (Table 2-3).

While the results of the surface area to volume ratio experiment do not explain the effects

of Wyeomyia spp. on A. albopictus, increased habitat complexity in this experiment reduced the

development time ofA. albopictus in the presence of Wyeomyia spp.. In the habitat complexity

experiment the addition of waterproof paper increased the foraging surface area This increase

provided additional surface area to the developing A. albopictus, a factor which was not included

in other competition experiments in this thesis. In this experiment, while more foraging area was

added to the treatments, the total foraging area between treatments was kept the same, because in

both treatments the size of the paper added, and the number of pieces were kept constant, the

paper was folded only to increase complexity. By the end of the experiment though, many of the

low complexity treatment squares had become appressed to the sides of the artificial bromeliad,

thus resulting in a decrease in the foraging surface area between the two habitat complexities.

Therefore, habitat complexity may have decreased the development time ofA. albopictus in the

presence of Wyeomyia spp., but the difference in foraging surface area between the habitat

complexities may have contributed to this decrease in development time. If this effect is due to

habitat complexity, its relevance under Hield conditions is uncertain. When Wyeomyia are

present in bromeliads, which is the case in most of southern Florida (O'Meara et al. 1995b), then









increased complexity could decrease the development time of A. albopictus. Whether increased

habitat complexity affects the developmental rate ofA. albopictus in the absence of Wyeomyia

spp. remains uncertain as no treatments without Wyeomyia were used in this experiment.

The results of the behavior experiment indicated that Wyeomyia spp. spent most of the

time foraging on the bottom of the container, especially during periods of low food

concentrations. When in contact with Wyeomyia spp., A. albopictus spent significantly less time

on the bottom of the container, and stayed the furthest away from Wyeomyia spp. by spending

significantly more time at the top of the container irrespective of food concentration. Such

behavior ofA. albopictus could indicate avoidance of Wyeomyia spp.

These experiments do not support chemical interference as a likely competitive

mechanism to explain negative effects of Wyeomyia spp. on A. albopictus. From the habitat

complexity experiment, encounter competition is the likely explanation for the increase in

development time ofA. albopictus in the presence of Wyeomyia spp., but, as mentioned before,

increased foraging area may have also contributed to this effect. Like other surface area to

volume ratio habitat experiments performed with mosquitoes, this surface area to volume ratio

experiment failed to explain the competitive interactions between A. albopictus and Wyeomyia

spp.. The behavioral experiment provided some indication that A. albopictus larvae change

location within the container in the presence of Wyeomyia spp. These changes in location could

be a result of encounter competition causing A. albopictus to change their location to avoid

encounters with Wyeomyia spp. This avoidance of Wyeomyia spp. may contribute to the

decrease in development rate observed among A. albopictus in the presence of Wyeomyia spp..










Table 2-1. Means and standard errors for the concentrations of ammonia and pH pre- and post-
expeniment.

pH (ppm) (mean & SE) Ammonia (mean & SE)

Pre-experiment 6.21 & 0.02 0.22 & 0.01

Post experiment 6.19 & 0.02 0.21 & 0.01

Paired t-tests showed no significant differences (alpha= 0.05) between pre- and post-experiment
means.










Table 2-2. Analysis of variance for A. albopictus average instar based on the presence or absence
of Wyeomyia spp. and the surface:volume ratio of the artificial plant and the
interaction of these two variables.


P-value

<0.01


Wyeomyia

Bromeliad Size

Wyeomyia x Size


162.38


2.01

4.56


0.11


<0.01










Table 2-3. Analysis of variance for A. albopictus average survival based on the presence or
absence of Wyeomyia spp., and the surface area:volume ratio of the plant (size) and
the interaction of these two variables.
F Df P-value

Wyeomyia 6.48 1 0.01

Bromeliad Size 5.41 3 <0.01

Wyeomyia x Size 1.56 3 0.20










Table 2-4. Multivariate analysis of variance table for feeding location.
Effect Wilks' Lambda Df P-value


Species

Alone


65.20

2.66

8.30

0.88

4.83

4.68


<0.01

0.02

<0.01

0.68

<0.01

<0.01

0.02


Food

Time


Species x Alone

Species x Food

Alone x Food


3.84


The species effect refers to the location of either A. albopictus or Wyeomyia spp.. Alone refers to
whether or not each species is alone or in the presence of the other species. The food category
variables were either 0. 1 g leaves (low treatment) or 0.5 g leaves (high treatment). Time is the
hour of observation from the start of the experiment.










Table 2-5. Analysis of variance for the effects of food level presence or absence of Wyeomyia
spp. on the location of A. albopictus.
F Df P-value

Combo High Food 15.40 3,63 <0.01

A. albopictus only High Food 3.49 3,63 0.02

Combo Low Food 12.24 3,63 <0.01

A. albopictus only Low Food 1.12 3,63 0.35

Combo indicates the treatments that had both Wyeomyia spp. and A. albopictus.









Table 2-6. Analysis of variance for the effects of food level and presence or absence of A.
albopictus on the location of Wyeomyia spp.
F Df P-value

Combo High Food 14.55 3,63 <0.01

A. albopictus only High Food 33.87 3,63 <0.01

Combo Low Food 39.58 3,63 <0.01

A. albopictus only Low Food 15.58 3,63 <0.01

Combo indicates the treatments that had both Wyeomyia spp. and A. albopictus.





























3cm


3cm


A




B


Figure 2-1. Construction of the squares for the habitat complexity experiment. A) the low
complexity square is unaltered before introduction to the bromeliad. B) the high
complexity square was cut along the lines indicated inside the square.


-1.5 cm































A BB C



Figure 2-2. The folded shape of the high complexity and low complexity treatments and the
orientation of the squares within the cones. A) in the low complexity experiment the
squares floated on the top, or laid on the sides of the cone. C) in the high complexity
cone, the altered squares create a lattice-like formation on the inside of the cone. B)
The diagram in the center shows the shape of the altered square after the cutting and
folding described in Fig. 2-1.









I


2. 05


~ I.9


1.95



1. 85
~ieomyia Absent o~myia Present

Figure 2-3. Average instars of A. albopictus after 48 h exposure to Wyeomyia spp. through a
dialysis membrane and without Wyeomyia in the control. The error bars are standard
error.












0.68


'$0.4

S0.2



FF)eomyia Ab~sent Wyeomyia Present

Figure 2-4. Mean survivorship of A. albopictus after 48 h exposure to Wyeomyia spp. through a
dialysis membrane and without Wyeomyia spp. in the control. The error bars are
standard error.












4.5a
a a



3.5 --

811b b b






< 1.5 -



0.5 -


NoWy Wy NoWy Wy NoWy Wy NoWy Wy
Size 1 Size 2 Size 3 Size 4

Figure 2-5. The average instar + SE ofA. albopictus in the absence or presence of Wyeomyia
spp. in four plant sizes. NoWy refers to the treatments with no Wyeomyia spp., and
Wy refers to treatments with Wyeomyia spp.. The size below these symbols refers to
the surface area to volume ratio in the artificial bromeliad, proceeding from smallest
(1) to largest (4), as quantified in Materials and Methods. Means with common
letters above the bars are not significantly different by (P<0.05).













rp bc


I I I I


8bC


0.7
O -




.r 0.6



0.3 -


be
r


0.2 -

0-


Nos~ Wy NoWy Wy NosWy Wy NO~Cy Wyy
TSize 1 Size 2 Size 3 Size 4


Figure 2-6. The average survivorship + SE ofA. albopictus in the absence or presence of
Wyeone i spp. in four plant sizes. NoWy refers to the treatments with no Wyeonglia
spp., and Wy refers to treatments with Wyeone ia spp.. The size below these symbols
refers to the surface area to volume ratio in the artificial bromeliad, proceeding from
smallest (1) to largest (4), as quantified in Materials and Methods. Mean values
without common letters above the bars are significantly different (P<0.05).














S3.8







3.1 --






L2 H2 L3 H3 L4 H4

Treatment


Figure 2-7. The average instar (+SE) of A. albopictus in two levels of habitat complexity and
three levels of surface area to volume ratio. L and H denote the low and high
complexity treatments. Numbers correspond to increasing surface area to volume
ratios, as indicated in Materials and Methods. Mean values without common letters
above the bars are significantly different by a Bonferroni adjusted multivariate
pairwise test.














