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The Effects of Various Carbohydrate Sources on Longevity and Nutritional Reserves of Culex quinquefasciatus Say, Culex n...

Permanent Link: http://ufdc.ufl.edu/UFE0024830/00001

Material Information

Title: The Effects of Various Carbohydrate Sources on Longevity and Nutritional Reserves of Culex quinquefasciatus Say, Culex nigripalpus Theobald and Culex salinarius Coquillett
Physical Description: 1 online resource (93 p.)
Language: english
Creator: Vrzal, Erin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: culex, longevity, mosquitoes, sugar
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Culex species are important vectors of diseases such as West Nile virus, Eastern Equine Encephalitis virus, St. Louis Encepahitis virus and lymphatic filariasis. While these mosquitoes feed on a wide range of nectar sources consisting of varying concentrations and amounts of carbohydrates and amino acids, little is known about the utilization of these different carbohydrates, their accompanying amino acids and their effect on longevity and deposition of nutritional stores in different species of mosquitoes. In this thesis, male and female Culex nigripalpus, Culex quinquefasciatus and Culex salinarius were fed a variety of single sugars, including monosaccharides (glucose, fructose, sorbose and mannose), disaccharides (sucrose and trehalose) and trisaccharides (melezitose and raffinose), as well as a water control and a Lantana camara nectar mimic with and without amino acids, all of which have been reported to support varying levels of survival. The effects of feeding these single carbohydrates as 5% (w/v) solutions or nectar mixtures (with or without amino acids) and water controls ad libitum had on 50% survival of each species was determined. Nutritional deposition in Culex quinquefasciatus males and females was also examined by feeding 5% (w/v) solutions of sorbose, mannose, melezitose and sucrose only. Overall, females lived longest on nectar sugars sucrose, glucose and fructose, but survival on melezitose, a honeydew sugar allowed accumulation of large amounts of lipid and glycogen and enhanced survival in Culex quinquefasciatus as well as the nectar sugars. Male survival was enhanced by raffinose, another honeydew sugar, and they also displayed increased survival when fed common nectar sugars with nutritional reserves of Cx. quinquefasciatus increasing as a result of feeding on nectar and honeydew sugars sucrose and melezitose. The addition of amino acids into the adult diet of female Cx. quinquefasciatus increased survival, but only when fed a low nutritient food diet as larvae. These results stress the importance of considering larval nutritional conditions, and recognizing their potential to result in decreased nutritional reserves upon emergence, decreased adult survival and dependence on a well-nutritioned adult diet to overcome larval deficits if larval conditions are poor.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Erin Vrzal.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Allan, Sandra A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024830:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024830/00001

Material Information

Title: The Effects of Various Carbohydrate Sources on Longevity and Nutritional Reserves of Culex quinquefasciatus Say, Culex nigripalpus Theobald and Culex salinarius Coquillett
Physical Description: 1 online resource (93 p.)
Language: english
Creator: Vrzal, Erin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: culex, longevity, mosquitoes, sugar
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Culex species are important vectors of diseases such as West Nile virus, Eastern Equine Encephalitis virus, St. Louis Encepahitis virus and lymphatic filariasis. While these mosquitoes feed on a wide range of nectar sources consisting of varying concentrations and amounts of carbohydrates and amino acids, little is known about the utilization of these different carbohydrates, their accompanying amino acids and their effect on longevity and deposition of nutritional stores in different species of mosquitoes. In this thesis, male and female Culex nigripalpus, Culex quinquefasciatus and Culex salinarius were fed a variety of single sugars, including monosaccharides (glucose, fructose, sorbose and mannose), disaccharides (sucrose and trehalose) and trisaccharides (melezitose and raffinose), as well as a water control and a Lantana camara nectar mimic with and without amino acids, all of which have been reported to support varying levels of survival. The effects of feeding these single carbohydrates as 5% (w/v) solutions or nectar mixtures (with or without amino acids) and water controls ad libitum had on 50% survival of each species was determined. Nutritional deposition in Culex quinquefasciatus males and females was also examined by feeding 5% (w/v) solutions of sorbose, mannose, melezitose and sucrose only. Overall, females lived longest on nectar sugars sucrose, glucose and fructose, but survival on melezitose, a honeydew sugar allowed accumulation of large amounts of lipid and glycogen and enhanced survival in Culex quinquefasciatus as well as the nectar sugars. Male survival was enhanced by raffinose, another honeydew sugar, and they also displayed increased survival when fed common nectar sugars with nutritional reserves of Cx. quinquefasciatus increasing as a result of feeding on nectar and honeydew sugars sucrose and melezitose. The addition of amino acids into the adult diet of female Cx. quinquefasciatus increased survival, but only when fed a low nutritient food diet as larvae. These results stress the importance of considering larval nutritional conditions, and recognizing their potential to result in decreased nutritional reserves upon emergence, decreased adult survival and dependence on a well-nutritioned adult diet to overcome larval deficits if larval conditions are poor.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Erin Vrzal.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Allan, Sandra A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024830:00001


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THE EFFE CTS OF VARIOUS CARBOHYD RATE SOURCES ON LONGEVITY AND NUTRITIONAL RESERVES OF Culex quinquefasciatus SAY, Culex nigripalpus THEOBALD AND Culex salinarius COQUILLETT By ERIN MICHELLE VRZAL 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 2009 1

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2009 Erin Michelle Vrzal 2

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To my m om and dad, Jocelyn Paul and Jeffrey Vrzal. I would also like to dedicate this to all of my friends and family who have provided love and support throughout my life and my educational career. Without you, this would not have been possible. 3

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ACKNOWLEDGMENT S I would like to thank my major advisor, Dr Sandra Allan for allowing me the opportunity to pursue my degree, while also working as her technician. I greatly appr eciate all of her help and guidance throughout my research and writing of this thesis. I would also like to thank my other committee members Drs. Dan Hahn and Dan Kline for their support and comments on my research and this thesis. Additionally, I am very grateful to Joy Diesel, Leslie Rios and especially Frank Wessels for all of their support and excellent advice. All of my friends in the Entomology and Nematology Department, including all of the ENSO participants, thank you for your support, advice and allowing me to le arn about bugs and life from all of you. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 1 INTRODUCTION................................................................................................................. .11 Mosquitoes ..............................................................................................................................11 Taxonomy ........................................................................................................................11 Biology and Distribution .................................................................................................11 Vectoring Capabilities .....................................................................................................12 Longevity ................................................................................................................................13 Sugar Feeding ..................................................................................................................13 Nutritional Reserves and Body Size ................................................................................15 Mating Status ...................................................................................................................15 Amino Acids in Nectar Sources ......................................................................................16 2 EFFECT OF CARBOHYDRATE SOURCE ON NUTRITIONAL RESERVES OF Culex quinquefasciatus ...........................................................................................................18 Introduction .............................................................................................................................18 Materials and Methods ...........................................................................................................20 Larval Rearing .................................................................................................................20 Mosquito Dry Weights ....................................................................................................20 Nutritional Cage Experiment ...........................................................................................21 Glycogen and Lipid Analyses .........................................................................................21 Statistical Analyses ..........................................................................................................22 Results .....................................................................................................................................23 Comparisons of Initial Dry Wei ght, Glycogen and Lipid Content .................................23 Glycogen Content ............................................................................................................23 Lipid Content ...................................................................................................................24 Discussion ...............................................................................................................................25 3 EFFECTS OF VARIOUS CARBOHYDRA TE SOURCES ON THE L ONGEVITY OF Culex nigripalpus Culex quinquefasciatus AND Culex salinarius .......................................33 Introduction .............................................................................................................................33 Materials and Methods ...........................................................................................................35 Larval Rearing .................................................................................................................35 Longevity Assays ............................................................................................................35 Statistical Analyses ..........................................................................................................36 5

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Results .....................................................................................................................................37 Male Survivorship Curves ...............................................................................................37 Female Survivorship Curves ...........................................................................................38 Comparison of Male Da ys to 50% Mortality ..................................................................39 Comparison of Female Days to 50% Mortality ...............................................................39 Discussion ...............................................................................................................................41 4 EFFECT OF NUTRITIONAL STATUS AND PR ESENCE OF AMINO ACIDS TO A SUGAR MIXTURE ON LONGEVITY OF C ulex quinquefasciatus ....................................54 Introduction .............................................................................................................................54 Materials and Methods ...........................................................................................................57 Mosquito Rearing ............................................................................................................57 Winglength and Dry Weight Measurements ...................................................................57 Glycogen and Lipid Analyses .........................................................................................58 Mating Assay ...................................................................................................................59 Amino Acid Longevity Assay .........................................................................................59 Statistical Analyses ..........................................................................................................60 Results .....................................................................................................................................61 Effect of Larval Nutrition on Adult Size, Nutrient Reserves and Mating .......................61 Survival Analyses ............................................................................................................62 Culex quinquefasciatus fe d treatments without sugar ..............................................62 Culex quinquefasciatus fed tre atments with sugar ...................................................62 Discussion ...............................................................................................................................63 5 CONCLUSIONS AND FUTURE RESEARCH....................................................................75 Longevity and Sugar Feeding .................................................................................................76 Nutritional Reserves and Mating Status .................................................................................77 Amino Acids in Nectar Sources .............................................................................................78 Future Directions ....................................................................................................................79 APPENDIX PROTOCOL FOR SULPHOSPHOVANILLIN AND HOT ANTHRONE ASSAYS......................................................................................................................... ........80 Mosquito Dry Weights ...........................................................................................................80 Preparation for Glycogen and Lipid Analyses ........................................................................80 Glycogen Analysis (Hot Anthrone Assay) .............................................................................81 Lipid Analysis (Sulphosphovanillin Assay) ...........................................................................82 Analysis Preparation ........................................................................................................83 LIST OF REFERENCES ...............................................................................................................84 BIOGRAPHICAL SKETCH .........................................................................................................93 6

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LIST OF TABLES Table page 2-1 Initial (day 1) glycogen, lipid and dry weights (LS Means SE) of unfed individual mal e Cx. quinquefasciatus ................................................................................................29 2-2 Initial (day 1) glycogen, lipid and dry weights (LS Means SE) of unfed individual femal e Cx. quinquefasciatus .............................................................................................29 2-3 Results of ANOVA on glycoge n levels displaying the effects of sugar diet, age and their interaction o n male or female Culex quinquefasciatus .....................................................30 2-4 Results of ANOVA on lipid levels displaying the effects of sugar diet, age and their interaction o n male or female Culex quinquefasciatus .....................................................30 3-1 Results of ANOVA on days to 50% morta lity of ma les fed water and sorbose and males fed the remaining treatments that supported survival. .......................................................47 3-2 Results of ANOVA on days to 50% mort ality of fem ales fed water and sorbose and males fed the remaining treatments that supported survival. .............................................47 4-1 Schedule of feeding for Cx. quinquefasciatus under a high or low food regime Larval food = 3% bovine liver powder (LP): 2% Brewers yeast (BY) (30 g bovine liver powder and 20 g Brewers yeast in 1L of water), 2% hogchow (36 g finely ground hog chow in 1800 ml of water). .........................................................................................67 4-2 Mean winglength (N=5) and dry we ight (N=10) measurem ents for adult Cx. quinquefasciatus males and females reared on high or low food diets as larvae. .............67 4-3 Mean glycogen and lipid cont ent (N=5) m easurements for adult Cx. quinquefasciatus males and females reared on high or low food diets as larvae. ..........................................67 4-4 Percent mated female Cx. quinquefasciatus when reared on high or low food diets as larvae and m aintained under conditions of survival assay. ................................................67 4-5 Days to 50% mortality (LS Means SE) of male and female Cx. quinquefasciatus fed high or low food diets as larvae. Adult di ets were no sugar treatme nts (water only, water + amino acids) and sugar treatments ( Lantana camara nectar mimic and L. camara mimic + amino acids). ..........................................................................................68 4-6 Results of ANOVA on the effect of larv al diet, adult diet a nd their interaction on survival of ma le or female Cx. quinquefasciatus ..............................................................69 7

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LIST OF FIGURE S Figure page 2-1 The effect of age and the interaction between sugar diet and age on glycogen content (g/mg ) of male and female Cx. quinquefasciatus .........................................................31 2-2 The effect of age and the interaction between sugar diet and age on lipid content (g/mg ) ...........................................................32 of male and female Cx. quinquefasciatus 3-1 Proportion survivorship of Culex ma les maintained on 5% solutions of different sugars.. ...48 3-2 Proportion survivorship of Culex fem ales maintained on 5% solutions of different sugars. ................................................................................................................................49 3-3 Effect of species and sugar on days to 50% mortality of ma le Cx. nigripalpus, Cx. quinquefasciatus and Cx. salinarius .................................................................................50 3-4 Comparison of days to 50% mortality of Cx. nigripalpus (CN), Cx. quinquefasciatus (CQ) and Cx. salinarius (CS) ma les maintained on 5% solutions of similar sugars. ........51 3-5 Effect of species and sugar on days to 50% mortality of fem ale Cx. nigripalpus, Cx. quinquefasciatus and Cx. salinarius .................................................................................52 3-6 Comparison of days to 50% mortality of Cx. nigripalpus (CN), Cx. quinquefasciatus (CQ) and Cx. salinarius (CS) fem ales maintained on 5% solutions of similar sugars. .....53 4-1 Photograph of mosquito wing with arrow in di cating measurements taken from the alular notch (A1) to the distal end of wing vein R and used to calculate mean wing 2lengths. ...............................................................................................................................70 4-2 Me ma le and female Cx. quinquefasciatus ...............................................................................71 4-3 Da diets with or without sugar as adults. .................................................................................72 4-4 Th quinquefasciatus when fed treatments without sugar. .......................................................73 4-5 Th quinquefasciatus when fed treatments with sugar. ............................................................74 an winglength (mm), dry weight (mg) and glycogen and lipid weights (g/mg) of ys to 50% mortality of males and females fed low or high food diets as larvae and e effect of larval diet and adult diet on days to 50% morta lity of male and female Cx. e effect of larval diet and adult diet on days to 50% morta lity of male and female Cx. 8

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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 THE EFFECTS OF VARIOUS CARBOHYD RATE SOURCES ON LONGEVITY AND NUTRITIONAL RESERVES OF Culex quinquefasciatus SAY, Culex nigripalpus THEOBALD AND Culex salinarius COQUILLETT By Erin Michelle Vrzal August 2009 Chair: Sandra Allan Major: Entomology and Nematology Culex species are important vectors of West Nile virus, Eastern Equine Encephalitis virus, St. Louis Encephalitis virus and lymphatic filari asis. While these mosquitoes feed on a wide range of nectar sources consisting of varyi ng concentrations and am ounts of carbohydrates and amino acids, little is known a bout the utilization of these different carbohydrates, their accompanying amino acids and their effect on longe vity and deposition of nutritional stores in different species of mosquitoes. In this thesis, male and female Culex nigripalpus Culex quinquefasciatus and Culex salinarius were fed a variety of single sugars, including monosaccharides (glucose, fructose, sorbose and ma nnose), disaccharides (sucrose and trehalose) and trisaccharides (melezitose and raffinos e), as well as a water control and a Lantana camara nectar mimic with and without amino acids, all of which have been report ed to support varying levels of survival. The effects of feeding thes e single carbohydrates as 5% (w/v) solutions or nectar mixtures (with or without amino acids) and water controls ad libitum had on 50% survival of each species was determined. Nutritional deposition in Culex quinquefasciatus males and females was also examined by feeding 5% (w/v) solutions of sorbose, mannose, melezitose and sucrose only. Overall, females lived longest on ne ctar sugars sucrose, glucose and fructose, but 9

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10 survival on melezitose, a honeydew sugar allowe d accumulation of large amounts of lipid and glycogen and enhanced survival in Culex quinquefasciatus as well as the nectar sugars. Male survival was enhanced by raffinose, another honeydew sugar, and they also displayed increased survival when fed common nectar suga rs with nutritional reserves of Cx. quinquefasciatus increasing as a result of feeding on nectar and honeydew sugars sucrose and melezitose. The addition of amino acids into the adult diet of female Cx. quinquefasciatus increased survival, but only when fed a low nutrient food diet as larvae These results undersco re the importance of considering larval nutritional conditions, and rec ognizing their potential to result in decreased nutritional reserves upon emergence, decrease d adult survival and dependence on a wellnutritioned adult diet to overcome larval deficits if larval conditions are poor.

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CHAPTER 1 INTRODUCTION Mosquitoes Taxonomy The genus Culex Linnaeus is in the family Culicidae with 29 species in North America north of Mexico and 15 species occurring in Florida (Darsie a nd Ward 2005). There are three subgenera, Culex, Melanoconion and Neoculex This thesis uses mosquitoes that are included in the subgenus Culex, that are found in the subtropical and tropical regions of the world. The species used in this thesis are Culex nigripalpus Theobald, Culex quinquefasciatus Say and Culex salinarius Coquillett. The three Culex species mentioned are the most common in Florida and have been previously grouped together as simply Culex spp. (Provost 1969). There have since been clear distinctions made between these species; includi ng variable habitats, be haviors and identifiable characteristics. Culex quinquefasciatus is a part of the Culex pipiens complex and was formerly known as Culex fatigans (Foster and Walker 2002). It lives in the southern range of the complex and readily hybridizes with Cx. pipiens (which has a northern range) in the mid-range of the U.S. where both species occur simultaneously. Biology and Distribution Culex nigripalpus display crepuscular behavi ors, feeding early in the morning and at dusk, as do many Culex spp., and are known to feed primarily on avian hosts duri ng the dry winter months and very opportunistically on everything fr om birds in dry winter months to horses, cows and armadillos to humans during wet su mmer months in Florida (Day 1997). Culex quinquefasciatus are also crepuscular mosquitoes and feed primarily on avian hosts in the U.S., but will feed on humans (Zinser et al. 2004). However, they are thought to be mostly 11

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anthropophilic in Africa and Indi a, thriving in urban areas wher e breeding conditions are optimal and hum an populations are dense (Chandler et al. 1975, Subra 1970, 1981). Similarly, Culex salinarius are also active at dawn and dusk and are very opportunistic when it comes to host feeding (Edman 1974, Molaei et al. 2004). All of thes e species lay their eggs in the form of rafts on the top of water, but Culex quinquefasciatus prefer polluted oviposition sites, whereas Cx. nigripalpus and Cx. salinarius prefer to lay their eggs in fresher water such as roadside pools. Additionally, Culex salinarius may sometimes prefer brackish water (Murphey 1961). Culex nigripalpus are found in Central and South Ameri ca, Mexico the Caribbean Islands and the southeastern United States from Texas to Kentucky and down into Florida, where they are most abundant in the southern part of the state (Provost 1969, Day 1997). Culex quinquefasciatus are widely distributed in the tropical and subtropical areas of the world, including Africa, Asia and in southern Japan a nd the United States (Subra 1981). In the U.S., it is restricted to the southe rn part of the country. Culex salinarius is most abundant along the Atlantic and Gulf Coasts of the United States where it is a severe nuisance species. Vectoring Capabilities Culex nigripalpus is the primary vector of St. L ouis encephalitis (SLE) in Florida (Chamberlain et al. 1964, Dow et al. 1964, Day 1997) and Eastern Equine encephalitis (EEE) has been frequently isolated in this mosquito (Wellings et al. 1972). Culex quinquefasciatus are the main vectors of Wuchereria bancrofti the cause of bancroftian f ilariasis (Subra 1981) in the neotropics and Asia (Hawking 1973), India (Sam uel et al. 2004), Aust ralia (Chow 1973), East Africa (Nelson et al. 1962) a nd reportedly in rural West Africa (Dossou-yovo 1995). Culex quinquefasciatus is also a known vector of SLE (Jones et al. 2002, Foster and Walker 2002) and West Nile virus (WNV) (Sardelis et al. 2001) in the southeastern United States. Culex salinarius is a potential vector of EEE (Scott and Weaver 1989) and has b een implicated an important 12

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bridge vector of WN V to humans in the North eastern U.S. due to its opportunistic feeding behaviors (Molaei et al. 2004, Zyzak et al. 2002). Longevity Survival in insects is often studied due to its connection with the ability of those insects to transmit disease. This is esp ecially true in mosquitoes wher e early studies of longevity in relation to malaria vectors showed that longer lived Anopheline mosquitoes tended to be vectors of the disease in particular ar eas where other, shorter lived Anophelines were not (Treillard 1938). In a field setting longevity is influenced by many factors, in cluding larval and adult diets, mating status, temperature, humidity, and predati on (Smith 1975). It has thus far been difficult to study survival of mosquitoes in field settin gs, though Reisen et al. (1991), Muir and Kay (1998) and others, have attempted mark-release-re capture studies, they are only successful at looking at daily survival times. Many studies have focused on longevity in the laboratory as a means for understanding the relationship between survival and vectoring capability of various species (Galun and Fraenkel 1957, Nayar and Sauerman 1971a, 1975, Eischen and Foster 1983, Briegel et al. 2001). Despite the many factors involved in the surviv al of mosquitoes in the field, the only way to answer many of these questions about vectoring capab ilities are to conduct laboratory studies mimicking as ma ny of the factors as possible. Culex mosquitoes are particularly long-lived mosquitoes which allo w them greater opportunities to contact multiple hosts, including humans as well as the ability to vector diseases which require a longer life-span. Sugar Feeding Many have recognized sugar feeding as an activity in which both males and females partake and a critical factor to increase survival and dispersal to the point of vectoring capability (Day 1997, Foster 1995, Bidlingmayer and Hem 1973, Nayar and Sauerman 1971a, 1971b and 1975). Feeding on sugar leads to the accumulatio n of lipid and glycogen, which are used for 13

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flight energy, survival and fecundity far beyond what would be possible from teneral reserves alone (Van Handel 1965b). Nectars are considered the prim ary sour ce for sugar feeding (Foster 1995), although alternative sources such as honeyde w, extrafloral nectaries, rotti ng fruit and even plant tissues are suitable (Joseph 1970, Grimstad and DeFolia rt 1974, Muller and Schlein 2005, Foster 1995). Generally, there have been some observations made of mosquitoes feedi ng on particular nectar and honeydew sources (Haeger 1955, Grimstad and DeFoliart 1974, Nayar 1982) and some identification of particular sugar in the crops of mosquitoes (Burkett et al. 1998, Shaefer and Muira 1972), but field observation is difficult, especially consid ering that feeding habits are crepuscular in most Culex mosquitoes (Magnarelli 1979). The primary sugars in nectar are sucrose, glucose and fructose, which are availabl e in various combinations and concentrations depending on the source (Baker and Baker 1983a, 1983b, 1975). Traces of mannose are also found in nectar, but are not a primary sugar (Wackers 2001). Honeydew contains many sugars known to sustain insect life including common nect ar sugars sucrose, glucose and fructose as well as sugars more specific to honeydew includi ng melezitose, raffinose a nd trehalose (Volkl et al. 1999, Baker and Baker 1983b). Studies have shown that some mosquitoes wi ll not even begin host seeking until they have obtained their first sugar meal (Foster 1995), al though some species of sugar fed mosquitoes seem to be less avid host-seekers (Foster and Eischen 1984). Despite sugar feeding deterring host seeking, Walker and Edman (1985) found that sugar fed mosquitoes are more persistent when they are host seeking, which combined wi th a long-life may allow them more successful blood meals. These findings raise important que stions about the effect of sugar feeding on behaviors such as host seeking and biting persistence, which ar e directly related to vector 14

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competence. These high ly specialized behaviors play an important role in mosquitoes as dangerous disease vectors and these factors are crit ical to why laboratory st udies of longevity in many insects are so important and well studied. Nutritional Reserves and Body Size Nutritional reserves have been directly correlated with larval diet and body size upon emergence in mosquitoes (Tela ng and Wells 2004). Glycogen is stored in the thoracic muscles and fat body and used for immediate energy when sugar is not available in the crop (Clements 1955, Nayar and Sauerman 1971a, 1971b), whereas lipids are used for long-term energy and survival (Foster 1995, Nayar 1982). Nayar a nd Pierce (1977) inferred that emergence triglycerides were used as a source for survival by connecting 50% surviv al times to rate of decline of lipids and that a poor larval diet resulted in low trig lyceride levels and decreased 50% survival time. Briegel et al. (2001) also were interested in the effects of body size and teneral reserves on longevity and recogni zed that larger mosquitoes c ontained more teneral lipid and glycogen and survived longer on a wa ter diet than smaller mosquito es. It was also determined that 50% survival times were higher for larger mosquitoes when fed a similar sugar diet as smaller mosquitoes. Additionally, small mosqu itoes, which are common in nature due to frequent poor larval nutr itional conditions, emerge with not onl y minimal nutritional reserves but also underdeveloped follicles, which without sugar feeding cannot accumulate yolk after bloodfeeding (Feinsod and Spielman 1980). Mating Status Mating is suspected to have an effect on longe vity of both male and female mosquitoes. For males and females, the ingestion of sugar is in tegral in their increased survival and ability to mate (Gary and Foster 2004). Without sugar their lifespans are not long enough to allow time for mating. Feeding on nectar or other carbohydrat e sources is a sufficient resource to extend 15

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life to the po int of mating capability. Liles a nd DeLong (1960) observed that when maintained together, males lived less time and females lived mo re than if reared separately. They concluded that mating likely reduced the lifespan in male Aedes aegypti, whereas females benefitted from the association with males by an increased life span. Increased mating instances have been implicated in reduced survival in male mosquitoes, and furthermore increased mating competition and number of matings can be a result of optimal larval rearing (Nghabi et al. 2005, 2008). Typically, males reared under optimal la rval conditions would live longer than those provided less optimal diets as larvae; however, this may not al ways be the case when taking mating into consideration. Amino Acids in Nectar Sources Since the discovery that nectar sources contained resources other than sugar, and that amino acids were second m ost abundant component of nectars, there has been a focus on understanding the role amino acids play in in sect life history (Baker and Baker 1973, 1978). Most butterflies do not obtain large amounts of protein as adults, t hus many of these studies have focused on butterflies and the eff ect small amounts of proteins from their adult food sources, such as nectar, dung and fruit or pollen feedi ng may have on fitness (Alm et al. 1990, MeviShutz and Erhardt 2003a, 2005). However, mosqu itoes also require pr otein for egg production and usually rely on those obtaine d from blood feeding. Their need to feed on sugar sources for survival makes nectar a relevant potential sour ce of amino acids alte rnate to a blood source, especially during times when a host is not pr esent or in between blood feeding events. Preference for nectars mixtures containing amino acids by honeybees and cabbage w hite butterflies has been shown (Alm et al. 1990) and feeding on amino acids in nectar has been known to increase longevity and fecundity in the map butterfly (Mevi-Shutz and Erhardt 2005). Pollen fed in a sugar solution to Aedes aegypti increased the longevity and fecundity of that 16

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17 particular mosquito species (Eischen and Foster 1983). However, the findings that longevity and fecundity were increased in th e ma p butterfly were only valid for larvae maintained on poor diets. Butterflies that were not challenged in larval life did not seem to need the added nutrition of amino acids in their adult diets. Wild mosqu itoes are often nutritionally challenged as larvae, and since nectar feeding is also an important part of their adult life amino acids contained within may increase life span and fecund ity in these insects as well.

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CHAPTER 2 EFFECT OF CAR BOHYDRATE SOURCE ON NUTRITIONAL RESERVES OF Culex quinquefasciatus Introduction Many adult insects rely on sugar as a source that can be used imme diately for energy or stored as glycogen and lipids that will be us ed for energy, survival and fecundity (Chapman 1998, Heimpel et al. 2004, Xue et al. 2008). Most mosquitoes require sugar as a source of energy for flight prior to host s eeking, and they likely depend on te neral reserves for the initial energy needed to locate a carbohydrate source. The body size, teneral rese rves, survivorship and flight potential of adult mos quitoes depend highly on nutritiona l conditions available to larvae such as amount and type of accessible food (N ayar and Sauerman 1970, Briegel et al. 2001). Females from nutritionally-deprived larvae are smaller, contain fewer teneral reserves, less fecund and live less time than females from larv ae that were reared unde r more optimal larval conditions (Nayar and Pierce 1977, Telang and Wells 2004, Feinsod and Spielman 1980). Adult mosquitoes feed on floral nectar, fru it and honeydew as sources of sugar, which can be metabolized for use as immediate energy for flight (Nayar a nd Van Handel 1971) or converted into glycogen and lipid and used for long-term energy and maintenance, survival and fecundity (Foster 1995, Nayar and Sauerman 1971a, 1971b, 1974). Without feeding on sugars, newly-emerged mosquitoes typically deplete thei r teneral reserves and die within 2-4 days (Nayar and Sauerman 1975, Nayar 1982, Van Handel 1965b). There is a direct correlation between 90% depletion of lipids and 50% mortality (Nayar 1982). Both Aedes aegypti and Anopheles gambiae have been reported to have increas ed survival feeding solely on human blood without feeding on sugar (Costero et al 1998, Harrington et al. 2001), however, there are other reports of increased survival when thes e species feed on sugar (Gary and Foster 2004, Impoinvil et al. 2004). Excess sugars, from nectar f eeding or from teneral re serves, are stored as 18

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lipid and glycogen within the mosquito fat body and glycogen in the flight m uscle (Clements 1992). Lipid stores may be used to provision the initial batch of eggs in anautogenous females (Briegel et al. 2002). Fructose, glucose and sucrose have been id entified as the main components of nectar (Percival 1961, Baker and Baker 1975, 1983a, 1983b) and are also present in honeydew (Wackers 2001). Other sugars, such as melezito se and raffinose are more specifically honeydew sugars and have been identified in the mos quito crop (Burkett et al. 1999, Wackers 2001). Mannose is less common, but is present in tr ace amounts in nectar and has been used, like sorbose, to examine the effects of various types of sugars on survival and deposition of lipids and glycogen when fed to mosquitoes (Galun and Fraenkel 1957, Ozalp and Emre 2001). Sorbose is a sugar that is found in the mountain ash berry and is a byproduct of th e breakdown of sugars (US Government 2008), but it has also been used experimentally and found to be very unsupportive of life in Aedes aegypti (Galun and Fraenkel 1957). Nectar is thought the be the primary sour ce of carbohydrates for mosquitoes (Baker and Baker 1983a, 1983b), though identification of honeyde w sugars in the crops of mosquitoes and increased survival of some species when fed these sugars suggests that feeding on honeydew is also very important (Foster 1995, Burkett et al. 1999, Galun a nd Fraenkel 1957, Vrzal Chapter 3 of this thesis). Questions about the importance of nectar sugars remain because field observation of mosquitoes feeding on carbohydrates sources have been difficult, although various authors have witnessed mosquitoes feed ing on nectar sources and even Cx. nigripalpus feeding on homopteran honeydew (Haeger 1955, McCrae et al. 1969, Nayar 1982, Grimstad and DeFoliart 1974). 19

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The objective of this stu dy was to examine the effect of different sugars on the accumulation of adult reserves of male and female Cx. quinquefasciatus We chose to evaluate a range of naturally-occurring sugars including nectar and honeydew suga rs that occur more commonly (sucrose and melezitose) and some sugars which are less common (mannose and sorbose). Reserves of lipid and glycogen were quantified through the first 15 days of adulthood to examine the effects of different sugars on reserve accumulation. Materials and Methods Larval Rearing The Culex quinquefasciatus colony was established from a Gainesville, FL collection in 1995. Adults were maintained at 27 1.0 C, ~80% relative humid ity and a 14:10 light: dark photoperiod. Adults were blood fed on defibrinated bovine blood and continuously provided a 5% sucrose solution. Larvae were reared in plastic trays (35.5 cm x 48.3 cm x 6.4 cm) containing 2.5 liters of well water. Approximate ly 10 egg rafts were set in each pan. Larval food was provided as a slurry of 25 ml of a 3:2 (g/ml of water) bovine liver powder: brewers yeast on day 1 and a finely ground 2% hog chow (Pur ina Mills, LLC, St. Louis, Missouri) slurry (50 ml on days 2 through 5, and 75 ml on day 6) until pupation. Mosquito Dry Weights Dry mass measurements were obtained from samples of mosquitoes 24 2 hours post emergence. Mosquitoes were sampled prior to being sugar-fed and these measurements provided the initial (emergence) levels for lipid and glycogen deposits. Experiments conducted using sorbose, sucrose or mannose/melezitose treatments used mosquitoes from different days. Therefore, it was necessary to establish a baseline measurement to confirm that these mosquitoes began at similar nutritional levels. 20