I


S0.4-
O








Wyeomyia No Wyoemyia
Location of Mosquet

[ ] Bottom
I ITop
I Wander


Figure 2-8. Location ofA. albopictus in the presence and absence of Wyeomyia. The bottom and
top locations are significantly different between Wyeomyia and no Wyeomyia
treatments (alpha<0.05) by a Tukey means comparison. The bars indicate SE.




























o II2
C







No A. abdcpictubS A. abdcpictubS

Locatio n of M osquito

I Bottom
I Top
I Wander


Figure 2-9. Location of Wyeomyia spp. in the presence and absence ofA. albopictus. No
locations were significantly different between A. albopicats and no A. albopicats
treatments (alpha<0.05) by a Tukey means comparison. The bars indicate SE.























c0.4
0 a 0


" 0.2

o- a a:







~0a a SideI








Fiur -1.Loato oA aboitu y od cocnrto adtepee cando absenc sof u~





Figue 2indiLcate igniofic anly diffretu menaogh food concentrations (high pesn and lbeeow)

and treatments (Wyeone ia spp. and No Wyeone ia spp.) for each location (side,
bottom, top and wander) (alpha=0.05).






















LU0.4 b b
o7 b b

a







ff 44Location of M ~osuito



go~ go Wander


Figure 2. 11: Location of Wyeomyia spp. by food concentration and the presence and absence of
A. albopictus. The bars indicate SE. Different letters over the same bar category
indicate significantly different means among the food concentrations (high and low)
and treatments (A. albopictus spp. and No A. albopictus spp.) for each location (side,
bottom, top and wander) (alpha=0.05)









CHAPTER 3
Wyeomyia spp. AS POTENTIAL PREDATORS OF Aedes albopictus IN BROMELIADS IN
SOUTHERN FLORIDA

Introduction

In mosquito populations predation can effect the survivorship (Griswold and Lounibos

2006, Blaustein et al. 1995, Fincke et al. 1997), and development time (Alto et al. 2005, Grill

and Juliano 1996) of the prey as predators consume prey or the prey become less active in the

presence of predators. These alterations in survivorship and development time can effect the

population and community structure of both the predator and prey species (Sih 1985).

Intraguild predation is the killing and consumption of competitors that consume similar

resources, which has an immediate energetic benefit and a beneficial reduction in exploitative

competition for the predator (Polis et al. 1989). Age and size dependent intraguild predation is

common throughout all ecological systems, and many predators are cannibalistic on smaller

sized or younger conspecifics (Polis et al. 1989). Often intraguild predation occurs between

competitors with the greatest resource overlap, and may be a result of the increased encounter

rates due to niche overlap (reviewed by Polis et al. 1989).

Mosquitoes that are predominantly browsers and filter feeders, such as Aedes and

Anopheles species (Clements 1999), may become cannibals (Hinman 1934, Reisen and Emory

1976, Koenekoop and Livdahl 1986, Koenraadt et al. 2003) or facultative predators (Edgerly et

al. 1999, Koenraadt et al. 2003) under certain conditions. Koenekoop and Livdahl (1986) and

Edgerly et al. (1999) found that cannibalism in Aedes triseriatus was resource dependent with

lower resource availability leading to higher cannibalism. Koenraadt et al. (2003, 2004) found

no increase in cannibalism or predation by Anopheles gambiae complex mosquitoes related to

resource availability. Increased habitat complexity also reduced predation by A. triseriatus on

congeners, but the opposite effect was seen with A. aegypti (Edgerly et al. 1999). Koenraadt et









al. (2004) also showed that higher densities of anopheline mosquitoes in small spaces increased

cannibalistic and predatory behavior.

Although facultative predation may influence the population dynamics of many Aedes and

Anopheles mosquitoes, W. mitchellii and W. vanduzeei have yet to be examined as potential

predators of A. albopictus in bromeliads. Because predation is an important mechanism shown to

effect the survivorship and development time of mosquito species in other studies, Wyeomyia

spp. larvae from Florida bromeliads were dissected after exposure to A. albopictus to determine

whether these Wyeomyia spp. may be facultative predators ofA. albopictus.

Materials and Methods

Testing for Density Dependent Predation by Wyeomyia spp.

In this experiment, three densities (0, 25, 50) of 1st instar A. albopictus were placed into

plastic containers containing 75 mL sieved bromeliad water and 0.5 g dried (680C for 48 h),

chopped Quercus virginiana leaves. Thirty-six containers were used, allowing 12 replicates of

each density. To each container thirty 4th instar Wyeomyia spp. were added after previously

being kept in tap water without food for 36 h prior to the experiment. The lack of food for

Wyeomyia spp. prior to the experiment was to ensure that all gut contents examined after the

experimentation resulted from the food obtained during the experiment, and not prior feedings.

After 12 h, 6 containers were removed from each density, and all live 4th instar Wyeomyia spp.

larvae were dissected. At 24 h, the remaining containers were removed and again all of the live

4th instar Wyeomyia spp. larvae were dissected. Dead larvae of Wyeomyia spp., and pupae were

not dissected.

Testing for Resource Dependent Predation by Wyeomyia spp.

In a second experiment, two densities of food resources (0. 1 g and 0.5 g) of dried (680C

for 48 h), chopped Q. virginiana leaves were placed into 24 plastic containers with 75 mL sieved









bromeliad water and 25 1st instar A. albopictus. Twenty-five 4th instar Wyeomyia spp. were

added to each container. Twelve hours and 96 h after the start of the experiment, 6 containers at

each food level were removed and their live 4th instar Wyeomyia spp. were dissected. As before,

dead Wyeomyia spp., and pupae were not dissected.

Predation Observation

Visual examinations of the behavior of A. albopictus and Wyeomyia spp. were conducted

in plastic containers. Ten 1st instar A. albopictus were added with either (1) 0. 1 g food and no

Wyeomyia spp.; (2) 0.1 g food and 10 4th instar Wyeomyia spp.; (3) 0.5 g food and no Wyeomyia

spp.; or (3) 0.5 g food and 10 4th instar Wyeomyia spp.. There were two replicates of each

combination. The food was dried, chopped Q. virginiana leaves as used in the two previously

described predation experiments. Two cups at each food level were selected, every hour for 10

h, to be examined for signs of predation by Wyeomyia spp. on A. albopictus. Predatory behavior

was classified as chewing, biting, grabbing or actually consuming another organism. Each

examination occurred for 1 min, and since light was necessary for visualization of the 1st instar

A. albopictus, a desk light kept the cups illuminated for the entire 10 h duration of the

experiment.

Results

No A. albopictus body parts were found in the guts of any of the 941 Wyeomyia spp.

dissected (Table 3-1). All guts of Wyeomyia spp., including the treatment without A. albopictus,

contained brown organic material, but no signs of head capsules, or any other body parts, ofA.

albopictus. There were also no signs of aggression upon A. albopictus by Wyeomyia spp.

throughout the entire duration of the predation observation experiment.









Discussion

From these experiments, it seems very unlikely that Wyeomyia spp. prey upon A.

albopictus. When field-collected larvae of W. vanduzeei were examined by Fish (1976), no

remains from any invertebrates were found; only organic particulate matter, protozoans and

pollen grains were found in the gut contents. In a study by Broberg and Bradshaw (1995), the

pitcher plant mosquito, Wyeomyia smithii, was found to not exhibit density dependent

cannibalism. Therefore, although other detritivores, such as some Aedes and Anopheles species,

can be cannibalistic or facultative predators, it seems that Wyeomyia spp. of North America do

not exhibit these characteristics, at least under the density and resource conditions examined in

the lab.

As an adaptation to the variable water and resource levels in a bromeliad, W. vanduzeei can

develop slowly on limited food resources (Frank and Curtis 1977), so the Wyeomyia spp. of

Florida bromeliads may not have adopted a predation strategy, because they have adapted to a

food limited environment. Most Aedes and Anopheles species that are cannibalistic or facultative

predators would starve at food levels that Wyeomyia spp. can subsist upon (Barrera and

Medialdea 1996).

As with any lab experiment, the applicability of these results to nature comes into question.

As mentioned in other chapters, bromeliad phytotelmata vary in size, structure, complexity,

water holding capacity, and faunal composition. All of these factors could influence the

expression of predation in Wyeomyia spp.. Variations of bromeliad phytotelm size, structure,

and complexity have been examined to some extent in this study. In this study, whether

conducted within an actual bromeliad or an artificial one, there was no significant reduction in

survivorship of A. albopictus that would indicate predation. In conclusion, if there is predation










by Wyeomyia spp. on A. albopictus it occurs so rarely as to be unimportant in overall

survivorship of A. albopictus.










Table 3-1. Number of Wyeomyia examined in predation experiments.
Density Dependent Resource Dependent

Time 0 Albo. 25 Albo. 50 Albo. Low food high food

12 h 111 123 128 74 111

24 h 117 126 151 n/a n/a

96 h n/a n/a n/a 87 158

This table compiles the number of dissected Wyeomyia spp. from two different experiments.
The time box indicates the number of hours after A. albopictus were added that Wyeomyia were
examined. The next three categories, O Albo., 25 Albo., and 50 Albo., correspond to the first
experiment in which the Wyeomyia were examined in the presence of 0, 25 and 50 1 st instar A.
albopictus. The next two categories, low food and high food, indicate the treatments in the
second experiment in which Wyeomyia spp. were examined after exposure to 0.1 g or 0.5 g of
food in the presence of 25 1 st instar A. albopictus.









CHAPTER 4
DO Wyeomyia spp.. LARVAE INTHIBIT EGG HATCH OF Aedes albopictus?

Introduction

Eggs of Aedes spp. hatch after submersion in water, and low oxygen levels caused by

nearby microbial growth stimulate hatching (Gjullin et al. 1941, Judson 1960, Fallis and Snow

1983). Most eggs will hatch in response to the initial stimulus, but some hatching is delayed,

awaiting later stimuli (Gillett et al. 1977, Livdahl and Koenekoop 1985, Andreadis 1990).

Suppression of egg hatch by larvae occurs when the larvae consume microbes on and near the

eggs, thus causing a reduction in the local microbial population, and a subsequent increase in

oxygen resulting in inhibition (Gillett et al. 1977, Edgerly and Marvier 1992) There are also

abiotic secondary determinants of hatch inhibition, such as temperature (Mallack et al. 1964),

photoperiod (Horsfall 1956, McHaffey and Harwood 1970, McHaffey 1972, Shroyer and Craig

1980), and variations in wet and dry periods or humidity (Andreadis 1990, Clements 1999),

which will also delay hatch by putting eggs into quiescence or diapause until conditions are

favorable for hatching .

Egg hatch inhibition is hypothesized to benefit larvae by delaying hatch when

competition for resources is high (Livdahl et al. 1984, Livdahl and Edgerly 1987), risk of

predation is high (Koenekoop and Livdahl 1986), or when abiotic conditions are unfavorable for

development (Shroyer and Craig 1980, Clements 1999). For many Aedes species, these

benefits are thought to outweigh the costs of egg hatch inhibition, which increases the time until

reproduction. Increasing the time to reproduction may lead to a reduction in population growth

and may increase mortality in the dormant egg stage of aedine mosquitoes (Livdahl and Edgerly

1987).









Because many Aedes mosquitoes exhibit some level of hatch inhibition (Edgerly et al.

1993), it is important to examine this role of competition on Aedes hatch. Within the exotic

bromeliads of southern Florida, larval competition between Wyeomyia spp. and A. albopictus is

thought to influence the mosquito community structure in this system (Lounibos et al. 2003),

although the mechanism for this competition has yet to be determined. Field collections from

exotic bromeliads in southern Florida show that high densities of 4th instar Wyeomyia spp. are

common in exotic bromeliads (Raban unpublished data). Additionally, Wyeomyia larvae

develop slowly, as an adaptation to the variable water and resources levels in a bromeliad (Frank

and Curtis 1977), so it is feasible that these large, later instar larvae are present for long

durations. Also, W. mitchellii and W. vanduzeei are found in the larval stage throughout the

year, while some A. albopictus overwinter as eggs. Thus, even an initial spring A. albopictus

cohort could experience egg hatch inhibition from the presence of Wyeomyia spp. Therefore, a

resource dependent egg inhibition experiment was conducted to determine whether older instar

Wyeomyia spp. inhibit the hatch of A. albopictus eggs and whether inhibition varies with larval

food level.

Materials and Methods

Fifty A. albopictus eggs on each of 56 papers inserted individually in plastic cups were

submerged in 75 mL of sieved bromeliad water. All A. albopictus eggs used in the study were

from one oviposition paper, resulting from approximately 3 days of oviposition from a Florida

colony of this species supplemented irregularly with wild mosquitoes. All eggs were counted

under a dissecting microscope to ensure no hatch or damage before the start of the experiment.

Twenty-eight of these cups were randomly assigned to a low food level of 0. 1 g of dried,

chopped, live oak leaves (Q. virginiana), and the remaining 28 were assigned a high food level

of 0.5 g of dried, chopped, live oak leaves. All leaves were dried at 410C for 72 h before the










experiment. At each food level, 20 field collected, 4th instar Wyeomyia spp. were added to 14 of

the cups. The other fourteen cups contained only the 50 A. albopictus eggs and served as

controls. After 48 h the experiment was terminated, and the egg hatch rates were recorded for

each cup. The number of eggs hatched was determined by counting the number ofA. albopictus

larvae present and the number of eggs that hatched on the paper. Whichever hatch number was

higher, number of larvae or the number of eggs hatched on the paper, was considered the hatch

number for that cup. In almost all cases, the number of eggs hatched on the paper exceeded or

equaled the number of larvae found in the cup.

The bleaching technique of Trpis (1970) was used to determine the viability of the

unhatched eggs. Eggs were considered viable if they had a fully developed embryo. Empty eggs

and partial or shriveled embryos were considered inviable and were omitted in the data analysis.

On the other hand, if eggs were not wholly encased and appeared to be crushed or damaged,

these eggs were classified as damaged.

Due to the high number of inviable eggs, an ANOVA was conducted on their incidence to

ensure their uniform spread across treatments. To ensure homogeneity of variances, hatch rates

were arcsine square root transformed before an ANOVA was performed in SAS (2002) with a

subsequent Tukey's post-hoc test. A Kruskal-Wallis test and a Dunnet' s test was performed on

the damaged egg data. Two separate treatments had one replicate each that was omitted from the

analysis because they both had a 0.0% hatch rate with only 2 to 4 viable eggs per replicate.

Results

Overall, 29.0% + 2.4% (mean & SE) of all eggs were inviable, with no difference among

treatments (F3,50= 1.88, P= 0.14). There was a significant difference between the hatch rates of

the treatments (F3,50=6.28, P=0.02), which was attributed to the high food with Wyeomyia spp.

treatment being significantly different than both food treatments without Wyeomyia spp. (Fig. 4-










1). There was a lower hatch rate for the treatments with Wyeomyia spp. as compared to the

treatments without Wyeomyia spp., although this difference was only significant for the higher

food concentration (Fig. 4-1). On the other hand, there was a significant variation (X2=8.06, df=3,

P=0.04) among treatments in the incidence of damaged eggs (Fig. 4-2). Significantly more

damaged eggs were observed in the low food treatment with Wyeomyia spp. compared to both

the food treatments without Wyeomyia spp. (Fig. 4-2).

Discussion

In this experiment, there was a significantly lower hatch rate ofA. albopictus in the

presence of fourth instar Wyeomyia spp at higher food concentrations. These Eindings are not

overly surprising as previous studies by Edgerly et al. (1993) showed reduced hatch of A.

albopictus in the presence of high densities of larger instar Aedes larvae at 24 h. While the hatch

rate of A. albopictus in the lower resource treatment with Wyeomyia spp. was lower than the

lower resource control, this difference was not significant, indicating that resource level may

influence the egg hatch inhibition in A. albopictus.

Even though in this experiment egg hatch inhibition by Wyeomyia on A. albopictus was

found to influence the hatch rate at high food concentrations, the effects on hatch rate were only

moderate. In this experiment, treatments with Wyeomyia experienced only an approximately 9%

decrease in hatch rate at 48 h, and 80% to 90% of viable eggs still hatched in all treatments.

Therefore, while there may be egg hatch inhibition, if the hatch rates seen in this experiment are

indicative of natural field hatch rates, then the influence of egg hatch inhibition on A. albopictus

development is not pronounced and does not explain the decrease in development rate of A.

albopictus in the presence of Wyeomyia spp. seen in the surface area to volume ratio experiment,

and the larval experiment in Lounibos et al. (2003).









The reduced effects of egg hatch inhibition on A. albopictus are not surprising, as Edgerly

et al. (1993) found that at higher larval densities A. albopictus egg hatch was inhibited, but at

reduced rates as compared to other Aedes spp. mosquitoes tested. So, in general it is possible

that A. albopictus does not experience pronounced egg hatch inhibition. Also, this experiment

was allowed to run for 48 h, while the Edgerly et al. (1993) experiment was terminated at 24 h,

so it is possible that most pronounced egg hatch inhibition occurs in the first 24 h, which was

missed in a 48 h experiment.

Livdahl et al. (1984) and Livdahl and Edgerly (1987) found that Aedes triseriatus hatch

rates are reduced by large densities of late instar conspecifics. These authors hypothesized that

large 4th instar larvae inhibit hatch through their elevated grazing intensity, which reduces

oxygen-depleting microbes. The significantly larger amount of egg damage seen in the presence

of Wyeongia spp. in this experiment may be a result of browsing by the late instar Wyeongia

spp. on the eggs ofA. albopictus. Because the percentage of eggs damaged was highest in the

low resource group (but not significantly different from high resources) with Wyeongia spp., it

suggests that with fewer leaf resources to graze upon, Wyeomyia spp. may have increased their

grazing upon A. albopictus eggs. The maj ority of egg damage probably occurred after hatch or

did not effect hatch rate, because the high resource with Wyeongia spp. treatment had lower

hatch rate than the lower resource with Wyeongia treatment, but the lower resource treatment

with Wyeongia spp. had higher egg damage. Likewise, the lower resource with Wyeongia spp.

treatment did not have a significantly different hatch rate from either of the controls at each

resource level, but it did have significantly more egg damage. Thus, there seems to be no strong

relationship between hatch rate and egg damage.