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Groups of 10 ma les or 10 females were placed in microcentrifuge tubes, frozen (-20 C/ 80C) and then freeze-dried for approximately 48 hours to remove all moisture. Immediately after freeze-drying, the samples were placed in a dessicator, weighed on a microbalance (Sartorius CP2T) and again stored at -20 C until biochemical analysis. Nutritional Cage Experiment Upon adult emergence, also referred to as day 1, 15 male and 15 female Culex quinquefasciatus were placed in 0.47 L paper containers (Solo Cup Company, Highland Park, IL) modified with screened tops to allow vi ewing and air movement. No sugar was provided prior to being set in cages. Half of a cotton dental wick (Richmond Dental, Charlotte, NC) measuring 3.8 cm in length was placed in a 1.5 ml microcentrifuge tube. The wick was saturated with either 1.0 ml of treatment solution. The tube contai ning the cotton wick was inserted through a hole (1.0 cm in diameter) in the side of the cage (15.0 cm from the bottom) to allow access to the solution on the moist cotton. They were provided with a 5% (w/v) solution of D(+)-mannose, D-(+) melezitose, sucrose, or D -(+)-sorbose (Sigma-Aldrich, St. Louis, MO) ad libitum and the cotton wicks were changed daily to reduce fungal growth. To describe the accumulation of lipid and glycogen, groups of 10 males and females were sampled on days 1, 3, 5, 10 and 15 post-eclosion. There were five replic ations per treatment and control. The groups of mosquitoes were frozen at -20 C for later analysis using th e sulphosphovanillin and hot anthrone assays. Glycogen and Lipid Analyses Initial nutritional reserves of the mosquitoes at the time of pupal emergence were determined by measuring the levels of glycogen a nd triglyceride. The hot anthrone (glycogen) (Van Handel 1965a, 1985) and sulphosphovanill in (lipid) (Van Handel 1965a, 1985, Hahn 2005) assays were conducted following the methods provided in detail in Appendix A. Three to five 21

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replicates were completed for each sex, larv al re aring regime (low and high food diets) and day (1, 3, 5, 10 and 15). All of the samples that were previously freeze -dried, weighed and refrozen were analyzed for glycogen and lipid levels using both the hot anthrone assay for glycogen and the sulphosphovanillin assays for lipids. Briefly, (full prot ocol is presented in the Appendix), the dried mosquitoes were homogenized in 100 l of saturated sodium sulfate, 200 l of methanol, 100 l ultrapure water and 500 l of 1:1 chloroform: methanol in the microcentrifuge tube. The glycogen and lipid samples were measured usin g the methods of Van Handel (1965a, 1985), as modified by Hahn (2005), including a chromatographi c step using silicic acids to remove polar lipids. A spectrophotometer was used to measur e glycogen and triglyceri de levels at 625 and 525 nm, respectively using standard curves. Statistical Analyses Dry weight measurements of 10 mosquitoes for each of the 5 replicates were used to obtain average weights of individual mosquitoes in each treatment group. Glycogen and triglyceride levels were calculated for 3-5 replicates of 10 individuals fed each sugar. Mannose and melezitose are from the same batch of mosquitoes, so they are considered together for initial dry weights and lipid and glycogen analyses. Sorbose did not su pport adequate survival beyond 2 days, so these samples were excluded from all stat istical analyses. Tenera l (day 1) dry weights and nutritional contents for all treatments (m annose/melezitose, sucrose and sorbose) were compared with Minitab 15 (Minitab, Inc) using a one-way ANOVA and Tukeys HSD to separate the means ( P <0.05). Separate analyses of lipid and glycogen contents throughout time (days 1-15) of males and females were performed with JMP (SAS, Inc) using multivariable ANOVAs with diet, age and their in teraction term as factors. 22

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Results Comparisons of Initial Dry Weight, Glycogen and Lipid Content Glycogen and lipid contents of newly emerged fem ales used for sucrose, mannose/melezitose and sorbose experiments did not differ significan tly, nor did glycogen contents of males (F3,19= 2.68, P=0.082; F3,18=2.24, P =0.126; F3,17=0.38, P =0.769, respectively) (Tables 2-1, 2-2). However, initial lipid contents of males used for the sorbose experiment were different from the other batches (F3,17=5.08, P =0.014). Dry weights of males or females used the different treatments were significantly different (F3,18=60.63, P <0.001; F3,19=90.01, P <0.001) (Table 2-1 and 2-2). Glycogen Content Sorbose-fed adults lived for only 2 days a nd glycogen content of males decreased from 27.86 5.26 g/mg initially to 6.06 2.03 g/mg by day 2. Females contained 6.36 0.49 g/mg of reserves initially and were reduced to 2.62 0.68 g/mg glycogen by day 2. Because of their short survival times, they were excl uded from further analysis due to their short lifespans. Analysis of the remainder of the sugar diet treatments for days 1, 3, 5, 10 and 15 revealed that the most significant effect on glycogen accu mulation by males and females was age not diet (Table 2-3). For both males and females, wi th diets pooled, there was a significant overall increase in glycogen content from teneral levels at day 1 to day 3 with a slight decrease until day 10 where the reserves increased again on day 15, though the increase was not significant for females (Figures 2-1a, 2-1b). The interaction between age and diet was also significant for males and females (Table 23), with both increasing their glyc ogen levels significantly beyond te neral reserves with all sugar diets (Figures 2-1c, 2-1d). F eeding on melezitose and sucrose by males and females resulted in 23

PAGE 24

significant increases in the amount of glycogen by days 3 and 5, with levels remaining above teneral levels for the period of the study. (F igures 2-1c, 2-1d). Le vels of glycogen for melezitose-fed males were significantly greater at day 15 than day 3 (Figure 2-1c). Females fed melezitose had glycogen levels that remained high until day 15, with a slight drop at day 10 (Figure 2-1d). By days 10 and 15, the glycogen content of sucrose-fed males and females were significantly lower than days 3 and 5 (Figure 2-1c). Mannose-fe d glycogen reserves of males never increased significantly a bove teneral levels (Figure 21b), although females fed mannose had glycogen levels that increased above teneral levels on all day except on day 10, when they decreased to teneral leve l amounts (Figure 2-1d). Lipid Content Sorbose-fed adults lived for only 2 days lip id content of males decreased from 68.20 7.29 g/mg initially to 47.75 5.47 g/mg by day 2. Females contained 52.05 7.26 g/mg initially which was reduced to 42.87 6.70 g/mg by day 2. Analysis of the remainder of the sugar diets indicated that age had the most significant affect on lipid accumulation in males and females (Table 2-4). However, males and females displayed opposite trends, with overall lipid content in male s continuously decreasing through time from the teneral reserves (Figure 2-2a) and females slowly deposited more lipid over time (Figure 2-2b). The interaction between age a nd diet was also signifi cant for both males and female for lipid content (Table 2-4). Mannos e-fed males contained mo re lipid reserves on emergence than for days 3-15 (Figure 2-2c) In contrast, mannose-fed females did not differ in lipid levels, except at day 10 (Fi gure 2-2d). Melezitose-fed male s did not differ in their lipid content from teneral reserves throughout the study period (Figure 2-2c), however females fed this diet had reserves that increased significantly on day 3 a nd remained steadily high for the entire 15 days (Figure 2-2d). For sucrose-fed males, there was no difference in lipid deposits 24

PAGE 25

between any of the ages tested (Figure 2-2c), how ever lipid reserves in fem ales slowly increased from days 3 to 15, resulting in large accumulations by day 15 (Figure 2-1d). Discussion Age of male and female Cx. quinquefasciatus had a significant eff ect on glycogen and lipid accumulation in this study. Nutritional reserves are critical to survival and flight stamina, which greatly impact the ability of mosquitoes to locat e hosts and transmit disease. However, age alone was not the only factor affecting gl ycogen or lipid content. The inte raction of adult diet with age also played a role in whether gl ycogen or lipid increased or re mained steadily low throughout the 15 day study. Briegel et al. (2002) showed with Aedes aegypti, that continuous feeding on sugar in between gonotrophic cycles allowed triglyceride levels to continue to increase for >40 days, even with the addition of some of the triglycerides synthesized going to egg production. Nayar and Pierce (1977) showed a correlation between depleting lipid level and death, and concluded that three different species had 50% survival times th at corresponded with 90% decrease of teneral lipid reserves. Additionally, death occurred in Cx. nigripalpus when their lipid reserves were completely used up (Nayar 1982). Without feed ing on a sugar source that supports survival, such as those sugars contained in nectar and hone ydew, teneral reserves ar e depleted in 2-4 days followed by mosquito death (Nayar and Sauerman 1975, Nayar 1982, Van Handel 1965b). We fed these male and female Cx. quinquefasciatus sorbose, mannose, melezitose and sucrose, which are derived from different sources and kno wn to support very low to maximum survival (Galun and Fraenkel 1957, Wackers 2001). Sorbose, a monosaccharide, did not support surv ival beyond 2 days in males or females. Lipid and glycogen decreased from teneral reserv es by day 2, and based on the findings of Nayar and Pierce (1977), we believe that reserves would be depleted completely by day 3, though we 25

PAGE 26

did not measure the contents of the d ead mos quitoes due to inaccuraci es associated with breakdown of nutritional reserves af ter death. These results are cons istent with the findings of Galun and Fraenkel (1957) when they fed this sugar to Ae. aegypti although we found no evidence that it is toxic. According to the U. S. Governments Code of Federal Regulations (2008) and Gardner (1943) this sugar is found in the mountain as h berry and is a byproduct of the breakdown of some sugars, in dicating that it is possible for mosquitoes to encounter this sugar in a field setting. There is also evidence that this sugar is palatabl e and readily ingested by the ant Lasius niger (Tinti and Nofre 2001) and the blow fly, but did not support adequate survival in the blow fly (Fraenkel 1940), or in Aedes aegypti, where survival was less than or equal to survival on water (Galun and Fraenkel 1 957). Because it is so unsupportive of survival but is readily ingested by other insects, it may be a good candidate as an attracticide for mosquitoes. Mannose, another monosaccharide, increased gl ycogen content of females slightly beyond teneral reserves, but the glycogen content of male s and lipid contents of males and females were all reduced from teneral reserves. Longevity re corded in various mosquito species feeding on mannose, which is found as traces in nectar (Wackers 2001), have indicated that it is a fairly poor source of food leading to decr eased survival when compared to more adequate nectar or honeydew sugars like sucrose and melezitose (Galun and Fraenkel 1957, Vrzal Chapter 3 of this thesis). Because this supports low survival a nd does not increase lipi ds and glycogen very far beyond teneral reserves and it is found only as traces in nectar, it would be interesting to find whether feeding on other, better sources of car bohydrates would counteract the negative affects of this sugar. Culex tarsalis, Ae. aegypti and other dipterans have been found to have salivary carbohydrases that seem to differ between fam ily and even species (Gooding 1975, Marinotti and 26

PAGE 27

Jame s 1990, Schaefer and Muira 1972). These are shunted to the cr op upon ingestion of carbohydrates (Gooding 1975) and it is possible that if an enzyme is lacking to break down this particular sugar, the mosquito imbibing it will not be able to use it for nutrition and survival. Melezitose, a trisaccharide, and sucrose, a disaccharide, allowed for the accumulation of moderate to large amount of lipid and glycogen in males and fema les. Melezitose-fed adults maintained high levels of glycogen throughout the study period, whereas sucrose-fed adult content tapered off on days 10 and 15. Lipid leve ls remained above teneral reserves in females when fed both diets, and male reserves remained steady and equal to tene ral reserves for all 15 days. Both of these sugars have been found to enhance survival significantly in mosquitoes (Galun and Fraenkel 1957, Vrzal, Chapter 3 of this thesis), with melezitose supporting survival as well or better than sucrose. Sucrose is a ne ctar sugar, but also occurs in honeydew and is often used for maintenance of insect coloni es that sugar feed (Baker and Baker 1983a, 1983b Wackers 2001). Melezitose, on th e other hand, is more specifica lly a honeydew sugar (Burkett et al. 1999, Wackers 2001). Me lezitase is a specific ca rbohydrase that was found in Cx. tarsalis (Schaefer and Muira 1972) and may provide an explanation for why mosquitoes are able to utilize this sugar so well. Burke tt et al. (1999) postulat ed that analysis of mosquito crops testing positive for fructose, a byproduct of the breakdown of melezitose, may be missing that these insects had been feeding on honeydew. Because of the increased survival on this sugar as well as the identification of honeydew sugars in the cr ops of various mosquito species (Burkett et al. 1999, Galun and Fraenkel 1957, Vrzal Chapter 3 of this thesis) and the variety of sugars that are unique to homopteran honeydew (Wei et al. 1996, Yee et al. 1996). Therefore, it may be possible that mosquitoes prefer specific honeydews and that th ey contain the carbohydrases to utilize them to their full advantage. 27

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The accumu lations of lipid and glycogen stores are important for in creased longevity in mosquitoes. Without feeding on them, mosquitoes would not be able to seek hosts or have the opportunity for multiple feedings and ultimately transmit disease. Some sugars found in nature that are palatable, but do not support life (Fraenkel 1940). Th e basis for why some sugars increase longevity and some do not remains understudied. The identificati on of species-specific carbohydrases may be an important step to assist in determining species-specific attracticides or finding attractive honeydew sugars to use for population monitoring. 28

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Table 2-1. Initial (day 1) glycogen, lipid and dry weights of unfed individual ma le Cx. quinquefasciatus. Treatments Initial nutritional status measurements Glycogen (g/mg)1 N Lipid (g/mg)1 N Dry Weight (mg) 2 Sucrose 29.58 3.81a 5 73.19 10.80ab 3 0.32 0.02a Mannose/Melezitose 33.38 4.25a 4 113.00 12.04a 5 0.45 0.01b Sorbose 27.86 5.26a 5 68.20 7.29b 5 0.34 0.00a Similar letters within a column indicated no significant differences (Tukeys HSD, P< 0.05). 1Means of groups of 10 mosquitoes/dry weights of 10 mosquitoes. 2Dry weights = approximate weights of individual mosquitoes. Table 2-2. Initial (day 1) glycogen, lipid and dry weights of unfed individual female Cx. quinquefasciatus. Treatments Initial nutritional status measurements Glycogen (g/mg)1 N Lipid (g/mg)1 N Dry Weight (mg) 2 Sucrose 11.64 2.13a 5 39.96 7.81a 4 0.51 0.01a Mannose/Melezitose 11.61 1.66a 5 60.58 4.98a 5 0.68 0.01b Sorbose 6.36 0.49a 5 52.05 7.06a 5 0.50 0.01a Similar letters within a column indicated no significant differences (Tukeys HSD, P< 0.05). 1Means of groups of 10 mosquitoes/dry weights of 10 mosquitoes. 2Dry weights = approximate weights of individual mosquitoes. 29

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30 Table 2-3. Results of ANOVA on glycogen levels di splaying the effects of sugar diet, age and their interaction on male or female Culex quinquefasciatus Trait Source df F P Males Whole model 14 48.57 <0.001* Diet 2 0.05 0.952 Age 4 42.28 <0.001* Diet x age 8 21.32 <0.001* Error 57 Total 71 Females Whole model 14 52.07 <0.001* Diet 2 0.00 1.000 Age 4 110.23 <0.001* Diet x age 8 8.85 <0.001* Error 60 Total 74 *Indicates significant term ( P <0.05). Table 2-4. Results of ANOVA on lipid levels displa ying the effects of sugar diet, age and their interaction on male or female Culex quinquefasciatus Trait Source df F P Males Whole model 14 13.39 <0.001* Diet 2 5.08 0.009* Age 4 9.38 <0.001* Diet x age 8 5.92 <0.001* Error 56 Total 70 Females Whole model 14 29.39 <0.001* Diet 2 1.77 0.180 Age 4 16.53 <0.001* Diet x age 8 15.79 <0.001* Error 56 Total 70 *Indicates significant term ( P <0.05).