In nature, egg hatch inhibition ofA. albopictus within bromeliads could differ from the

laboratory results. In the typical bromeliad habitat many other organisms could also be

influencing the bacterial content of the area around the eggs and, thus, the hatch rate of A.

albopictus eggs. Many other bacteria- and detritus-consuming macroinvertebrates are known to

inhabit bromeliads (Fish 1976, Frank 1983).

Additionally, the bromeliad itself can influence the chemical composition of its contents

based on its physiological needs (see bromeliad as a habitat section in Chapter 1 of this thesis).

For instance, Fallis and Snow (1983) determined that an increase in water nitrogen induced hatch

in Aedes punctor, and nitrogen is one of the main chemicals that is absorbed by bromeliads

(Benzing 2000), and thus may influence the hatch ofA. albopictus. Fallis and Snow (1983) also

found that the change in oxygen content, and not the concentration of oxygen was the hatching

trigger for A. punctor, which may also be regulated by the bromeliad. So, although Wyeomyia

spp. may inhibit the hatch ofA. albopictus eggs at high resource levels, it is possible that other

organisms or processes within the bromeliad can alter egg hatch inhibition in A. albopictus.

With more macroinvertebrates in the aquatic community, there could be more grazing

and an increase in localized oxygen levels near the eggs, and thus more inhibition. In contrast,

Edgerly and Marvier (1992) hypothesized that at a certain density, the number of organisms

surrounding eggs can be great enough to deplete the overall oxygen levels in the water, and thus

stimulate egg hatch. Although as mentioned before, Edgerly et al. (1993) found that at high

densities A. albopictus had a low level of hatch inhibition compared to congeners, so higher

densities of mosquitoes may have little effect on the overall hatch ofA. albopictus.

In this experiment the eggs of A. albopictus were submerged below the water line in a

cluster on the side of the container. In the location study from Chapter 2 of this thesis










Wyeomyia spp. larvae were located more often at the bottom and at the top of the container and

less on the sides. If A. albopictus does not oviposit its eggs on the inner axil walls of bromeliads,

then this experiment may misrepresent the influence of Wyeomyia spp. on egg hatch inhibition

of A. albopictus. There is no evidence that A. albopictus lays its eggs on the sides of the

bromeliads, nor is it probable that their eggs are laid in one cluster as in this experiment. Further

investigation into the location and distribution of oviposited eggs of A. albopictus within

bromeliads is needed to determine the relevance of this experiment to natural conditions.















r I


0.7 -

0.6 -
0 -



0.4 -

0.3 -


0.2 -


- -

- -




- -


- -

- -


-

-




-


-


-

-




-


-


High Food High Food Low Food Low Food

No Wvy Wy No WvyW



Figure 4-1. Proportion hatch of viable eggs + SE in each treatment. High Food refers to high

food treatments, and Low food refers to low food treatments. No Wy indicates

treatments with no Wyeomyia spp. and Wy indicates treatments with Wyeomyia spp..

Treatments with the same letter above the SE bar are not significantly different based

on a Tukey test.









0.35b

0.3








HihFodHg Fo owFo LwFo
NoN

Fgur 4-2.PootoegsdmgdSEprtetetHihForeesthghfd
tramns an o odrfrst o odteamns oW ndctstetet






Tiue42 rpretmonents wihthamaed letter trabove theS a areh noot sinfiatliferen basedhf


on a Dunnett' s Test.









CHAPTER 5
FIELD STUDIES ON Aedes albopictus AND Wyeomyia spp. INT EXOTIC BROMELIADS OF
SOUTHERN FLORIDA


Introduction

In a study conducted by O'Meara et al. (1995b), Wyeomyia spp. were the most abundant

mosquitoes in bromeliads throughout southern Florida, but in northern Florida where W.

vanduzeei and W. mitchelii are absent, A. albopictus was the most common mosquito species in

bromeliads. In the southern sites with Wyeomyia spp., A. albopictus was often found in great

numbers in nearby artificial containers. For example, when vases were placed near bromeliads

in Vero Beach, Florida, A. albopictus larvae were subsequently collected from all the vases but

from only 40 percent of the bromeliads. In the study of Lounibos et al. (2003), A. albopictus was

also found in bromeliads in greater numbers in the absence of Wyeomyia spp..

Wyeomyia spp. vary in larval abundance and ovipositional preference based on the species

of bromeliad. In collections by O'Meara et al. (1995b), Wyeomyia spp. were less abundant in

Neoregelia spectabilis than in Aechmea fasciata. Frank and O'Meara (1985) also found that W.

vanduzeei showed an oviposition preference for the native bromeliad Tillandsia~~ll1~~~~111~~~ utriculata over

another native bromeliad, Catopsis berteroniana.

The purpose of the current study was to further explore the differences in abundance and

distribution of Wyeomyia spp. and A. albopictus within bromeliads based on the location within

the plant axill or central tank), by bromeliad species, and by macrohabitat. Neoregelia

spectabilis and B. pyramnidalis are two exotic bromeliad species commonly featured in the

residential landscaping of many homes in the cities of Vero Beach and Fort Pierce, Florida.

These two plants were chosen for further study because they were common and distinctly

different in size, color, and shape, but are often grown in the same location. In the current study,










N. spectabilis and B. pyra~nidalis were sampled to compare the abundances of Wyeonzyia spp.

and A. albopictus in these two bromeliad species. Due to the differences in their physical

characteristics these bromeliads may have different abundances of mosquitoes. These samples

were further divided by location within the plant, i.e. lateral axil or central axil, to determine

whether the abundance of Wyeonzyia spp. or A. albopictus varied by location within the plant. It

is also hypothesized that the abundance of mosquitoes will not vary by location within the

bromeliad, because bromeliad studies by Frank and Curtis (1977) have indicated that during

rainfall eggs may be washed to other locations within the plant. Thus, if rainfall occurs

frequently enough there should be a rather homogeneous distribution of larvae throughout the

plant. To further describe habitat preferences of bromeliad-inhabiting mosquitoes, mosquitoes

were collected from the Hield to determine whether mosquito abundance varied between two

canopy types, mainly oak tree and palm tree. Frank and O'Meara (1985) determined that W.

naitchellii preferred shady habitats and, thus, different canopy types could also provide shade

variations that influence the abundance of bromeliad mosquitoes.

Materials and Methods

Differences in the Density of A. albopictus and Wyeomyia spp. within N. spectabilis and B.
pyramidalis

The aquatic contents of B. pyra~nidalis and N. spectabilis bromeliads were collected with a

meat baster monthly from September 2005 to July 2006 at eight sites in Vero Beach and Fort

Pierce, Florida, and from two sites in Orlando, Florida (Fig 5-1). On a few occasions plants in

Tampa Bay and Washington Oaks Park were also sampled (Fig 5-1). At each site, Hyve to

twenty-Hyve plants of each species were sampled every 4 to 8 weeks. To avoid taking repeated

samples from the same plants, at each sampling the approximate locations of the samples were

recorded, although repeated sampling may have occurred at two of the smaller Vero Beach, FL









sites due to a reduced number of plants. The sites where re-sampling of plants was suspected

were omitted from all analyses.

From each plant two samples were taken by removing all extractable fluid contents with

the baster. One sample consisted of the contents of the central axil of the plant, while a separate

sample was taken of the lateral axil contents. The central axil sample was extracted from the

area of water held in the center of the plant (photo A in Fig. 2.2), and the lateral axil sample was

extracted from the spaces created by the lateral axils of the plant (photo B in Fig. 2.2).

Due to the fact that B. pyramnidalis with inflorescences held very little, if any, water, only

B. pyramnidalis that were not flowering were sampled in this study. Conversely, since N.

spectabilis holds very little if any water in its central tank when it is not flowering, only

flowering N. spectabilis were sampled in this study. The species and development stage of each

mosquito immature collected were recorded. To aid in the identification of 1st instar larvae, all

samples were initially examined in trays under a dissecting scope.

Studies by Frank et al. (1977) have shown a correlation between bromeliad size and the

number of mosquito larvae and pupae. After February 2006, in the current study the amount of

water in each bromeliad was also measured to determine whether a correlation exists between the

amount of water in a bromeliad and the abundance of either species of mosquito and their total.

Thus, the analysis of the data from these collections is broken into two categories 1) all

collections, with analyses being based on total mosquitoes per plant, and 2) collections after

February 2006, which were analyzed by numbers of mosquitoes per volume of water. ANOVA

calculated from type III sums of squares was applied to detect significant variations in the

dependent variables densityy of mosquitoes per mL and per plant) in relation to the independent

variables (bromeliad species). Means comparisons were conducted with Tukey's tests. The









density means (mos/mL and mos/plant) were also compared by a paired t-test in relation to the

location within the plant (lateral vs. central axil). A repeated measures ANOVA, with

subsequent means comparisons by Tukey's tests, was applied to detect significant variations in

the dependent density variables by the month of collection. Correlations were performed using

Spearman' s p between the volume of water in bromeliads and the numbers of A. albopictus and

Wyeomyia spp. found within.

Canopy Effects on Density

A study was also conducted on the differences in density of Wyeomyia spp. and A.

albopictus in bromeliads based on the surrounding habitat. Preliminary observations during

collections showed that there may be fewer Wyeomyia spp. and A. albopictus in bromeliads with

palm trees overhead versus in bromeliads shaded by oak trees. These habitats differ in amount

of sun exposure, leaf litter, and throughfall input into the bromeliads below.

The largest sample site in Vero Beach, FL (VB-5) contained four clumps of interspersed N.

spectabilis and B. pyramnidalis under palm trees and seven clumps of interspersed N. spectabilis

and B. pyramnidalis under oak trees. In both habitats B. pyramnidalis was the more common of the

two bromeliad species. Once in April 2006 and once in June 2006 two samples, one from the

central axil, and one from the lateral axil, of fifteen B. pyramnidalis and nine N. spectabilis were

taken from under either an oak tree or a palm tree. In order to avoid sampling the same plants

twice, the location of the first collection was different from the location of the second collection.

The mosquitoes collected were counted and recorded as described in the previous section.

Significant effects of canopy type on the numbers of Wyeomyia spp. in bromeliads were tested

by nested ANOVA with bromeliad species (B. pyramnidalis vs. N. spectabilis) nested within

canopy type (Oak vs. Palm). Aedes albopictus was omitted from ANOVA analysis as no larvae

of this species were found in the oak location.









Results

Both mosquitoes per plant (mos/plant) and per unit volume (mos/mL) varied significantly

between plant species (Table 5-1 and Table 5-2). Aedes albopictus had a higher density

(mos/mL and mos/plant) in N. spectabilis, but Wyeomyia spp. was found in significantly higher

densities in B. pyramnidalis (Tables 5-3 and 5-4). Aedes albopictus had higher densities

(mos/plant) in the central axils of N. spectabilis (t217= 2.22, P=0.03), but showed no difference in

density between the central and lateral axils in B. pyramnidalis (t242= 0.86, P=0.39) (Table 5-4).

Aedes albopictus also showed no difference in density (mos/mL) between the central and lateral

axils in either N. spectabilis (tl72= -0.70, P= 0.49) or B. pyramnidalis (tl93= 1.00, P= 0.32).

Wyeomyia spp. showed no differences in densities (mos/plant) between the lateral and central

axils of B. pyramnidalis (t242= 1.38, P= 0. 17) or N. spectabilis (t217= 1.60, P= 0.11). However,

Wyeomyia spp. did have higher mean densities (mos/mL) in the lateral axils of both N.

spectabilis (tl72= 2.27, P= 0.02) and B. pyramnidalis (tl93= 2.52, P= 0.01) (Table 5-4).

The densities (mos/plant) of Wyeomyia spp. and A. albopictus varied by the month of

collection (W.- F6= 8.74, P<0.01 and A. albo.- F6= 12.36, P<0.01) and by the site of collection

(W.- Fs= 10. 18, P<0.01and A. albo.- Fs= 2. 15, P=0.03). The density of A. albopictus in B.

pyramnidalis was relatively constant throughout the year, but in N. spectabilis the density at each

site of A. albopictus increased in September and in April through July (Fig. 5-3). In B.

pyramnidalis the density of Wyeomyia spp. increased in September, decreased in November

through March, and increased in April and in July. Aedes albopictus also experienced similar

changes in density (mos/mL) over time, but Wyeomyia spp. density (mos/mL) did not differ

significantly among months (Tables 5-5, 5-6, Figs. 5-4, 5-5,).

There were also distinct differences in the water holding capacity between the plants

(Table 5-7). More water was extracted on average for N. spectabilis than B. pyramnidalis (Table










5-7). When the data were analyzed separately by the location within the plant there was

significantly more water in N. spectabilis than B. pyramnidalis in both lateral and central axils

(Table 5-7). There were positive correlations between the number of Wyeomyia spp. (p400= 0.12;

P=0.01), the number of A. albopictus (p400= 0.26; P<0.01), and the total mosquitoes (p400= 0.18

P<0.01) and the amount of water extracted from the bromeliad.

Wyeomyia spp. differed in densities between the palm and oak sites (Table 5-8), and by

plant species in the palm and oak sites (Table 5-8). Wyeomyia spp. were less abundant in the

palm sites, and in both the oak and palm sites Wyeomyia spp. was found in a significantly greater

densities in B. pyramnidalis plants (Table 5-9).

Discussion

In the current surveillance Wyeomyia spp. immatures were denser in B. pyramnidalis than in

N. spectabilis. O'Meara et al. (1995b) showed that Wyeomyia spp. were more abundant in

Aechmea. fasciata than in N. spectabilis. On the other hand, A. albopictus had much higher

mean densities in N. spectabilis than in B. pyramnidalis. The differences in abundances in each

plant could indicate that A. albopictus is avoiding contact with Wyeomyia spp. by ovipositing in

bromeliads not occupied by Wyeomyia spp., but in an experiment conducted by Lounibos et al.

(2003), A. albopictus showed no difference in ovipositional preference between B. pyramnidalis

with Wyeomyia spp. present or absent. The Lounibos et al. (2003) experiment indicates that

ovipositing A. albopictus are not responding to cues from Wyeomyia spp. larvae. The

Washington Oaks site had the greatest abundance of A. albopictus, and the lowest abundance of

Wyeomyia spp. The studies by O'Meara et al. (1995b) and Lounibos et al. (2003) indicate that

more northern sites like Washington Oaks have more A. albopictus in their bromeliads than in

the southern sites. These two studies also indicated that lower abundances of Wyeomyia spp.

occurred in northern Florida, which was likely due to the intolerance of Wyeomyia spp. to










temperatures below freezing, or to the rarity of native bromeliad phytotlemata in northern

Florida.

There were differences in the densities of Wyeonyia spp. and A. albopictus by location

within the plant. Wyeonzyia spp. had higher densities (mos/mL) in the lateral axils of B.

pyra~nidalis and N. spectabilis, so either Wyeonzyia spp. oviposits more frequently in lateral

axils, or if Wyeonzyia spp. oviposit more eggs into the central axil, then some eggs or larvae are

washed by rainfall into the lateral axils. Frank et al. (1976) observed that female W. vandueezi

oviposit more frequently into the central axils of 7: utriculata, but were still found in the lateral

axils of that bromeliad species. In a Frank and Curtis (1977) study, W. vandueezi were found to

be easily washed out of the central tank of the bromeliad during rainfall. Therefore, it is

unknown whether the lateral axils and central axils have the same number of mosquitoes due to

oviposition preference or due to egg and larval movement during rainfall.

The Frank et al. (1977) study determined that there was a correlation between the size of

the bromeliad and the number of mosquitoes within the bromeliad. The Frank et al. (1977)

experiment used the total water holding capacity of the bromeliad as the indicator of the size of

the bromeliad. In the current research, the actual amount of water within the bromeliad, not the

total holding capacity, was used to measure the densities of mosquitoes per milliliter of extracted

fluid. While the quantity of water actually present is probably not as good an indicator of size as

the total water holding capacity of the bromeliad, it gives an approximation of the habitat

available to the mosquito at the time of sampling.

There was positive correlation between the amount of water present in the bromeliad and

the number of mosquitoes extracted, although its strength varied between mosquito species.

Aedes albopictus had a stronger positive correlation with water volume than Wyeonzyia spp.,










which is possibly due to the fact that Wyeomyia spp. are bromeliad specialists, while A.

albopictus is a container generalist. Wyeomyia spp. are probably better adapted to the variable

bromeliad environment, and thus can thrive under a variety of bromeliad conditions, including

reduced water. For instance, Frank and Curtis (1977) hypothesized that the prolonged larval

development of W. vanduzeei under reduced food conditions was an adaptation to the variable

habitat in bromeliads. Aedes albopictus on the other hand, can also have prolonged larval

development with reduced resources (Barrera and Medialdea 1996), but probably cannot prolong

its life as long as Wyeomyia spp.. Thus, under variable conditions A. albopictus females have to

either be more selective for environments with more water for their oviposition, or risk a

reduction in survivorship due to desiccation or decreased space. Conversely, ovipositing

Wyeomyia spp. females do not have to be as selective for the amount of water in the bromeliad

habitat, as their larvae can tolerate alterations in water conditions.