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1(Unfed)351015 0 50 100 150 200 250 Mannose Melezitose Sucrose 1(Unfed)351015 0 50 100 150 200 250 Mannose Melezitose Sucrose c) d)a ab bc bc bc cd de ef f f f efa a ab ab bc bc cd d de de de efDaysf f f f f f 0246810121416Glycogen Content (g/mg) (LS Means) 0 50 100 150 200 250 a a b aa)c Age 0246810121416 0 50 100 150 200 250 a a b b cb) 31 Figure 2-1. The effect of age of males (a ) and females (b) and the interaction betwee n sugar diet and age on male (c) and fema le (d) Cx. quinquefasciatus on glycogen content (g/mg) (SE) (LS Means). Treat ments with similar letters are not significantly different (Tukeys HSD, P <0.05).

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Figure 2-2. The effect of age of males (a) and females (b) and the inter action between sugar diet and ag e of male (c) and female (d) Cx. quinquefasciatus on lipid content (g/mg) (SE) (LS Means). Treatm ents with similar letters are not significantly different (Tukeys HSD, P <0.05). Days 1(Unfed)351015 0 50 100 150 200 250 Mannose Melezitose Sucrose c bc Age 0246810121416 0 50 100 150 200 250 bc a ab ab)c Days 1(Unfed)351015 0 50 100 150 200 250 Mannose Melezitose Sucrose c)ab ab ab ab ab abc ab cd bc de cde de eea a abc dead)a a a a a a bc bc bcb b bc 0246810121416Lipid Content (g/mg) (LS Means) 0 50 100 150 200 250 ab bc bc ca)a 32

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CHAPTER 3 EFFECT S OF VARIOUS CARBOHYDRA TE SOURCES ON THE LONGEVITY OF Culex nigripalpus Culex quinquefasciatus AND Culex salinarius Introduction Adult male and female mosquitoes feed on carbohydrates, which are converted to glycogen for immediate energy (Nayar and Van Handel 1971) and to lipids for long -term energetic needs and survival (Clements 1955, Nayar and Sauerm an 1971a, 1971b, 1975). Nectar is considered the primary source of carbohydr ates (Foster 1995, Yuval 1992, Grimstad and DeFoliart 1974, Haeger 1955), but Magnarelli ( 1983) found very few mosquitoes actually feeding on floral nectaries compared to the numbe r found with sugar in their crops This suggests that other sources may be used to meet carbohydrate needs, but how frequently these sources are used is unknown. Additional sources include honeydew (B urkett et al. 1998, Haeger; unpublished in Nayar 1982, Haeger 1955), extrafloral nectaries (Foster 1995), and even plant tissues (Mller and Schlein 2005). Floral nectar is often composed primarily of sucrose, fructose and glucose (Baker and Baker 1983a, 1983b), while traces of mannose ma y also be present (Wackers 2001) it is considered a primary source of carbohydrates for mo squitoes (Foster 1995). As well as sucrose, glucose and fructose, honeydew can contain other unique sugars su ch as raffinose, melezitose (Volkl et al. 1999) and trehalose (Baker and Baker 1983a), all of which have been identified in the crops of mosquitoes (Burkett et al. 1999). Identification of sugars in mosquito crops proves that mosquitoes are encountering these in nature and likely using them for survival. Sorbose is rare in nature (Gardner 1943), but is found in moun tain ash berries and can result from microbial breakdown of sugars (United States Govern ment 2008, Gardner 1943). Previously it was evaluated in longevity studies with Ae. aegypti (Galun and Fraenkel 1957). Sorbose has been implicated as having deleterious effects on su rvival and may possibl y be toxic (Galun and 33

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Fraenkel 1957). In the laboratory, mosquitoes ar e often ma intained on a 5-10% sucrose solution, which is sufficient to maintain su rvival for long periods of time, such as greater than 2 months for Cx. nigripalpus (Nayar and Sauerman 1973). Physiological and behavioral studies, as well as studies relating to mosquito di sease transmission, require prolonge d survival in the laboratory. Culex species play an important role in the tran smission of vector-borne disease in Florida, throughout the United States, and around the world. Culex nigripalpus are considered the main vectors of West Nile virus (WNV ) and St. Louis encephalitis virus (SLE) (Day 1997) in Florida. In Asia, Africa, the Western Pacific and South America, Culex quinquefasciatus are urban vectors of lymphatic filariasis caused by Wuchereria bancrofti (Subra 1981). Culex quinquefasciatus are also known vectors of SLE (Jones et al. 2002) and competent and potential vectors of WNV (Sardelis et al. 2001, Molaei et al. 2007) in the southeastern United States. Culex salinarius are potential vectors of Eastern Equine encephalitis virus (EEE) (Scott and Weaver 1989) and considered a possible bridge v ector of WNV to humans in the northeastern U.S., mainly due to its opportunistic f eeding behaviors (Molaei et al. 2004). Transmission of mosquito-borne diseases relies heavily on the survival of mosquitoes and the ability of the pathogen to survive and repl icate in the field (Patz et al. 1998, Craig et al. 1999). Factors affecting mosquito longevity are critical as the longer lived the mosquito, the greater the opportunity to obtain multiple blood meal s and to acquire and transmit disease. Sugar feeding is imperative to the extended survival of mosquitoes and adult feeding on nectar, honeydew and other carbohydrate sources is necessary for disease transmission. Culex species differ in their life history parameters and may pot entially utilize carbohydrate sources differently. The objective of this study was to examine the effect of various sugars on the longevity of Cx. 34

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nigripalpus Cx. quinquefasciatus and Cx. salinarius Additio nally, comparisons were made to determine sex-specific patterns of surviv al on various carbohydrate sources. Materials and Methods Larval Rearing Mosquitoes used in this study were from colonies maintained at the USDA, ARS (Gainesville, FL) and included Culex nigripalpus (Vero Beach, 1999) Culex quinquefasciatus (Gainesville, 1995) and Culex salinarius (Hartford, 2001). All colonies were maintained at 27 1.0C, ~80% relative humidity and a 14:10 light: dark photoperiod. Adults were blood-fed on manually defibrinated bovine blood or chicke n blood (University of Florida IACUC D469). Adults were provided continuous access to a 5% sucrose solution on saturated cotton balls. All larvae were reared in plastic trays (35.5 cm x 48.3 cm x 6.4 cm ) containing 2.5 liters of well water. The rearing of Cx. nigripalpus entailed placing 4 egg rafts in each tray and the subsequent larvae were fed a 0.08 g (on day 1) and 0.24 g (from day 2 until pupation) portion of dry food mixture (1:6 bovine liver powder: brewe rs yeast) (MP Biomedicals, Solon, OH)) daily for 6 days until pupation. An increased number of egg rafts resulted in higher larval density and increased death or incomplete pupation. Culex quinquefasciatus and Cx. salinarius were reared with 10 egg rafts per tray and we re fed a slurry of 25 ml of a 3:2 bovine liver powder: brewers yeast (g/ml of water) on day 1 and a 2% finely ground hog chow (Purina Mills, LLC, St. Louis, Missouri) slurry (50 ml on days 2 through 5 and 75 ml on day 6) until pupation. Longevity Assays For longevity assays, mosquitoes were allo wed to emerge into a cage with no access to sugar. Within 24 hours post-emergence mosquitoes were chilled, and 35 males and 35 females were placed into 0.47 L paper containers (Sol o Cup Company, Highland Pa rk, IL). Container lids were modified with transparent tulle fabric to allow viewing inside the cages. An access 35

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hole (1.0 cm) was cut in the side of the contai ner approxim ately 15.0 cm from the bottom. A 1.5 ml microcentrifuge tube, containing a cotton dental wick ( 3.8 cm long) (Richmond Dental, Charlotte, NC) saturated with 1.0 ml of a 5% s ugar solution, was inserted into the hole to allow the mosquitoes to feed ad libitum The mosquitoes were provided with new wicks saturated with a 5% sugar solution or water cont rol every other day. Sugars te sted include monosaccharides; D(-)-fructose, D-(+)-gluco se, D-(+)-mannose and D-(+)-sorbose, disaccharides; sucrose and D-(+)trehalose, and trisaccharides; D-(+)-melezitose and D-(+)-raffinose (Sigma, St. Louis, MO). Between six and ten replicates of each treatm ent were conducted for each species, depending on the availability of mosquitoes. The cages were maintained in incubators at 28 1.0 C and 80 0.44 % RH with a 14:10 light: dark photoperiod. The dead mosqu itoes were counted daily and recorded. For each container, 50% mortality was determined to be the time (day) at which 50% of the mosquitoes in the cage were dead. Statistical Analyses Statistical analyses were conducted using JMP (SAS, Inc. Cary, NC). Kaplan-Meier survival analysis using log-rank was used to crea te survivorship curves. Pairwise comparisons using log-rank tests were made of the survival cu rves for each species and sex to determine if the curves differed. Additionally, the number of days to 50% mortality was determined for each cage (Briegel et al. 2001, Nayar 1986, Nayar and Sauerman 1975) and these averaged for each treatment. Analyses were conduc ted for males and females separa tely because they are known to differ in their survival (Liles and DeLong 1960, Br iegel and Kaiser 1973) as well as for water and sorbose treatments and all other treatments du e to very low survival and variances when fed water and sorbose (Galun and Fraenkel 1957). Multivariable ANOVA models were constructed with species and sugar as fixed effects and th eir interaction as fact ors. The time to 50% mortality for each species was examined separa tely using a one-way ANOVA or Kruskall-Wallis 36

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nonparame tric analysis when necessary and th en means were separated using Tukeys HSD ( P <0.05) in all analyses. Assumptions of normality and homogeneity of variance were confirmed using graphical representations of the data and Levenes tests. Results Male Survivorship Curves Culex nigripalpus. Feeding different sugars to male s had a significant effect on survival curves ( 2=2987.62, df=8, P <0.001) (Figure 3-1). Pairwise comparisons allowed for the separation of the survival curves ( P <0.05). Survival curves of ma les feeding on melezitose and raffinose indicated significantly l onger survivorship than any of the other sugars. Males feeding on trehalose and fructose fed males produced cu rves that indicated high survivorship, both supporting longer survivorsh ip than sucrose. Whereas, mannose and glucose fed adults lived a very short period of time compared to the other tr eatments thus far. The only diets that were less supportive than mannose and glucose were sorbose and the water control. The sorbose curve indicated a significantly shorter lifespan resulted from feeding on this treatment than when feeding on the water control. Culex quinquefasciatus. Males fed different sugars differed in their survival significantly ( 2=3803.29, df=8, P <0.001) (Figure 3-1). For this spec ies, fructose and raffinose curves indicated the greatest survivorship, followed by survival on glucose. Moderate survival was indicated by the curves of melezitose, sucrose and trehalose. Very poor survival was obtained by those males fed mannose. Again, however, water and sorbose were the poorest diets, with sorbose fed adults living less time that those fed the water control. Culex salinarius. Feeding different sugars led to signi ficantly different survivals of the males feeding on them (2=3050.15, df=8, P <0.001) (Figure 3-1). Gr eatest survival was achieved by adult males fed raffinose, with fructose glucose, melezitose and sucrose-fed adults 37

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surviving almost as well. Mannose and trehalosefed ma les did not survive as long as the other treatments, but they did increase survival over that of water a nd sorbose. For this species, sorbose-fed males lived longer th an those fed the water control. Female Survivorship Curves Culex nigripalpus. Survival differed significantly depending on the diet fed to female Cx. nigripalpus ( 2=3378.61, df=8, P <0.001) (Figure 3-2). Glucose-fed females survived significantly longer than females fe d any other diet. Females fed fructose, melezitose, trehalose and raffinose were moderately long-lived in comparison. Sucrose and mannose provided the nutrition that allowed for only moderately low survival, although the mosquitoes fed these treatments did live significantly longer than thos e fed sorbose and the water control. Females fed sorbose again lived a shorter amount of time than those fed the water control. Culex quinquefasciatus. Females fed different sugars su rvived significantly different amounts of time depending on treatment ( 2=4607.95, df=8, P <0.001) (Figure 3-2). Fructose-fed females lived longer than females fed any othe r treatment, with glucos e and melezitose-fed females living significantly less time. Females fed sucrose and raffinose lived moderately long lives. However, trehalose and ma nnose-fed adults did not survive as long as any of the previous treatments. They did outlive sorbose and water-fe d adults, but survival was fairly low. Waterfed adults lived longer than those fed sorbose diets. Culex salinarius. Varying sugar treatments resulted in differing survival rates of female Cx. salinarius ( 2=3491.58, df=8, P <0.001) (Figure 3-2). Females fed fructose and glucose surpassed the rest of the females in survivorsh ip. Melezitose, sucrose and raffinose-fed adults clustered behind them and had long lives as we ll. Supporting only moderately low survival, trehalose and mannose-fed females did live l onger than those fed the sorbose and water treatments. Water did not exte nd life beyond that of sorbose. 38

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Comparison of Male Days to 50% Mortality Sorbose and water supported minimal survival and were partitioned fr om other sugars in analysis. For males fed on water and sorbose, ther e were significant effects of sugars and species with a significant sugar x species interaction (Table 3-1). A si milar pattern was seen for males fed the other sugar treatments (Table 3-1). Overall, males differed in the time to 50% mortality based on species with Cx. quinquefasciatus males living the longest, followed by Cx. salinarius and the shortest lived were Cx. nigripalpus (Figure 3-3a). The longest-living males were fed on raffinose and fructose followed by glucose and su crose, then melezitose (Figure 3-3b). Trehalose and mannose supported p oor survival, though they did s upport survival significantly longer than water controls. Male s that were fed sorbose lived le ss time that those fed the water control (Figure 3-3b). Survival of Cx. nigripalpus males was similar when fed fructose, glucose, melezitose, raffinose, sucrose and trehalose wi th significantly lower survival on mannose (Figure 3-4). Male Cx. quinquefasciatus lived longest on raffinose, fructose glucose, followed by sucrose then melezitose and trehalose. Mannose supported low survival. Survival of male Cx. salinarius were supported the longest on raffinose, sucrose, fructose, glucose a nd mannose, with trehalose-fed males living the least amount of time. Wate r and sorbose both supported poor survival for Cx. quinquefasciatus and Cx. salinarius and survival length was not different ( t =0.12, df=15, P =0.905). Survival of Cx. nigripalpus and Cx. quinquefasciatus males, however was less when fed sorbose than water ( t =707.11, df=18, P <0.001; t =2.44, df=8, P =0.040, respectively) (Figure 3-4). Comparison of Female Days to 50% Mortality Similar to males, all species of females maintained very low survival on water and sorbose and multivariable ANOVAs were c onducted on water and sorbose-fed females 39

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separately from those fed th e other sugars (Table 3-2) For females fed water and sorbose, there was a significant effect of species and species x sugar interaction, but not of sugar alone (Table 3-2). Females fed on other sugars displayed si gnificant effects of sp ecies, sugars and the interaction of species and sugars (Table 3-2). Overall, female Cx. nigripalpus and Cx. salinarius had similar lifespans and Cx. nigripalpus lived significantly longer than Cx. quinquefasciatus (Figure 3-5a). With species pooled, fructose, glucose a nd sucrose supported the greatest survival (Figure 3-5b). Raffinose and melezitose fed female s lived as long as those fed sucrose, which is commonly used to rear laboratory adults (Figure 3-5b). Again, as seen in males, trehalose and mannose supported only moderate su rvival, but significantly higher than the wateror sorbosefed adults. Waterand sorbose-fed female s did not differ in their survival times. The longest survival of each specie s was achieved on a different sugar, Cx. quinquefasciatus on fructose, Cx. nigripalpus on glucose and Cx. salinarius on sucrose (Figure 3-6). Survival of Cx. nigripalpus females was greatest on glucose, fructose, melezitose, raffinose, sucrose and trehalose, with moderate survival on mannose. Female Cx. quinquefasciatus survived the longest feeding on sucrose, fructose, glucose and raffinose, with moderate to poor survival on trehalose and mannose. Culex salinarius females lived the longest when fed sucrose, fructose, glucose and raffinose, with moderate to low survival on trehalose and mannose (Figure 3-6). Variation in survival was seen on all the sugars, each supporting longevity better or worse depending on the species. However, longevities were not significantly different when provided any of the s ugars, except for trehalose, which Cx. nigripalpus lived significantly longer than the other two species and fructose, which allowed Cx. quinquefasciatus to live significantly longer than Cx. nigripalpus. Female Cx. salinarius fed water and sorbose lived less time on water (t =4.49, df=15, P <0.001), but Cx. nigripalpus and Cx. quinquefasciatus 40

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fem ales lived less time on sorbose or showed no difference in survival between the two treatments, respectively ( t =8.99, df=18, P <0.001; t =0.00, df=8, P =1.000) (Figure 3-6). Discussion The length of adult Culex survivorship was clearly affected by sex, dietary sugar and species. Much research has been dedicated to examining the ma in sugar components of nectars present in common trees and shrubs (Percival 1961, Baker and Baker 1975, 1983a, 1983b). Nectar is widely known to be fed upon by mos quitoes (Grimstad and DeFoliart 1974, Foster 1995) and consists primarily of sucrose, glucose and fructose (Baker and Baker 1975), with mannose found in trace amounts (Wackers 2001). Honeydew as a source of carbohydrates for mosquitoes contains several unique sugars, including melezitose, raffinose and trehalose (Wackers 2001, Foster 1995), which were also exam ined. Water was fed to mosquitoes as a control and sorbose is a sugar that has been used in previous studies and has been implicated as having toxic effects on the insects that feed on it. The effect of various sugars on longevity for insects such as mosquitoes and flies (Gal un and Fraenkel 1957, Liles and DeLong 1960, Nayar and Sauerman 1971a, 1971b and 1975), parasitoids (Jacob and Evans 2000), phorid flies (Fadamiro et al. 2005) and butterflies (Hill and Pierce 1989, Mevi-Shutz and Erhardt 2003a, 2005) have been widely studied. Other complex s ugar sources for mosquitoes that are suspected to provide nutrition include rotting fruit, pl ant tissues (Muller and Schlein 2005) and aphid honeydew (Burkett et al. 1998). The role of hon eydew sugars on adult survival has been investigated with An. gambiae (Gary and Foster 2004) and with parasitoids (Lee et al. 2004, Jacob and Evans 2000). Male and female survival curves in all speci es were mostly reverse sigmoidal in shape, similar to curves produced when Ae. aegypti were fed a 10% sucrose solution (Liles and DeLong 1960) or pollen (Eischen and Foster 1983) and a phorid fly, Pseudoacteon tricuspis was fed 41

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various sucrose solutions (Fadamir o et al. 2005). Males and f emales of all species in this study showed similar trends when fed the common nect ar sugar components. Survival was increased far beyond that seen in individua l provided only water, and in mo st cases, they were among the most suitable sugars provided. These data corres pond with many of the studies of this type with various insects (Galun and Fraenkel 1957, Wacker s 2001) and support the findings that nectar sugars play an important role in the su rvival of mosquitoes and other insects. However, feeding sorbose, mannose and trehal ose did not follow the same pattern of survival and were unsuitable diets and that di d not increase survival much above the water control. For both males and females, water a nd sorbose did not support survival over 4 days. Male and female Cx. nigripalpus lived significantly less time when fed sorbose than when fed the water control, while Cx. salinarius females lived longer when fed sorbose. All others lived as long on sorbose as on the water control, but this sugar was very unsupportive of survival when compared to any of the other treatments. Gal un and Fraenkel (1957) postu lated that sorbose may have possible toxic effects to mosquitoes, which may explain the low survival seen in Cx. nigripalpus males and females when fed that sugar alone. Mannose is a sugar that is found in nature as traces in plant nectar (Wackers 2001). Mannose was found previously to be moderately supportive of longevity in the parasitoid Cotesia glomerata (Wackers 2001) and barely increased survival beyond that of sorbose in Ae. aegypti (Galun and Fraenkel 1957). In this study, survival of male Cx. quinquefasciatus and Cx. nigripalpus males maintained on mannose was low, but s lightly higher than the water control. Culex salinarius survival, however, was as long on mannose as when fed fructose, glucose or melezitose. Clearly differences exist in the u tilization of this suga r between species. 42

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Finally, trehalose, a known component of aphid honeydew suga r (Baker and Baker 1983a), only supported m oderately low survival of Culex Similar trends were seen in the survival of the parasitoids Cotesia glomerata (Wackers 2001) and Bathyplectes curculionis (Jacob and Evans 2000) and the mosquito Ae. aegypti (Galun and Fraenkel 1957). In contrast, Cx. nigripalpus males and females maintained on this sugar had equivalent survival when fed another disaccharide, sucrose. Honeydew is a carbohydrate-rich product of aphids feeding on plant phloem (Holldobler and Wilson 1990). Composition and amount of honeydew produced by aphids is variable depending on species (Volkl et al. 1999). Honeydew sugars include common nectar sugars sucrose, fructose and glucose, but also contain a variety of other sugars including xylose, maltose, melezitose, raffinose (Volkl et al. 1999) and tr ehalose (Baker and Baker 1983a). Sugars associated with honeydew have been identified in the crops of many field-caught insects such as parasitoids (Heimpel et al. 2004), sandflie s (MacVicker et al. 1990) and mosquitoes, Culiseta melanura and Anopheles quadrimaculatus (Burkett et al. 1999). Additionally, feeding on honeydew from the green aphid (Haeger 1955) and from Coccus viridis, by Cx. nigripalpus in the field has been documented (Nayar 1982). Feeding on honeydew has been implicated as a possible alternative source for mo squitoes to obtain sugars not found in nectar or found in different concentrations from nectar sources (Foster 1995). Because mosquitoes are known to feed on honeydew (Burkett et al. 1999), but obser vation of this is complicated (Foster 1995), survival on the associated sugars equaling the surviv al seen in nectar sugars in our study suggests that the importance of honeydew as a sugar source for Culex mosquitoes has been greatly underappreciated. 43

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Our results reveal a trend of increased su rvival on sugars commonly found in aphid honeydew, with the exception of trehalose, which was only a moderate diet for male and female Cx. nigripalpus. Males of all three species survived as long on raffinose as on the three nectar sugars mentioned previously, which is the ma in sugar component of bahiagrass and which Cx. nigripalpus has been observed feeding on around homes in Florida (Burkett et al. 1999). Melezitose was moderately supportive for males, increased survival significantly beyond that of water and sorbose, and displayed similar survival to that of Cx. quinquefasciatus fed sucrose and Cx. salinarius fed glucose and fructose. In addition, fe males fed raffinose and melezitose also survived a similar amount of time to those fed th e three nectar sugars. Raffinose did not increase survival over that of water when fed to Cotesia glomerata (Wackers 2001) and induced only moderate survival in Ae. aegypti over nectar sugars fed to th at species (Galun and Fraenkel 1957). The different type and amount of sugars pr esent in honeydew may be nefit different insect species according to their efficiency in utilization of these sugars. Because particular species survived overall longer than others when treatments were pooled, there was an interest in comparing the diffe rent effects that a similar diet would have on each of the three species. For example, male Cx. quinquefasciatus lived the longest on fructose, glucose and raffinose, Cx. salinarius on mannose, and there were no significant differences in survival between species when males were mainta ined on melezitose or sorbose. Females also differed in survival depending on species and diet. Female Cx salinarius survived the longest on sucrose and trehalose fed mosquitoes displaye d significant differences in survival among all three species, with Cx. nigripalpus living the longest and Cx. quinquefasciatus the shortest. These differences may be explained by a separa tion in space and time providing one species of male or female with the resour ces allowing them to survive bette r on one diet than another. 44

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Culex nigripalpus occur throughout the year in the extreme southern portion of the U.S. (Carpenter and LaCasse 1955), but peak season is from late summ er into the fall. In collections of larvae from an oviposition si te in Florida, all three Culex species in this study were found in association with each other throughout the year (Nayar 1982). Seasonal abundances of the species were similar for Cx. quinquefasciatus and Cx. nigripalpus. Culex salinarius has a larger range in the U.S., which extends from the southeas t into the northeast and west to Utah and even into Canada, but it is likely most abundant in the Atlantic and Gulf Coasts. In the extreme south, this species can be present all year, but overwinte rs as adults in the nor thern range (Carpenter and LaCasse 1955). It can be assumed that di fferent plants are fl owering and different homopteran species may be abunda nt, introducing varying ratios of sucrose: glucose: fructose and different honeydew sugars all together depending on what specie s is present. These species may differ in their preference for honeydews pro duced by certain aphids depending on the sugars contained therein. The relationship between mosquitoes and sugar f eeding is critical because of the associated survival (Foster 1995, Nayar 1982, Nayar and Sa uerman 1975) and subsequent host-seeking behavior (Nasci 1991, Foster 1995) which relate to vector potential and disease transmission. Although findings that sugar-fed mosquitoes are le ss avid host-seekers than starved or water-fed mosquitoes (Foster and Eischen 1984), there is ev idence that they are more persistent at blood feeding attempts (Walker and Edman 1985), which coupled with long life can be important to vector potential. Studying longevity of Culex species in a laboratory se tting may provide a better understanding of the poten tial of these insects to survive on particular sugars found in natural sources as well as the effect of different sugar sources on mosquito physiology, such as deposition of nutritional reserves. Furthermore, sugar feeding studi es can be used as a way to 45

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improve species-specific trappi ng and population m onitoring by impr oving attractants or baiting systems, which is important for anticipa ting the potential for disease outbreak. 46

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Table 3-1. Results of ANOVA on da ys to 50% mortality of ma les fed water and sorbose and males fed the remaining treatments that supported survival. Treatment Source df F P Water and Sorbose Whole model 5 8.67 <0.001* Species 2 8.10 0.001* Sugars 1 13.19 0.001* Species x sugars 2 5.63 0.007* Error 41 Total 46 Other sugars Whole model 20 39.52 <0.001* Species 2 44.01 <0.001* Sugars 6 71.66 <0.001* Species x sugars 12 21.33 <0.001* Error 165 Total 185 *Indicates significant terms (P <0.05). Table 3-2. Results of ANOVA on da ys to 50% mortality of fema les fed water and sorbose and males fed the remaining treatments that supported survival. Treatment Source df F P Water and Sorbose Whole model 5 28.04 <0.001* Species 2 17.75 <0.001* Sugars 1 0.39 0.539 Species x sugars 2 48.88 <0.001* Error 41 Total 46 Other sugars Whole model 20 29.43 <0.001* Species 2 4.83 0.001* Sugars 6 76.79 <0.001* Species x sugars 12 9.67 <0.001* Error 167 Total 187 *Indicates significant terms (P <0.05). 47

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Cx. nigripalpus 0.0 0.2 0.4 0.6 0.8 1.0 Water (e) Fructose (b) Glucose (d) Mannose (d) Melezitose (a) Raffinose (a) Sorbose (f) Sucrose (c) Trehalose (b) Cx. quinquefasciatus Proportion Alive 0.0 0.2 0.4 0.6 0.8 1.0 Water (f) Fructose (a) Glucose (b) Mannose (e) Melezitose (c) Raffinose (a) Sorbose (g) Sucrose (c) Trehalose (d) Cx. salinarius Days 02 04 06 08 0 0.0 0.2 0.4 0.6 0.8 1.0 Water (e) Fructose (b) Glucose (b) Mannose (c) Melezitose (b) Raffinose (a) Sorbose (d) Sucrose (b) Trehalose (c) Figure 3-1. Proportion survivorship of Culex males maintained on 5% solutions of different sugars. Treatments with similar letters are not significantly different (Kaplan-Meier Survival Curve, pairwise tests, P <0.05). 48

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Cx. salinarius Days 02 04 06 08 0 0.0 0.2 0.4 0.6 0.8 1.0 Water (f) Fructose (a) Glucose (a) Mannose (d) Melezitose (b) Raffinose (b) Sorbose (e) Sucrose (b) Trehalose (c) Cx. quinquefasciatus Proportion Alive 0.0 0.2 0.4 0.6 0.8 1.0 Water (e) Fructose (a) Glucose (b) Mannose (d) Melezitose (b) Raffinose (c) Sorbose (f) Sucrose (c) Trehalose (d) Cx. nigripalpus 0.0 0.2 0.4 0.6 0.8 1.0 Water (g) Fructose (b) Glucose (a) Mannose (f) Melezitose (c) Raffinose (d) Sorbose (h) Sucrose (e) Trehalose (c) Figure 3-2. Proportion survivorship of Culex females maintained on 5% solutions of different sugars. Treatments with similar letters are not significantly different (Kaplan-Meier Survival Curve, pairwise tests, P <0.05). 49

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50 Species CN CQ CSDays to 50% Mortality ( SE) 10 15 20 25 30 35 40 Sugars F r uc t os e G l uc os e M annos e M el ez i t os e R af fi nos e S uc r os e T r eh al os e W a te r S or bos e 0 10 20 30 40 a b c a ab bc bc c d e A Ba) b) Figure 3-3. Effect of species (a ) and sugar (b) on days to 50% mo rtality ( SE) (LS Means) of males. Columns with similar lowercase (Tukeys HSD, P <0.05) or uppercase ( t -tests, P <0.05) letters are not significantly different.

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Sugars F r uc t o s e G l u c o se Ma n n o se Me l e z it o s e R a f f i n o se S u cr o se Tre h a l o s e W a t e r S or b o seDays to 50% Mortality ( SE) 0 10 20 30 40 50 CN CQ CS A A A B A A a a ab a ab bc abc cd bc a bc a a aa c a d d b eANOVA; F 6,59 =10.98, P<0.001 ANOVA; F 6,66 =84.08, P<0.001 ANOVA; F 6,58 =15.48, P<0.001 51 Figure 3-4. Comparison of da ys to 50% m ortality of Cx. nigripalpus (CN), Cx. quinquefasciatus (CQ) and Cx. salinarius (CS) males maintained on 5% solutions of similar sugars. Columns for each species with similar letters are not significantly different (Tukeys HSD, P<0.05).

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Species CN CQ CSDays to 50% Mortality ( SE) 25 30 35 40 45 50 Sugars F r uc to s e G l u c o s e M a n n o s e M e l e z i t o s e R a ffi n o s e Su c r o s e T r e h a l o s e W a t er So r b o s e 0 10 20 30 40 50 a ab bc c c d e a ab b A Aa) b) Figure 3-5. Effect of species (a ) and sugar (b) on days to 50% mo rtality ( SE) (LS Means) of females. Columns with similar lowercase (Tukeys HSD, P <0.05) or uppercase (t tests, P <0.05) letters are not significantly different. 52

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Sugars F ruc t os e Gl u c os e M ann os e M el e z i t os e R a f f inos e S uc ros e T re hal o s e W a t e r S orb os eDays to 50% Mortality ( SE) 0 10 20 30 40 50 60 CN CQ CS aaa a ab ab ab abb bbbbb bbcc c c c A A A B B AKruskall-Wallis; H=31.05, df=6, P<0.001ANOVA; F 6,66 =42.06, P<0.001 ANOVA; F 6,59 =38.86, P<0.001 53 Figure 3-6. Comparison of da ys to 50% m ortality of Cx. nigripalpus (CN), Cx. quinquefasciatus (CQ) and Cx. salinarius (CS) females maintained on 5% solutions of similar sugars. Colu mns for each species with similar letters are not significantly different (Tukeys HSD, P <0.05).