In the habitat study Wyeomyia spp., but not A. albopictus, differed in mean densities under

different canopy types. It is possible that with more samples, A. albopictus would have also

differed in mean densities, because the total number ofA. albopictus recovered from all plants

was small. Since the palm trees offered less protection from the sun, the bromeliads under palms

were exposed to more insolation. In a study by Frank and O'Meara (1985), W. mitchellii showed

a preference for shaded habitats. In the current study, on two occasions the larvae collected from

most sites were identified to species. During both examinations about 80-85% of the Wyeomyia

spp. collected were W. mitchellii with the remaining being W. vanduzeei. Thus, the decrease in

density of Wyeomyia spp. in the palm habitat was most likely due to the preference of Wyeomyia

mitchellii for shaded areas.









Even though Wyeomyia spp. had the higher mean density in all plants, Wyeomyia spp. had

a higher mean density in B. pyramnidalis, while A. albopictus had a higher mean density in N.

spectabilis. In the following chapter, oviposition and larval competition experiments were

conducted to explain the causes of the differing relative abundances of Wyeomyia spp. and A.

albopictus in these two bromeliad species.









Table 5-1. Analysis of variance for the density of mosquitoes per plant based on month (month
of collection), site, and bromeliad species (N. spectabilis vs.B. pyramidalis).

df F-stati stic P-value

Month 6,472 0.27 0.53

Site 7,472 12.36 <0.01

Bromeliad species 1,472 7.92 <0.01

A repeated measures ANOVA was applied to the month variable.










Table 5-2. Analysis of variance for the density of mosquitoes per mL based on month (month of
collection), site, and bromeliad species (N. spectabilis vs. B. pyramidalis).
df F-stati stic P-value


Month


4,378

7,378

1,378


4.50


<0.01

<0.01

<0.01


Site


Bromeliad species


42.92


A repeated measures ANOVA was applied to the month variable.










Table 5-3. Analysis of variance for the density of each mosquito species per mL of water in each
bromeliad species.
Mosquito Bromeliad Mean & SE (mos/mL) F- Stati sti c df P-value

A. albopictus N. spectabilis 0.0310.11 7.16 1,399 <0.01

B. pyramidalis 0.000310.0003

Wyeomyia spp. N. spectabilis 0.0910.01 42.53 1,399 <0.01

B. pyramidalis 0.2610.02

These means are representative of the data taken after February 2006. All samples
including those with no mosquitoes were included in this analysis.











Table 5-4. Mean densities of mosquitoes (mos/plant) in B.pyramidalis and N. spectabilis based on location within the plant and plant
species.
Species Location albopictus Wy. spp. Total Total Wy spp. Total mosquito


abundance

(mos/plant)

Mean & SE


abundance

(mos/plant)

Mean & SE


A. albopictus

Abundance

(mos/plant)

Mean & SE

2.58 & 0.39



0.11 & 0.04


abundance

(mos/plant)

Mean & SE


abundance

(mos/plant)

Mean & SE


N. spectabilis

N. spectabilis

B. pyramnidalis

B. pyramnidalis


central axil

lateral axil

central axil

lateral axil


1.67 & 0.32

0.92 & 0.16

0.06 & 0.03

0.05 & 0.02


2.53 & 0.44

3.13 & 0.41

7.87 & 0.54

8.62 & 0.60


5.67 & 0.77



16.48 & 1.01


8.30 + 0.89



16.60 & 1.02


:. F1,472=42.53, P<0.01; 2: F1,472= 18.07, P<0.01; 3: F1,472= 18.51, P<0.01.










Table 5-5. Analysis of variance table for the effects of month and site on mos/mL and
mos/plant.
Mosquito Effect F -stati sti c df P-value

mos/mL A. albopictus Month 9.17 4,378 <0.01

mos/mL Wyeomyia spp. Month 2.07 4,378 0.07

mos/plant A. albopictus Month 12.36 6,472 <0.01

mos/plant Wyeomyia spp. Month 8.74 6,472 <0.01

mos/plant A. albopictus Site 2.15 8,472 0.03

mos/plant Wyeomyia spp. Site 10.18 8,472 <0.01










Table 5-6. Analysis of variance results for the effect of month and sites on mosquito abundances
(mos/plant) by bromeliad species.


Bromeliad


Mosquito


Effect

Month

Site

Month

Site

Month

Site


Month

Site

Month

Site

Month

Site


F-stati stic df P-value


B. pyramnidalis



N. spectabilis



B. pyramnidalis




N. spectabilis



B. pyramnidalis



N. spectabilis


Total Mosquitoes



Total Mosquitoes



Wyeomyia spp.




Wyeomyia spp.



A. albopictus



A. albopictus


25.58

7.53

7.14

13.47

24.13

7.30


3.86

16.86

6.37

0.64

12.99

2.02


6,252

7,252

6,225

8,225

6,252

7,252


6,225

8,225

6,252

7,252

6,225

8,225


<0.01

<0.01

<0.01

<0.01

<0.01

<0.01


<0.01

<0.01

<0.01

0.73

<0.01

0.05










Table 5-7. Average (+SE) amount of water extracted from each plant species.
N. spectabilis B. pyramnidalis N. spectabilis B. pyramnidalis

lateral axil lateral axil central axil central axil


Total water

N. spectabilis


109.12 & 5.80


Total water

B. pyramnidalis


79.09 & 2.90


Mean water 56.04 & 2.96 39.03 & 2.36 53.08 & 3.81

(mL) & SE

ANOVA results F1,198= 3.02; P<0.01- by plant species


33.28 & 1.45


F1,198= 47.59; P<0.01- by plant species










Table 5-8. A nested ANOVA table for effects of Oak vs. Palm canopy and bromeliad species (B.
pyramnidalis vs. N. spectabilis) on Wyeomyia spp. densities by mos/plant.


F df


P-value


Wyeomyia spp.


Oak vs. Palm


26.61 1,32

8.77 1,32

6.73 1,28


<0.01

<0.01

0.01


B. pyramnidalis vs. N. spectabilis

Oak vs. Palm

(B. pyramnidalis vs. N. spectabilis)










Table 5-9. Means and SE of densities ofA. albopictus and Wyeomyia spp. by location within
plants under oak and palm canopies.
Mean & SE

Wyeomyia spp. Oak B. pyramnidalis central axil 15.73 & 3.20

B. pyramnidalis lateral axil 7.80 & 1.16

N. spectabilis central axil 3.85 & 0.92

N. spectabilis lateral axil 4.08 & 0.73

Wyeomyia spp.Palm B. pyramnidalis central axil 1.00 + 0.50

B. pyramnidalis lateral axil 0.75 & 0.41

N. spectabilis central axil 0.57 & 0.43

N. spectabilis lateral axil 0. 14 & 0. 14

A. albopictus Oak B. pyramnidalis central axil 0

B. pyramnidalis lateral axil 0

N. spectabilis central axil 0

N. spectabilis lateral axil 0

A. albopictus Palm B. pyramnidalis central axil 0.13 & 0.13

B. pyramnidalis lateral axil 0.38 10.26

























































Figure 5-1. Map of collection sites. Figure adapted from O'Meara et al. 1995b, Fig. 1, Pg. 218.








86







































Photo A Photo B

Figure 5-2. Location of water samples. The white arrow indicates the location where water samples were taken from the central axil
(Photo A) and the lateral axils (Photo B) of a N. spectabilis.







87



















, 0 -\








10-




-* eo l
MothNe

-7 -Bi l

-6 Bi l

Fiue53 h otl endeste fWemi p.adA.abpcu nN pcaii
an .prmdlsa ahste ro asrpeetS f h en tec ie o
B.prmdlsn 4 lns(28 .5 lnssmldprmnha ahst
(ma S),adfrN pcaii =28pat 95 .0pat ape e
mot tec it ma E)






































I I I I


-* A. albopictus
-o Wyeomyia spp.


S35


30 -3






O

10-
O
a b
15-




b bd d
C


Mont


b bd


bdc


Figure 5-4: The monthly mean densities of Wyeone ia spp. and A. albopictus in N. spectabilis at
each site. Error bars represent SE of the means at each site. Means without a
common letter written above indicate significant differences for Wyeone ia spp..
Means without a common letter written below indicate significant differences for A.
albopictus.













co50






S30-

SQ I b b
S20-
v, b


0- 10 _c

S0


n a b b b b bb





-*- albopictus
Month
-o Wyeomyia spp.