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CHAPTER 4 EFFECT OF NUT RITIONAL STATUS AND PR ESENCE OF AMINO ACIDS TO A SUGAR MIXTURE ON LONGEVITY OF C ulex quinquefasciatus Introduction ino acids are the second most abundant component of nectars, as been a keen interest in the role that th ey play in the fitness of the insects that feed on Baker and Baker 1973). Some insects rely on nectar as a primary source for amino acids, ers feed on fruit, dung, pollen or even bl ood to obtain supplements Ingestion of amino n sugar solution or by feeding on pollen has been reported to enhance longevity and ity in females and males of several of sp ecies of butterflies (O Brien et al. 2003, Meviand Erhardt 2005, Beck 2007). Providing pollen in sugar solutions to Aedes aegypti also ed longevity and fecundity in that species (Eischen an d Foster 1983). urce of flight energy as well as to enhance ity (Nayar and Sauerman 1971a, 1971b, 1973, 1975; Gary and Foster 2004). When not g on nectar, anautogenous species may al so feed on blood as a source of amino acids, are used to make vitellogenin for egg production (OMeara 1987). Eischen and Foster demonstrated some egg production from anautogenous Ae. aegypti fed only pollen. this is not typical, and in the absence of a sufficient amino acid source, most ous mosquitoes will not be able to obtain enough provisions for egg production. In s, blood feeding is the primary source of amino acids; however, when vertebrate hosts ilable, amino acids from nectar or pol len could enhance longevity and fecundity. ny physiological attributes can affect the longevity of mo squitoes in the field and es reared in the laborat ory. Larvae collected from the fi eld can be exposed to a wide ety of nutritional conditions, and those from suboptimal conditions may result in smaller y sizes (McCann 2006). Field-co llected mosquitoes from natural rearing conditions may Since the discovery that am there h them ( but oth acids i fecund Shutz increas Mosquitoes feed on nectar and honeydew as a so longev feedin which (1983) However anautogen most case are unava Ma mosquito vari bod 54

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contain nutritional reserv es that are mu ch eir counterparts reared in a laboratory under optimal cond ined in the laboratory solely on sucrose can attain high levels of lipid an d glycogen deposition (OMeara 1987; s eas t and potential vecto studie ker er smaller than th itions (Day and Van Handel 1986). Adult mosquitoes mainta Day and Van Handel 1986), with levels typi cally greater than fi eld caught individuals (Day and Van Handel 1986). Culex quinquefasciatus are the primary vector of Wuchereria bancrofti in Asia, Africa, the Western Pacific and sporadically in the Americas (Samuel et al. 2004). Lymphatic filariasis is caused by W. bancrofti and is a debilitating disease associated with a large number of death each year. It may even be a contributing factor in maintaining poor economic conditions in ar where it is endemic (WHO 1994). In the United States, Cx. quinquefasciatus is also a known vector of St. Louis encephalitis virus (SLE) (Jones et al. 2002) and a competen r of West Nile virus (WNV) (Sardelis et al. 2001, Molaei et al. 2007) in the southeastern United States. The ability of these mosquitoes to vector such diseases relies heavily on their length of life, potential to feed on multiple hosts, and successful incubation of the various pathogens. Amino acids in nectar could be an important source of energy and survival with potential to optimize the mosquitos ability to survive and, thus, to vector disease more effectively. Levels of amino acids in nectar have been us ed to broadly classify flowers into different groups based on the preferences of their potential pollinators ( e.g. butterfly flowers, bee flowers) (Baker and Baker 1973). Butterfly flowers contain high levels of amino acids, and have been d as a potential critical source for proteins contributing to repr oduction (Baker and Ba 1973). Common lantana ( Lantana camara L. (Verbenaceae)) is an ex ample of a butterfly flow that contains a high amino acid concentration ( 16 moles/ml) as well as the sugars, fructose, 55

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glucose and sucrose (Alm et al. 1990). Lantana camara is a widely used orname ntal in many temperate regions (Morton 1994), including Florid a and extending throughout the southeaste United States. Nectar mimics of this species have been used in various feeding preference studies with butterflies (Mev i-Shutz and Erhardt 2003b, Alm et al. 1990) and are a known attractant for the dipteran vect o rn r, tsetse fly, in Africa (Syed and Guerin 2004). Additionally, Haeg ns s dd were when fed an adult diet containing amino acids (Mev i-Shutz and Erhardt 2005). For this e In er (unpublished data, cited in Nayar 1982) has observed Cx. nigripalpus feeding on Lantana in the field, so a critical question is whet her mosquitoes feeding on nectar amino acids such as those present in Lantana nectar will also display enhanced survival. The objective of this study was to evaluate the effect of ami no acids in nectar on survival of adult mosquitoes. To determine if amino acids alone enhanced longevity, the mosquitoes were not blood fed at any time. In the field, mo squito larvae may experience stressful conditio such as overcrowding or poorly nutritioned diets. Rearing inse cts on high and low food diets a larvae mimic conditions mosquitoes may encounte r in the laboratory or nature. Day and Van Handel (1986) determined that individuals re ared in the laboratory, presumably under high nutrient conditions, contained increa sed levels of glycogen and lipid over those that were fiel caught. Adults of the map butterfly that were fed poor diets as larvae ha d longer lives an more fecund xperiment, glycogen and lipid analys es were performed on unfed newly emerged mosquitoes to illustrate differences between deposition of glycogen and lipid in larvae reared on low and high food larval diets. Additionally, measures of winglength and dry weights were obtained to compare body size between rearing regimes. Previous experiments have shown that females outlive males (Liles and DeLong 1960, Briegel and Kaiser 1973) and that a water diet only supports survival for a few days (Nayar 1986, Nayar and Sauerman 1971a, 1971b, 1975) 56

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our study, fema les and males from both larval diets were compared separately and treatments were compared as without sugar (water on ly, water + amino acids) or with sugar ( L. camara nectar mimic only, L. camara nectar mimic + amino acids). Materials and Methods Mosquito Rearing Culex quinquefasciatus were reared at the USDA, CMAVE facility in Gainesville, F Larvae were reared in plasti c trays (35.5cm x 48.3cm x 6.4cm) with 2.5 liters of water. Approximately 5 egg rafts were set in each pan, resulting in about 500 larvae. L. The colony was establ n s e number subsample of 10 males and 10 females were removed from cages and frozen (-20 C) for later ished from a Gainesville light trap coll ection in 1995. The protocols of Telang and Wells (2004) were followed to differentiate between th e negative effects of crowding and to focus o the effect of larval nutrition. Keeping larval density consistent and feeding some larvae more than others allowed us to test specifically for effect of nutrition on la rvae and examine those effects on size and nutritional state of adults. When rearing larvae on a high food diet, food wa provided as a slurry of 50 ml of a 3:2 bovine liver/brewers yeast powder (MP Biomedicals, Solon, OH) (added as 40 g to 1.0 L of water) on day 1 and 50 ml of finely ground 2% hog chow slurry (Purina Mills, Gray Summ it, MO) thereafter on days 3, 4 a nd 5 (Table 4-1). When rearing larvae on a low food diet, food was provided as a slurry of 50 ml of a 3:2 bovine liver/brewers yeast powder on day 1 and 25 ml of 2% hog chow slurry on day 4 only (Table 4-1). Th of days from egg to pupation for the larvae reared on low food diets was equal to that of the larvae reared on high food diets. Winglength and Dry Weight Measurements To evaluate the effect of rearing regimes on the size of resulting adults, we quantified winglength and dry weight. Upon pupal emergen ce, with no prior e xposure to sugar, a 57

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measure ment of winglengths a nd dry weights. Wings were removed, mounted, viewed under a microscope and photographed using ScopePhoto 1.0 (Scopetek, Hangzhou). Wings were measured from the alular notch to the distal end of the R gvein, fringehairs excluded (Figure 4-1) (Sigma Scan Pro 5.0 (SPSS, Inc are was calibrated with a 5.0 mm g measuremen ts based on that calibration. hese ) s for e homoge nized in microcentrifuge tubes with 100l of 0 l ultrapure wa ter and 500 l of 1:1 chloro h glass 2 win ., Chicago, IL). The softw steel pin and the win To measure dry weights, groups of 10 male and female mosquitoes from each larval feeding regime were frozen at -20 C prior to being set up in the longevity experiment. T mosquitoes were stored at -20 C until they were moved to -80C in preparation for freezedrying. The groups of mosquitoes were freezedried for 48 hours and dry weights were obtained with a microbalance (Sartorius Germany). The mosquitoes were stored again at -20 C until used for lipid and glycogen analyses. Glycogen and Lipid Analyses Initial nutritional reserves of the mosquitoes at the time of pupal emergence were determined by measuring the levels of glycogen a nd triglyceride. The hot anthrone (glycogen (Van Handel 1985) and sulphosphovanillin (lip id) (Van Handel 1985, as modified by Hahn 2005) assays were conducted following the methods provided in detail in the Appendix. Five replicates were completed for each sex and larval rearing regime. All of the samples that were previously fr eeze dried, weighed and refrozen were analyzed for glycogen and lipid using the hot anthrone assa y for glycogen or sulphosphovanillin assay lipids. The dried mosquitoes wer saturated sodium sulfate, 200 l of metha nol, 10 form: methanol with a motorized homogenizer. The glycogen and lipid samples were measured using the methods of Van Handel (1985) Lipid solutions were washed throug 58

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pipette columns of 0.2 g silicic aci d and rinsed with 4 m L of chloroform to extract only neutral lipids (Hahn 2005). Mating Assay The association of male and female mosquitoes has been reported to shorten the life of males and lengthen the life of female mosquitoes beyond that of females r eared alone. Liles and DeLo ently he mosquitoes lived long enough to mate. The setup of he longevity assays (described in more detail later), in which the mosq le and leted e 20C until dissection. Upon dissec ontainers where a 1.5 ml microcentrifuge tube could be in serted. The tube was filled with 1.0 ml of ng (1960) reported that mated parous female Aedes aegypti lived as long as unmated nulliparous females, indicating a possible benefit in longevity of females from being mated. Additionally, larger male An. gambiae have been reported to compete for mates more effici than smaller males (Nghabi et al. 2005). Theref ore, it is important to determine whether mating occurred successfully under th e experimental conditions. We hypothesized that mating would occur under our conditions, provided t this assay was similar to that of t uitoes were maintained on a 5% sucrose solution via a cotton wick. Thirty-five ma female mosquitoes were placed in the containe rs and maintained in an incubator at 28.0 1.0C 81.2 0.1% relative humidity and 14:10 light: dark photoperiod. Five replicates were comp for each larval rearing regime. The tests were conducted for fifteen days, to ensure enough tim to complete mating. The mosquitoes were removed and frozen at tion and examination of the spermathaeca unde r a microscope, the number of females that contained sperm and those that did not were counted. Amino Acid Longevity Assay Upon adult emergence, 35 males and 35 females were placed in 0.47 liter paper c (Solo Cup Company, Highland Park, IL). The lid s were modified with white tulle to allow viewing inside the cages. The cages were also modified by cutting an access hole in the side 59

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treatment solution and a cotton dent al wick previously saturated with the sam e solution (3.8 cm) (Richmond Dental, Charlotte, NC). Th is allowed the mosquitoes to feed ad libitum on the d. Treatments included a Lantana camara sugar mixture mimic only, Lantana cama were solution provide ra mimic with amino acids added, amino acids in water and a water only control. The nectar mimic contained 0.547 M sucrose, 0.282 M D-(+)-glucose, and 0.316 M D-(+)-fructose (Sigma, St. Louis, MO). The nectar mimic with amino acids additionally contained the nonessential amino acids L-alanine (0.718 mM), L-asparagine (0.421 mM), L-glutamic acid (0.326 mM), L-glutamine (0.931 mM), glycine ( 2.371 mM), L-proline (2.23 mM), and L-serine (1.37 mM) and the essential amino acids L-ar ginine (0.201 mM), L-threonine (0.672 mM), Ltyrosine (0.221 mM), and L-valin e (0.137 mM) (Sigma, St. Louise, MO ). This particular nectar mimic mixture has been used in previous studie s evaluating effects of am ino acids on fitness of various species of butterf lies (Alm et al. 1990, Mevi-S hutz and Erhardt 2005). Lantana camara is also a known nectar source of the tset se fly in Africa (Syed and Guerin 2004) and Culex nigripalpus in the U.S. (Haegar; in Nayar 1982), and is widely abundant as an ornamental and naturalized plant in many areas of the Southeastern United St ates, including Florida (Morton 1994). Ten replicates of each treatment and five re plicates of the water c ontrol were completed. Mosquitoes were maintained at 28.12 1.0 C, 81.2 0.1% relative humidity and 14:10 day: night photoperiod. The number of dead males a nd females were counted daily and the day of 50% mortality determined. Statistical Analyses All statistical analyses were conducted usi ng Minitab 15.1 (Minitab, Inc., State College, PA) or JMP (SAS Inc., Cary, NC). Assumptions of normality and homogeneity of variance confirmed using graphical representations of the data and a Levenes test ( P <0.05). 60

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One-way ANOVAs were used to determine di ffe rences in winglength and dry weight among females or males from each larval reari ng regime. Mean triglyceride and glycogen weights and their standard errors were obtaine d for each group of 10 mosquitoes. Nutritional reserve weights were calculated by dividing glycogen or lipid we ights by the dry weights of t mosquitoes in order to correct for weight diffe rences. Data for glycogen analyses were Ln transformed as necessary. A one-way ANOVA was used to determine differences in the lipid or glycogen weights, and Tukeys HSD ( P <0.05) was used to locate differences between the means. Previous experiments have shown that there ar e differences in the survival of males and females (Liles and DeLong 1960, Briegel and Kaiser 1973) and that providing a water only treatment will not support surviv al beyond a few days, whereas trea tments containing sugar will likely increase survival e xponentially beyond that (Nayar and Sauerman 1971a, 1971b, Nayar 1986). Therefore, days to 50% mortality for each replicate were dete rmined for males and females separately and treatme nts without sugar (water only and water + amino acids) and treatments with sugar ( Lantana camara nectar mimic and L. camara nectar mimic + amino acids) separately and used to build 4 multivariable ANOVA models containing all explanatory variables (adult diet, larval diet a nd their interaction). Tukeys HSD (P <0.05) or two-sample tests ( P <0.05) were used to separate the means. he mean tResults ition on Adult Size, Nutrient Reserves and Mating A ared on high food diets than adults than from larvae rear ed on low food diets (Table 4-2). Feeding a high Effect of Larval Nutr dult males and females had significantly larger winglengths ( t= 6.90, df=15, P <0.001; t= 8.37, df=12, P <0.001, respectively) (Table 4-2, Figure 4-2) and greater body mass ( t =14.02, df=4, P <0.001; t =27.77, df=6, P< 0.001, respectively) (Table 4-2, Figure 4-2) when re 61

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food di r arly lycogen and 3 tim es more triglycerides when fed a high food diet as larvae as oppose y most et y c, 4-4d). Culex quinquefasciatus fed treatments wit t achieved by treatm et resulted in adults that weighed nearly 2 times more in males and were 2.6 times large in females. High food diets as larvae result ed in larger glycogen and lip id reserves in both males (t=6.35, df=4, P=0.003; t=20.64, df=5, P<0.001, respectively) and females (t=16.73, df=4, P<0.001; t=26.24, df=7, P<0.001, respectively) (Table 4-3, Figure 4-2). Male s contained ne 9 times more glycogen and 4 times more trigly cerides when fed a high food diet than males reared on a low food diet. Females showed a similar trend, accumulating approximately 13 times more g d to a low food diet. Adult females that were fed high food and low food diets as larvae were nearly all mated (98% and 96%, respectively). Survival Analyses Culex quinquefasciatus fed treatments without sugar Adult diets lacking sugar, either water only or water + amino acids did not sufficientl support survival, with all individual living less than 5 days (Table 4-5). Larval diet had the significant effect on survival; males and female s resulting from larvae reared on a high food larval diet lived significantly l onger than those adults from larvae reared on a low food larval di (Table 4-6) (Figures 4-3, 4-4a, 4-4b). Amino acids added to the water did not increase longevit of males or females (Table 4-6) (Figures 4-3, 4-4 h sugar The addition of sugar to the adult diet in creased survival far beyond tha ents without sugar for both males and female s, with all adults living >25 days or 5 times longer than individuals not receiv ing sugar (Table 4-5) (Figure 43). The two sexes differed in the effects of larval diet on adult longevity (Table 4-6) (Figures 4-5a, 4-5b). Adult males fed sugar lived longer when fed a low food diet as la rvae (Figure 4-5a). Females that were fed a 62

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high food diet as larvae continue d to live longer than those that were fed a low food diet (Figure 4-5b). Adding amino acids to the adult diet of males did not increase longevity (Table 4-5, Figur ining les tly f female Cx. quinquefasciatus significantly er females fed only a L. camara nectar mimic when larval diets were n fed an adult diet containing amino acids, om n ingestion of amino acids and evi-Shutz and Erhardt 2003a, Molleman 2008) at an e 4-5c), but there was a sli ght increase in longevity when fe males were fed a diet conta amino acids (Table 4-5, Figure 4-5d). The intera ction between adult and la rval diet of fema revealed that those fed a low food diet as larv ae benefitted from the addition of amino acids to their adult diet, but females from larvae fed a high food diet did not. They were able to sligh overcome a poor larval diet and live as long as fe males that were fed a high food larval diet (Figure 4-5d). Discussion The addition of amino acids in to the adult diet o increased lifespan ov pooled, but had no effect on male survival. Fema les, whe overcame their poor larval diet and survival was equivalent to those females resulting fr optimally fed larvae. Although the increase in surviv al is small, it is statistically significant and is supported by similar research with Ae. aegypti fed pollen in their adult diet (Eischen and Foster 1983) and the map butterfly fed amino acids in their adult diets (Mevi-Shutz and Erhardt 2005). Beck (2007) indicated that longevity wa s increased only in some species of male butterflies, whereas other authors did not find a link betwee increased fitness in insects (Liles and De Long 1960, M Considering the research conducted on the map butterfly a nd our study, it appears th important factor that has been overlooked is the effect that am ino acids in the adult diet may have on survival of adults that were nutriti onally challenged as larvae Larval insects are exposed to many challenges in nature, including overcrowding and inadequate nutrition, with larval nutritional deprivation common in mosquitoes (Day and Van Handel 1986). Preference 63

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for adult diets containing amino acids com pared to those without has also been reported with butterflies (Alm et al. 1 990) with stronger effects reported in butterflies nutr itionally deprive larvae (Mevi-Shutz and Erhardt 200 3b). Examining the effects of the addition of amino acids to the adult diet of laboratory-reared mosquitoes may mask a resulting increase in longevity if we consider that optimally-reared mosquitoes may contain sufficient nutritional reserves acquired from the larval stage. There are differences re ported on the possible eff ect of amino acids on the adult diets of various insects (Mevi-Shutz and Erhardt 2005, 2003a, Eischen and Foster 1983 and even amon d as ) g species living under similar conditi ons (Beck 2007). Thus, it is possible that the role of amino acids in nectar may differ be ito species and should be taken into consi dy d diet re tw een mosqu deration. The effect of a larval diet on adult longevity was the most significant factor in this stu for both sexes. Male and female Cx. quinquefasciatus both emerged with larger winglengths, dry weights and glycogen and lipid reserves when reared on a high food diet. In studies that have used laboratory-reared mosquitoes, fema les exposed to high food resources likely have greater glycogen and lipid reserv es for flight and increased su rvival and would more closely resemble those fed high food diets in this st udy, in contrast to fiel d-caught individuals. However, unlike females, males in our study live d significantly longer when fed a low foo as larvae. The association of males and female s has been shown to positively affect female longevity and negatively impact survival of ma les (Liles and DeLong 1960). Nearly all of the females in this study were mated, which we think may play a role in their increased longevity. However, males seem to suffer from their associa tion with females, particularly those that we reared on a high food diet as la rvae. It has been shown that males that were larger upon emergence, and fed high food diets as larvae, were more likely to be better competitors and to 64

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mate f irst than those males that were smaller as adults due to nutritiona l challenges as larvae (Nghabi et al. 2005). Additionally, increased mating occurrences reduced survival of male Saltella sphondylli (Martin and Hosken 2004) and may explain why the more sexually competitive, male Cx. quinquefasciatus reared on a high food larval diet in our study survived less time than adults from thei r low food reared counterparts. It is important to understand how larval and ad ult nutrition affect longevity, flight potential (Nayar and Sauerman 1971a, 1971b, 1975), biti ng persistence (Nasci 1991) and, most importantly, disease transmission. Arboviruses transmitted by these Culex species have lengthy amplification periods th at require long lifespans (Turell et al. 2005). The survival of mosqui is highly depen toes dent on their abil ity to locate and feed on nectar honeydew or other sources of carbo f hich is ir al hydrates and amino acids. We found that tene ral reserves are an im portant predictor of adult life span in male and female Cx. quinquefasciatus. Similar to the findings of Mevi-Shutz and Erhardt (2005), amino acids adde d to the adult diet contributed to the enhanced survival o females reared on poor larval diet regimes. Th ese females were able to survive as long as females resulting from a high food larval diet. Not only are substant ial teneral reserves important for increased longevity, but also they ar e crucial for immediate flight energy w essential for location of carbohydrate sources when teneral reserves are depleted. There is evidence that some mosquitoes will not even be gin host-seeking until they have obtained the first sugar meal (Foster 1995) a nd that adults with low nutritiona l reserves are better vectors of WNV due to their reduced ability to avoid infection compared to adults with high nutritional reserves (Vaidyanathan et al. 2008). Becau se amino acids are the second most abundant component of nectar, and mosqu itoes feed on nectar frequently to increase their nutrition reserves, future studies may reveal additional roles that amino acids play in the diet of females. 65

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Future research ma y examine the effect of larv al nutrition on the establishment and transmissi of pathogenic agents, and the mitigat ion of this by adults feeding on diets rich in amino acids on 66

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67 Larval food = 3% bovine liver powder (LP) : 2% Brewers yeast (BY) (30 g bovine ground hog chow in 1800 ml of water). Days After Hatch Amount of food given per diet level (ml) Table 4-1. Schedule of feeding for Cx. quinquefasciatus under a high or low food regime. liver powder and 20 g Brewers yeast in 1L of water), 2% hogchow (36 g finely High Low 1 50 ml; 3% LP:2% BY 50 ml; 3% LP:2%BY 3 50 ml; 2% hogchow 4 50 ml; 2% hogchow 25 ml; 2% hogchow 5 50 ml; 2% hogchow Table 4-2. Mean winglength (N=5) and dry weight (N=10) measurements for adult Cx. quinquefasciatus males and females reared on high or low food diets as larvae. Winglength (mm) Dry Weight (mg) Males Females Males Females Low food 2.41 0.04a 2.64 0.06a 2.70 0.05a 3.49 0.18a High food 2.74 0.03b 3.16 0.03b 5.07 0.16b 9.11 0.10b Means within columns with a similar le tter are not significantly different ( t -test, P <0.05). Table 4-3. Mean glycogen and lipid content (N=5) measurements for adult Cx. quinquefasciatus males and females reared on high or low food diets as larvae. Glycogen ( g/mg) Lipid ( g/mg) Males Females Males Females Low food 9.38 1.67a 7.15 0.56a 42.64 2.83a 33.84 3.71a High food 82.61 9.70b 96.44 5.12b 113.31 1.43b 108.37 2.41b Means within columns with a similar letter are not significantly different (t-test, P <0.05). Table 4-4. Percent mated female Cx. quinquefasciatus when reared on high or low food diets as larvae and maintained under conditions of survival assay. Larval Feeding Regime N Percent Mated Females High food N=55 98% Low food N=50 96%

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68 Table 4-5. 0l( mand female uetu d hig larvae. s e no sugts (water on water + amand sugartments (Lantana cama ectar ara mi ino acids). Adult Die sugar val diet et 50% Mortality (SE) (LS Means)a Cx ino qu aci Day inq ds) s to fas cia s fe trea Da Ad mi t wi ys t ult mic th o 5 diet and % m wer L. cam Lar orta ity L S ar tr mi A Me eat c + dul ans men am t di SE) of ale l y, h or low food diets as ra n mui N N No osq of toes No. of osquitoes m No w nl .20 0.14 175 3.00 0.12 5 Lo Water o y 2 5 175 No amino .33 0.13 210 3.00 0.11 6 No h ater only .00 0.105 4.80 0.12 5 No ater + amin cids .00 0.1410 4.90 0.09 10 ra .00 1.04 32.7 7 Yes ra mimic amino acids .00 1.12 280 35.2 8 camara nectar .33 0.9910 350 36.70 0.82 10 Yes ra ic acids .60 0.94 038.00 0.82 10 Hig Yes Low Yes High W a W W a L m L + L m L + at cid er + s 2 5 5 30 32 27 27 6 210 175 350 245 280 350 350 175 350 45 o tar 7 8 1 5 ca im ca ma ic ma nec 2 0.9 0.9 8 2 im ca am ic ma ino mim 10 35 a full m d iet, lar and their ). odels (adult All LS Means derived from val diet interaction

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Table 4-6. Results of ANOVA on the effect of larv al diet, adult diet a nd their inte raction on survival of m Cx. quinquefasciatus e nt Sex u f F P ale or fe male So Treatm rce d No sug Males ar Whole model 2 251.43 <0.001* No sug Sugar Sugar Larval diet 1 492.37 <0.001* dult die 0.23 0.634 ror 3 ta 5 aFe hole m 148.40 <0.001* Larval diet 1 289.98 <0.001* Adult diet 1 0.25 0.625 ror 3 tal 5 Males Whole model 2 4 0.067* Larval diet 1 3 0.002* ult die 0.478 ror 1 t 3 Fe hole m 0.001* Larval di 3 0.001* ult die 0.044* ror tal A Er To W t odel 1 2 2 2 l r males Er To 2 2 52.4 11.3 0.51 9.16 14.4 4.40 Ad Er To W t odel 1 3 3 2 al males et t 1 1 31 34 Ad Er To Indicates a signifi ). cant effect ( P <0.05 69

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70 A1 R2 Figure 4-1. Photraph of mosquito wing with arrndicating ments taken from the alularotch (A1) to th l end of wiein R2 and used to calculate mean wing lengths. og ow i easurem n e dista ng v

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Measures of body size WinglengthDry WeightMeans (mm or ug/mg) 0 4 6 8 10Low food males Low food females2 High food males High food femalesa b c d a b c d Nutritional measures Glycogen Lipid Weight (ug/mg)0 20 40 60 80 100 120 140 a a b b a a b ba) b) Figure 4-2. Mean winglength (mm), dry weight (mg) (a) and glycogen and lipid weights (g/mg) (b) of male and female Cx. quinquefasciatus Treatments with similar letters are not significantly different (Tukeys HSD, P <0.05). 71

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Adult Diets without Sugar Males Females 72 Daysto50%Mortality(SE) 0 10 20 30 40 50Water only low food Water + aa low food Water onlylow food Water + aa high food b b a a b b a a Adult Diets with Sugar L. camara mimic onlylow food L. camara mimic + aa high food L. camara mimic onlylow food L. camara mimic + aa high food Males Femalesa ab b b a ab b b Figure 4-3. Days to 50% mortality of male s and females fed low or high food diets as larvae and diets with or without sugar as adults. Letters within a sex and adult di et` that are similar are not sign ificantly different (Tukeys HSD, P <0.05).

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Larval Feeding Regime 73 High food Low food 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 High food Low foodDays to 50% Mortality (SE)2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 a) Amino Acid OnlyWater OnlyAdult Feeding Regime Amino Acid OnlyWater Only 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 c)a a a a a bd)a bb) Figure 4-4. The effect of larval diet on days to 50% mortality (LS Means) of males (a) and females (b) and adult diet on males (c) and females (d) fed treatments without sugar. Treatments with similar letters are not significantly different ( ttests, P <0.05).

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Larval Feeding Regime High food Low food 20 25 30 35 40 45 High food Low foodDays to 50% Mortality (SE) 20 25 30 35 40 45 Adult Feeding Regime Sugar Mix + AA Sugar Mix 20 25 30 35 40 45 High food Low food a ba) b) d)a b Sugar Mix + AASugar Mix Only 20 25 30 35 40 45 a ac)a a b ab 74 Figure 4-5. The effect of larval diet on days to 50% mortality (LS Means) of males (a) and females (b) and adult diet on males (c) and females (d) fed treatments with sugar. Treatments with similar letters are not significantly different ( ttests or Tukeys HSD, P <0.05).

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CHAPTER 5 CONCLUSI ONS AND FUTURE RESEARCH Sugar feeding is an important behavior of Culex mosquitoes resulting in acquisitio carbohydrates and amino acids, which increase nutriti onal stores after emer gence and potentially enhance longevity and fecundity (Mevi-Shutz an d Erhardt 2005, Beck 2007). The poten disperse, host seek and become deadly disease vectors increases with s ugar feeding (Nayar and Sauerman 1971b). Without sugar feeding, all species in these studies died within 4 days, which corresponds with previous studies where various mosquito species fed only water died r (Nayar 1982, Galun and Fraenkel 1957, Gary and Foster 2004). Previous studies in the laboratory focusing on longevity of mosquitoes are numerous, however, most of them h focused on An. gambiae, Ae. aegypti or Ae. taeniorhynchus (Galun and Fraenkel 1957, Eischen and Foster 1983, Nayar and Sauerman 1971a 1971b, 1974, 1975, Briegel et al. 2001, Liles and DeLong 1960) with only some including Culex species (Briegel and Kaiser 1973, Nayar Sauerman 1973, Nayar 1986, Nayar a nd Pierce 1977). Few studies examined survival o and females (Liles and DeLong 1960, Briegel and Kaiser 1973, Nayar 1986) and only Galun and Fraenkel (1957) have fed mosquitoes various sugars that appear in all types of natural conditions, including nectars, fr uits and honeydew. Culex mosquitoes transmit West Nile virus (WNV) (Sardelis et al. 2001, Molaei e 2004), St. Louis Encephalitis virus (SLE) (Dow et al. 1964, Day 1997, Jones et al. 2002, Foster and Walker 2002), Eastern Equine Encephalitis virus (EEE) (Wellings et al. 1972, Scott Weaver 1989) lymphatic filari asis (LF) (Subra 1981, Hawking 1973, Chow 1973, Nelso 1962, Dossou-yovo 1995), dog heartworm (Villavaso and Steelman 1970) and many mo Because they are important disease vectors in Florida and throughout the world, understanding their biology and physiology is important to pr event disease outbreaks by working to improve n of tial to apidly ave and f males t al. and n et al. re. 75

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population monitoring or more effe ctive b Because sugar feeding involves both males and females and is critical for increasing energy reserves which make possible, dispersal, increased survival and vectoring capability, understandi ng this behavior and how it may affect the different sexes and species is important. Longevity and Sugar Feeding Often overlooked in favor of research on e ffects of blood feeding, sugar feeding is an important activity for male and female mosqu itoes. Feeding on carbohydrate sources increases longevity far beyond what would be possible w ithout obtaining the addi tional carbohydrates provided by nectar, rotting fruit, honeydew and ot her identified sources. The main components of nectar are sucrose, glucose and fructose, which also are found in honeydew along with unique sugars such as melezitose, raffi nose and trehalose. Sorbose is also a naturally occurring sugar (US Government 2008), which we used in these studies and has been previously found to be unsupportive of life in mosquitoes and ot her dipterans (Galun and Fraenkel 1957). These studies focused on the effect that sugar feeding had on the l ongevity of male and female Culex nigripalpus, Culex quinquefasciatus and Culex salinarius. Survival was variable depending on species and adult sugar diet; however, some overall trends occurred. Sugars that are commonly found in nectar and honeydew (sucro se, fructose and glucos e) supported long-life, in males and females of all three species. A dditionally, sugars that are exclusively found in honeydew (melezitose and raffinose) were as su pportive as nectar sugars commonly thought to support the greatest life sp an in these mosquitoes. Other sugars such as mannose and trehalose supported moderate to poor survival when comp ared to highly supportive sugars. Sorbose was overall a very poor sugar that did not increase survival over that of the water control. In some cases, survival was less than water, although ther e was not clear evidence that this sugar was toxic, only very poorly utilized. aiting sy stems 76