Figure 5-5. The monthly mean densities of Wyeongia spp. and A. albopictus in B. pyramnidalis at
each site. Error bars represent SE of the means at each site. Means without a
common letter written above indicate significant differences for Wyeongia spp..
Means without a common letter written below indicate significant differences for A.
albopictus.









CHAPTER 6
OVIPOSITION AND LARVAL DEVELOPMENT OF Aedes albopictus IN TWO EXOTIC
SPECIES OF BROMELIAD IN SOUTHERN FLORIDA

Introduction

Oviposition behavior by the female determines the aquatic habitat of the mosquito' s larval

stages (Clements 1999). Thus, it is not surprising that most ecological theories on oviposition

selection are generally based on the assumption that adults choose oviposition sites that

maximize the survivorship and reproductive output of their offspring and minimize their

development time (Srivastava and Lawton 1998). At least for some herbivorous insects, there is

some indication that fitness considerations of ovipositing females are also important in choice of

oviposition sites (Mayhew 2001). Whatever the ecological significance of oviposition

preference, numerous experiments have explored the cues that insects use to select oviposition

sites.

Culicids select oviposition sites based on visual, olfactory and tactile cues (Bentley and

Day 1989). The most common visual ovipositional stimuli of studied culicids are color cues.

Members of the genus Toxorhynchites are often attracted to black colored containers (Hilburn et

al. 1983, Jones and Schreiber 1994, Collins and Blackwell 2000), as are many species of the

genus Aedes (Beckel 1955, Wilton 1968). Culex mosquitoes are most often found to be attracted

to black and red (Dhileepan 1997), although color preference cannot be generalized by genus.

Aedes triseriatus are attracted to darker colors in the blue spectrum (Williams 1962, McDaniel et

al. 1976), and A. aegypti responded most strongly to black artificial bromeliads (Frank 1985).

There are also many chemical ovipositional cues originating from the habitat and from

conspecifics, predators, parasites and other species. There are numerous studies which indicate

that mosquitoes orient to cues from the organic material within the habitat (reviewed by

Clements 1999). For instance, in a laboratory study conducted by Wilton (1968), A. triseriatus










oviposited significantly more eggs in water collected from a treehole than in distilled water of

similar color. Culex spp. have also been found to respond to habitat specific chemical cues

derived from bacteria or the organic matter of the habitat (Ikeshoji et al. 1967, Beehler et al.

1994). Species of other mosquito genera also orient to chemical oviposition cues derived from

the habitat (Ikeshoji and Mulla 1970b, Bentley et al. 1979, Millar et al. 1992, Beehler et al.

1994). Habitat humidity (Kennedy 1942), salinity (Navarro et al. 2003), or algal content (Bond et

al. 2005) also influenced the oviposition site selection of mosquitoes.

Mosquitoes often respond to the presence of conspecifies and other species when choosing

oviposition sites. Some of the first olfactory studies conducted on Culicidae involved the

discovery of chemical oviposition cues in the egg rafts Culex spp. (Hazard et al. 1967, Ikeshoji et

al. 1967, Osgood 1971, Bruno and Laurence 1979, Laurence and Pickett 1982). In the Culex egg

raft experiments, female mosquitoes were attracted to oviposition sites that contained conspecific

eggs. Other mosquito species have shown a preference for ovipositing in the presence of

conspecific larvae (Soman and Reuben 1970, Bentley et al. 1976, Allan and Kline 1998, Mokany

and Shine 2003), or larvae of other species (Bentley et al. 1976, Allan and Kline 1998).

Mosquitoes are hypothesized to be attracted to oviposit in habitats with conspecifics, or other

species, because the presence of other mosquitoes is a possible indication of a successful larval

habitat (Clements 1999).

Some culicids have ovipositional preferences related to the presence or absence of

predators. In a study conducted by Munga et al. (2006), Anopheles gamnbiae avoided ovipositing

in containers that had been preconditioned with the waste of either of two predators. Avoidance

of oviposition in the presence of predators has also been seen for Ochlerotatus australis










(Mokany and Shine 2003), Culiseta longiareolata (Blaustein et al. 2004), and Aedes

taeniorhynchus (Ritchie and Laidlawbell 1994).

Once eggs are laid, the conditions within the habitat influence larval development. Since

A. albopictus is a container-inhabiting mosquito, variations in natural and artificial container

types occupied by this species influence larval development and survivorship. In treehole

communities, resource quantity within the habitat can influence larval development and

survivorship (Fish and Carpenter 1982, Leonard and Juliano 1995, Walker et. al 1997) and also

influence the community structure (Srivastava and Lawton 1998). The permanence of the habitat

also influences the development of larvae (Blaustein and Chase 2007), with drying of the habitat

often decreasing larval survivorship.

In southern Florida, A. albopictus is common in artificial or natural containers (O'Meara et

al. 1995a) and is occasionally found inhabiting bromeliads (O'Meara et al. 1995b). Within these

bromeliad habitats A. albopictus often co-occurs with other species such as W. mitchellii and W.

vanduzeei, and occasionally with A. aegypti, Aedes baha~nensis, Culex quinquefasciatus and

ulex biscaynesis (Frank 1985, O'Meara et al. 1995b, O'Meara et al. 2003). Past studies on

oviposition by A. albopictus have demonstrated a preference for darker colors (Gubler 1971, Yap

1975), and a lack of response to the presence of the bromeliad specialists W. vanduzeei and W.

naitchellii (Lounibos et al. 2003). Wyeonzyia vanduzeei showed a preference for oviposition into

flowering Tillandsia~~ll1~~~~111~~~ utriculata (Frank and O'Meara 1985).

During the field survey discussed in Chapter 5 of this thesis, there was a significant

difference between the densities of A. albopictus larvae in different species of bromeliads. Of

the two species of bromeliads that were the focus of the field survey, N. spectabilis had

significantly higher densities ofA. albopictus than B. pyra~nidalis. In order to understand the










differences in mosquito densities in these two plant species, an oviposition experiment was

implemented to determine whether oviposition was a factor that contributed to the higher

densities ofA. albopictus in N. spectabilis, and a larval development and survivorship

experiment was conducted to see whether the growth and survivorship ofA. albopictus differed

between N. spectabilis and B. pyramnidalis.

Materials and Methods

Oviposition Experiment

To determine whether A. albopictus preferentially oviposits into N. spectabilis in

comparison to B. pyramnidalis, an experiment was conducted in which 15 gravid A. albopictus

females were added by mouth aspirator to each of ten cages containing both one N. spectabilis

and one B. pyramnidalis bromeliad.

In accordance with approved animal-care protocol (VB-17 proj ect of the University of

Florida), each female had been bloodfed on a chicken five days before the start of the experiment

and held without an oviposition site. Only mosquitoes that appeared fully engorged were

removed from the main colony cage, and then transferred to a cage kept at 260C in a climate-

controlled insectary, with access to a 20% sucrose-water solution for the five days prior to the

start of the experiment. The colony from which the females were selected originated from

collections from southern Florida.

The N. spectabilis plants in the experiment were collected from one residence in the city

of Gotha in central Florida, and the B. pyramnidalis plants were collected from multiple

residences in Vero Beach and Fort Pierce, Florida. The N. spectablis plants chosen for the

experiment all contained partially submerged, nidulate inflorescences in the central tank

(Benzing 2000) and, thus, held larger amounts of water than non-flowering individuals. Because

N. spectabilis with inflorescences were the plants sampled in the field survey conducted in










Chapter 5, using flowering individuals for experiments may help to explain some of the findings

in that chapter. On the other hand, the B. pyra~nidalis in this experiment did not have

inflorescences, as their formation often destroys the water holding capacity of this species of

bromeliad. Non-flowering B. pyra~nidalis were also the only B. pyra~nidalis sampled in the field

surveys of Chapter 5.

Each plant was thoroughly washed by first rinsing in a bucket of water and then by

spraying with a high powered hose. Due to the fact that it is very difficult to remove all of the

leaves and organisms from the bromeliads, all plants were allowed to dry in an air-conditioned

laboratory from 8 to 10 days to ensure the death of all organisms due to desiccation. The plants

were then rewashed with a hose to remove any remains resulting from the drying.

Each replicate was enclosed in a 1.0 m wide and 0.76 m high pyramidal cage located

within a outdoor screened enclosure constructed for studies of mosquito flight behavior

(Bidlingmayer 1977). Within each cage, specimens of each bromeliad species were paired,

albeit the N. spectabilis were always slightly bigger. 1.0 g of chopped, dried (48 h at 750C) Q.

virginiana oak leaves, and 150 mL of sieved bromeliad water collected from both B. pyra~nidalis

and N. spectabilis bromeliads from the Vero Beach and Fort Pierce, Florida areas were added to

the central tank of each bromeliad. The water of each species was pooled, so the water added to

each plant was a mixture from both bromeliad species. The water was sieved through a 130 Clm

mesh to remove macroinvertebrates and detritus. The number of water holding leaves was

counted as an estimate of plant water holding capacity.

The experiment was run for 7 days, after which the remaining water in each bromeliad

was removed by a pipette, and each leaf containing water was rinsed with tap water into a metal

rearing pan. Each pan was kept for 10 days at 260C in a climate-controlled rearing room and










covered with a sheet of glass to prevent possible oviposition from stray mosquitoes in the rearing

room. On each day, the pans were checked for newly hatched A. albopictus, and the sums of

hatched larvae in the two bromeliad species were regarded as products of recent oviposition and

compared by a paired t-test.

Larval Development and Survivorship Experiment

Fourteen plants, 7 B. pyra~nidalis and 7 N. spectabilis, from the previous experiment

were washed in the manner as described above, and 1.0 g of chopped oak leaves (Q. virginiana),

and 150 mL of sieved bromeliad water were added to the central tank of each bromeliad. Then

after 20 4th instar Wyeonzyia spp. and 20 1st instar A. albopictus had been added into the center

tank, the plants were placed into metal cages in a 260C climate controlled rearing room. After 3

days, 50 mL of water was added to the plants to compensate for evaporation. After 6 days, all of

the contents of the bromeliads were rinsed into a pan in the manner as the oviposition

experiment. Variations between plant species in the number and developmental stage of

surviving A. albopictus were analyzed by ANOVA in SAS (2002). The survivorship data was

arcsine square root transformed to meet the homogeneity of variances assumption of ANOVA.

Results

Oviposition Experiment

Aedes albopictus oviposited significantly more (t9= 2.95, P<0.01) eggs into N.

spectabilis (mean & SE=33.5016.38) than B. pyra~nidalis (mean & SE=15.80 & 5.29).

Neoregelia spectabilis had significantly more (t9= 3.33, P<0.01) (mean & SE= 9.60 + 0.70)

water-holding axils than B. pyra~nidalis (mean & SE= 7.1 & 0.46).

Larval Development and Survivorship Experiment

A. albopictus had a significantly higher (F1,12= 67.41, P<0.01) average instar number

within N. spectabilis (mean & SE=3.96 & 0.22) than in B. pyra~nidalis (mean &










SE=2.3110.08).The mean survivorships of A. albopictus in N. spectabilis (0.56 & 0.08 (SE) )and

B. pyra~nidalis (0.67 & 0.04 (SE)) were not significantly different (F1,12= 1.43, P=0.25).

Discussion

While there was significantly more oviposition by A. albopictus in N. spectabilis, this

experiment did not consider the cause of this ovipositional preference The difference could be

based on coloration differences between N. spectabilis and B. pyra~nidalis. N. spectablis is dark

green with dark purple tips as compared to the entirely light-green B. pyra~nidalis. In an

experiment conducted by Frank (1985), other species of mosquitoes occasionally found in

bromeliads, C. quinquefasciatus, A. aegypti, and Toxorhynchites rutilus, preferentially oviposited

in darkly colored artificial bromeliads, as opposed to lighter green artificial bromeliads. In this

same study, Wyeonzyia spp. preferred to oviposit in the lighter green artificial bromeliads.

Therefore, it seems possible that A. albopictus, like other container-generalist mosquito species,

has a preference for the darker colored bromeliad N. spectabilis.

Another visual cue besides color that may have affected the experiment was the size of the

plants. Generally in the field, and in this experiment as well, N. spectabilis is a horizontally

larger plant with much longer leaves. B. pyra~nidalis tends to be smaller overall with shorter

leaves and a more vertically dominant stature than horizontally dominant. The paired t-test

demonstrated that the N. spectablis also has more water holding axils per plant than the B.

pyra~nidalis plants in this experiment.

Bentley and Day (1989) stated that specialist mosquitoes, those which have more

restricted ovipositional sites, such as crab holes, bromeliads and natural or artificial containers,

tend to rely strongly on visual cues to aid in the identification of ovipositional sites. While this

statement may also favor the color difference hypothesis for the observed ovipositional

preference of A. albopictus for N. spectablis, the fact that there was a difference in the size of the










two plants could also be a factor. Neoregelia spectabilis, the larger of the two bromeliad species,

may be visually more attractive to A. albopictus, regardless of color. This larger size is not an

artifact of the experiment, because in nature N. spectabilis are commonly larger than B.

pyra~nidalis. Therefore, although the difference may be due to size, there are still important

ecological implications to this difference.

On the other hand, visual cues may be of little importance to the ovipositional decision of

A. albopictus in bromeliads. Aedes albopictus could be responding to habitat-based chemical

cues unique to each plant species. For instance, the submerged nidulate inflorescence ofN.

spectaibilis may provide additional nutrients or chemical compounds to the central tank water,

while B. pyra~nidalis does not receive this input, because it creates tall, stalky inflorescenses.

Since the water from both species was pooled, each species received water originating from both

B. pyra~nidalis and N. spectabilis. Therefore, it is unlikely that chemical compounds within the

water contributed to the oviposition preference ofA. albopictus for N. spectabilis in this

experiment. The experiment ran for 7 days, so only chemical cues that accumulated in this short

period of time could have affected the preference of A. albopictus for N. spectabilis.

The larval competition experiment within N. spectabilis and B. pyra~nidalis showed that

A. albopictus can develop at different rates based on the species of bromeliad. The multitude of

structural differences in size and shape between the two plant species could contribute to the

developmental differences of A. albopictus within the two plants. In Chapter 2 of this thesis, and

in treehole microcosms study by Broadie and Bradshaw 1991, surface area to volume ratio was

not a factor influencing the development rate ofAedes mosquitoes. So, it is possible that other

structural differences account for the increased development rate ofA. albopictus in N.

spectabilis.









Neoregelia spectabilis has a sunken inflorescence which was decaying during this

experiment, while B. pyramnidalis did not have this decaying inflorescence. Therefore, it is

possible that this sunken inflorescence provided additional nutrients that increased the food

resources available to A. albopictus. Further experiments are necessary to elucidate the causes of

the developmental differences ofA. albopictus seen in these two bromeliad species.

This experiment shows the importance of bromeliad species in the distribution of A.

albopictus. So far in North America, A. albopictus has been found to be infected with LaCrosse

(Gerhardt et al. 2001), Eastern Equine Encephalitis (EEE) (Mitchell et al. 1992), and dengue

viruses (Ibafiez Bernal et al. 1997), and transmits Dengue (DEN) in other areas of the world

(Hawley 1988). As this mosquito species is a health concern, then the understanding of its

ovipositional preferences within bromeliads can be beneficial to determining the risk of

transmission of these viruses to human populations. In Brazil, bromeliad eradication projects

have been implemented to eliminate these supposed development sites of dengue vectors

(Benzing 2000). With the higher populations of A. albopictus in northern Florida where

Wyeomyia spp. is absent (O'Meara et al. 1995b), further studies on the ovipositional preferences

of A. albopictus in bromeliad species could help reduce risk by applying control measures only

to bromeliad species that are of highest concern.









CHAPTER 7
CONCLUSIONS

In aquatic container habitats, high densities of mosquito larvae can increase development

time and reduce survivorship and pupal weight (e.g. Frank and Curtis 1977, Mogi 1984, Livdahl

1982, Broadie and Bradshaw 1991). In the surface area to volume ratio experiment of Chapter

2, and a larval competition experiment conducted by Lounibos et al. (2003), A. albopictus

suffered reduced growth and survivorship in the presence of larger 4th instar Wyeomyia spp..

Resource competition is common among mosquito larvae, and interference competition in

the forms of chemical interference (Dye 1984, Sunhara and Mogi 2002, Bedhomme et al. 2005),

encounter competition (Dye 1984, Roberts 1998, Broadie and Bradshaw 1991), and facultative

or intraguild predation (e.g. Reisen and Emory 1976, Koenekoop and Livdahl 1986, Koenraadt et

al. 2003) also influence larval mosquito communities. The results of this study suggest that

encounter competition, not chemical interference or predation, causes the reduced growth and

survivorship of early instar A. albopictus in the presence of 4th instar Wyeomyia spp. in

bromeliads. Increasing the habitat complexity in an artificial bromeliad increased the

developmental rate ofA. albopictus in the presence of Wyeomyia spp.. However, because the

effect of habitat complexity on the development time ofA. albopictus in the absence of

Wyeomyia spp. was not explored, the relationship of habitat complexity to A. albopictus

development may be unrelated to interactions with Wyeomyia spp.

Even though large numbers of mosquitoes were used in the chemical interference

experiments, excretory products of Wyeomyia spp. did not affect the growth or survivorship of

A. albopictus in bromeliads. Neither the pH nor ammonia concentration of bromeliad water

differed before and after the experiment. Because bromeliads assimilate ammonia from their