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Nutritional Reserves and Mating Status Teneral nutr itional reserves are a result of larv al diet and larval rearing conditions (Telang and Wells 2004). A crowded larval environment or poor nutritional conditions result in adults that have small body sizes, low nutritional reserv es and shorter lifespans (Briegel et al. 2001, McCann 2006). Feeding on carb nu tritional reserves, such as glyco r ng adult emerg e it seemed critical to determ ine whether the assay conditions ohydrat es as adults can increase gen and lipid. Glycogen is stored in the fat body and flight muscles and can be used fo immediate flight energy (Nayar and Sauerman 1971a, 1971b). Lipids are important because they are used for long-term energy, maintenance a nd survival (Foster 1995, Nayar 1982). Increasi these reserves is critical to the survival and f light potential of a mosqu ito and its potential to transmit disease. These studies determined that larval diet play ed a large role in survival of both male and female Cx. quinquefasciatus. Mosquitoes that were fed a larv al diet that consisted of lower amounts of food had significan tly smaller body sizes, less glycogen and lipid upon ence and lived significantly less time than a dults that were fed a high food larval diet. Because mosquitoes that have been collected from the field have been found to be smaller and have lower nutritional reserves than those that were reared under assumedly more optimal laboratory larval condit ions (Day and Van Handel 1986), we c onclude that these results more closely represent natural conditions. Mating has also been reported to have an effect on survival. Liles and DeLong (1960) discovered that females lived longer when housed in proximity with males. Males maintained with females lived a shorter amount of time, indicating that females benefitted from their association with males. Mosquitoes in the longevity assays of our st udies were housed in close proximity and du to the potential effect on longevi ty 77

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supported natural behavior. o exam ined by determ on dies have also examined the af fect of amino acids on them (Eischen and Foster 1983) schen dult ile d a poor diet as larvae, the leng th of survival was only increa dt we believe that the benefit of amino acids in the adult diet may be mask The effect of larval diet on mating was als ining whether females fed a low or high food diet as larvae contained sperm in their spermathaeca. We found that ne arly all of the females in our assay were mated, indicating no negative effect of assay e nvironment or larval diet. Amino Acids in Nectar Sources Amino acids are the second most abundant component of nectar and are also present in honeydew (Baker and Baker 1973). Their presence in nectar has elicited much interest in the effect they have on the insects that feed on nect ar. Generally, research has focused on butterflies (Mevi-Shutz and Erhardt 2003a, 2003b, 2005, Alm et al 1990), but because mosquitoes feed nectar as well, stu Mevi-Shutz and Erhardt (200 5) discovered that when fed a poor diet as larvae, the map butterfly lived longer an d was more fecund when fed an adult diet containing amino acids. Similarly, Aedes aegypti survived longer and fecundity was in creased when fed pollen (Ei and Foster 1983). We determined that there was an effect of amin o acids in the adult diet on the survival of Culex quinquefasciatus females. Females that were fed a low food larval diet and had an a diet with amino acids added lived as long as females that were fe d a high food larval diet. Wh this effect was true if females were fe sed by 2 days and further research of this topic to ensu re the effect is biologically significant is suggested. Various studies have not found any connection between feeding on a sugar diet with the addition of amino acids a nd increased longevity (M evi-Shutz and Erhar 2003a, 2003b), however, ed by studies using optimally-f ed laboratory reared insects. 78

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Future Directions Studies focused on longevity of m osquitoes will continue to be important because of t association between survival of vectors and dise ase transmission. We found that differences in survival between species exist depending on ad ult d he iet. Because each of the species we examined have such different li ita ts, seasonal distribution, sex-d cts fe-histories (e .g. varying larval hab ifferentiated distribution), it may be increasi ngly important to further understand the effe of different adult diets on indivi dual species. Increased knowledge of the effects of sugars that did not support long life, such as mannose, treh alose and especially so rbose could lead to advances in the development of attracticides. Sorbose has been found to be attractive to and readily imbibed by the blow fly (Fraenkel 1940), but it will be important to examine the phagostimulatory effects this and other poorly supportive sugars have on mosquitoes and how that will direct those used as attracticides. 79

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APPENDIX PROTOCOL FOR S ULPHOSPHOVANI LLIN AND HOT ANTHRONE ASSAYS Mosquito Dry Weights For dry weight analysis, previously frozen gr oups of ten males or ten females were frozen further at -80 C for one hour. The caps of the microcentrifuge tubes were opened and the samples were freeze dried for approximately 48 hours to ensure that all moisture was removed. Immediately after freeze drying, the samples were placed in a dessicator to avoid absorption of moisture in the air. The samples were weighe d on a microbalance (Sartorius, Germany) and dry weights for the 10 mosquitoes in each sample we re recorded for later use in the nutritional analyses. After weighing, the samples were again stored at -20 C for later use. Preparation for Glycoge n and Lipid Analyses Nutritional reserves at the time of eclosion were determined by measuring the levels of sulphosphovanillin (lipid ) (Van Handel 1965a, 1985, as modified by Hahn 2005) assays were conducted. To prepare for these assays, 100 l of saturated sodium sulfate was added to the microcentrifuge tube while homogenizing the mo squitoes with a motorized homogenizer. Pestles were changed between homogenization of samples. During homogenization, 200 l of methanol and 100 l ultrapure water were also added. When the mosquitoes were sufficiently ground into a solution, 500 l of 1:1 chloroform: methanol was added. This solution was then decanted into a 20 ml glass centrifuge tube labele d with the sample number and the name of the test (glycogen). The remnants of the 1.5 ml micr ocentrifuge tube were then rinsed with another 500 l of 1:1 chloroform: methanol, which was adde d to the same glass centrifuge tube with the first portion of the sample. This process was pe rformed for each sample. The contents of the glass centrifuge tubes were placed in the centrifuge and run at 2500 rpm for 5 minutes. The glycogen and triglyceride. The hot anthrone (glycogen) (Van Handel 1965a) and 80

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crushed insect remnants and the glycogen d a sm all pellet on the bottom of the tube and the supernatant, or lipid por tion, was then transferred to a s econd 20 ml glass centrifuge tube labeled with the sam ensure removal of all lipids. The extraction wa s repeated by adding 1000 l of 1:1 chloroform: samples were returned to the centrifuge for 5 minutes and afterward the lipid portion was assay. The tubes labeled glycogen now contain the precipitated glycogen and some other sugars. These sugars were removed by adding 1000 l of 66% sulfate in water. The pelle ples were then te assays to conduct, a glycogen assay and a lipid assay. Prior to beginning the glycogen assay, anthrone reagent and a glucose standard were ma de. The reagent was 150 mg anthrone added to water). The glucose standard was used in pl ace of glycogen and was a 1.0 g/l solution (50 The samples were redissolved in 2.0 ml ultrapure water by stirring, and allowed to partially settle. Aliquots of the redissolved glycogen (100 l) from each sample were pipetted into two interpretation of the results. The same wa s done with glucose, using 0, 5, 10, 25, 50, 100 and portion forme ple number and the type of assay (lipid). The pellet was then washed to methanol. The glycogen portion was resuspe nded into solution by vortexing. Again, the decanted into the same tube with the previous portion. The completed lipid extract was placed in the freezer for later use in the sulphosphovanillin ethanol saturated with sodium t was resuspended into soluti on, centrifuged for 5 minutes and the supernatant discarded. This process was repe ated to remove all excess sugars. Glycogen sam stored for later use in the hot anthrone assay. Glycogen Analysis (Hot Anthrone Assay) After fractionating the glycogen portion from the lipid portion, there were two separa 100 ml of sulfuric acid /ultrapure water solution (358 ml sulfuric acid and 141.5 ml ultrapure mg/ml ultrapure water). separate tubes (A and B). Duplicating each sample and taking the mean, allowed proper 81

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125l as standards. To each tube, 2.4 m l of anthrone reagent was added and the tubes were heated to 90 C in a water bath for 17 minutes. After addition of the reagent, the tubes were covered to protect from light reactions. After he ating, the tubes were plac ed in an ice bath an kept in the dark for 2 minutes. The samples were vortexed and 125 l of each sample and its duplicate pipetted into a 96-well microtiter plate. The plate was placed in the spectrophoto (model, company, city, state)) and ab sorbance determined at 625 nm. Lipid Analysis (Sulphosphov d meter anillin Assay) triglycerides, while excluding charged cell membrane lipids. After th ly triglycerides and were ready to he 10 minutes in a 95 C water bath. They were held at room temperature to coo llow for Prior to the lipid analysis, several steps re quired completion. The samples were dried to complete dryness with nitrogen to remove all traces of methanol One milliliter of chloroform was added to each sample to resuspend the lipid. The samples were placed on previously made columns of 0.2 g silicic acid and glass wool. The n, the columns were washed with 4 washes of 1.0 ml of chloroform to separate the non-polar from the polar lipids. This is critical for examining the nectar lipid pool, which is <90% is separation, the lipi d samples contained sole for analysis. The total volume of each sa mple was measured and recorded. Aliquots of 200 l from each sample were pipetted into 100 ml glass centrifuge tubes; each was replicated reduce pipetting error. A 1.0 g /l triolein standard (50 mg/m l chloroform) was used as the control, using 0, 5, 10, 25, 50, 75, 100 and 125 l sta ndards. All samples and standards were dried under nitrogen comple tely and 200 l of sulfuric acid a dded to the tubes and vortexed. T samples were placed for l and develop for 5 minutes. A 2.5 ml aliquot of vanillin reagent (0.6 g vanillin, 100 ml ultrapure water and 400 ml of 85 % phosphoric acid) was added to each sample and standard. The samples were briefly vortexed and placed in a dark cabinet for 10 minutes, again to a 82

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83 e t a reaction to occur. Samples of 125 l were tr ansferred into a microtite r plate and absorbanc determined, as in the glycogen analysis, in a spectrophotometer, at 525 nm. Analysis Preparation Glycogen and lipid contents were measured as g of glycogen or lipid/ mg of dry weigh per group of 10 mosquitoes. The mean glycogen or lipid content was used in statistical analyses.

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LIST OF REFERENCE S Alm, J., T. Ohnmeiss, J. Lanza, and L.Vriesenga. 1990. Preference of cabbage white butterflies and honeybees for nectar that c ontains amino acids. Oecologia. 84: 53-57. Amino acids in nectar and thei r evolutionary significance. University of Texas Press, Austin, TX. Baker, H. G., and Baker, I. 1983a. A brief historical review of th e chemistry of floral nectar, pp. 126-152. In B. Bentley and T. Elias (eds .), The Biology of Nectars. Columbia University Press, New York, NY. Baker, H. G., and Baker, I. 1983b. Floral nectar sugar constitu ents in relation to pollinator type, pp. 117-141. In C. E. Jones and R. J. Little (eds.), Handbook of Experimental Pollination Biology Scientific and Academic Edition. Van Nostrand Reinhold, New York, NY. Baker, H. G., P.A. Opler, and I. Baker. 1978. A comparison of the ami no acid complements of floral and extrafloral nectars. Bot. Gaz. 139:322-332. Beck, J. 2007. The importance of amino acids in the a dult diet of male tropical rainforest butterflies. Oecol ogia. 151: 741-747. Bidlingmayer, W. L., and D. G. Hem. 1973. Sugar feeding by Florida mosquitoes. Mosq. News 33: 535-538. Briegel, H., M. Heft i, and E. DiMarco. 2002. Lipid me tabolism during sequential gonotrophic cycles in large and small Aedes aegypti J. Insect Physiol. 48: 547-554. Briegel, H., I. Knsel, and S. E. Timmerman. 2001. Aedes aegypti : size, reserves, survival and flight potential. J. Vector Ecol. 26: 21-31. Briegel, H., and C. Kaiser. 1973. Life-span of mosquitoes under laboratory conditions. Gerentologia 19: 240-249. Burkett, D. A. Baker, H. G., and I. Baker. 1973. Nature. 241: 543-545. Baker, H. G., and I. Baker. 1975. Nectar constitution and p ollin ator-plant coevolution, pp. 100140. In L. E. Gilbert and P. H. Ravens (eds.), Coevolution of animals and plants. D. A. Carlson and D. L. Kline 1998. Analysis of composition of sugar meals of wild mo squitoes by gas chromatography. J. Am. Mosq. Control Assoc. 14: 373-379. Burkett, D. A., D. L. Kline, and D. A. Carlson. 1999. Sugar meal com positions of five north central Florida mosquito species (Diptera: Culicidae) as determined by gas chromatography. J. Med. Entomol. 36: 462-467. Carpenter, S. J., and W. J. LaCasse. 1955. Mosquitoes of North America. University of California Press, Berkeley, CA. 84

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Eischen, F. A., and W. A. Foster. 1983. Life span and fecundity of adult fem ale Aedes aegypti (Diptera: Culicidae) fed aqueous extracts of pollen. Ann. Entomol. Soc. Am. 76: 661-663. Fadamiro, H. Y., L. Chen, E. O. Onagbola, and L. Graham. 2005. Lifespan and patterns of Feinsod, F. M., and A. Spielman. 1980. Nutrient-mediated juvenile hormone detection in Foster, W. A. 1995. Mosquito feeding and reproductive en ergetics. Ann. Rev. of Entomol. 40: Foster, W. A., and F. A. Eischen. 1984. Frequency of blood feeding in relation to sugar m. 80: 103-108. len and (eds.), Medical and Veterinary Entomology. Academic Press, San Diego, CA. Biol. Galun, R., and G. Fraenkel. 1957. Physiological effects of car bohydrates on the nutrition of a 111Gary, R. E., and W. A. Foster. 2004. Anopheles gambiae feeding and survival on honeydew Grimstad, P. R., and G. R. DeFoliart. 1974. Nectar sources of Wisconsin mosquitoes. J. Med. Gooding, R. H. 1975. Digestive enzymes and their control in hematophagous arthropods. Acta Trop. 32: 96-111. Haeg accumulation and mobilization of nutrien ts in the sugar-fed phorid fly, Pseudoacteon tricuspis Physiol. Entomol. 30: 212-224. mosquitoes. J. Insect Physiol. 26: 113-117. 443-474. availability in Ae. aegypti and An. quadrimaculatus (Diptera: Culicidae). Ann. Entomol. Soc. A Foster, W. A., and E. D. Walker. 2002. Mosquitoes (Culicidae), pp. 203-262. In G. Mul L. Durden Fraenkel, G. 1940. Utilization and digestion of carbohydrates by the adult blowfly. J. Exp. 17: 18-29. mosquito, Aedes aegypti and two flies, Sarcophaga bullata and Musca domestica. J. Cell. Comp. Physiol. 50: 1-23. Gardner, T. S. 1943. The problem of carbohydrate formation in nature. J. Org. Chem. 8: 120. and extra-floral nectar of peridomestic plants. Med. Vet. Entomol. 18: 102-107. Entomol. 11: 331-341. er, J. S. 1955. The non-blood feeding habits of Aedes taeniorhynchus (Diptera: Culicidae on Sanibe ) l Island, Florida. Mosq. News 15: 21-26. icana Hahn, D. A. 2005. Larval nutrition affects lipid stor age and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca amer J. Insect Physiol. 51: 1210-1219. 86

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Harrington, L. C., J. D. Edman, and T. W. Scott. 2001. Wh y do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood? J. Med. Entomol. 38: 411. Hawk Pap. 1: 104. gar m ethods originally developed for biting flies. Int. J. Pest Manage. 50: 193-198. Hill, C. J., and N. E. Pierce. 1989. The effect of adult diet on the biology butterflies. Oecologia. 81: 249-257. Holld Impoinvil, D. E., J. O. Kongere, W. A. Foster, B. N. Njiru, G. F. Killeen, J. I. Githure, J. C. itoid Bathyplectes curculionis (Hymenoptera: Ichneumonidae). Environ. Entomol. 29: 1088-1095. Jones 001. J. La. State Med. Soc. 154: 303-306. m. Assoc. 57: 25-131. Appl. 111: 189-199. Liles, J. N., and D. M. DeLong. 1960. The longevity and productivity of adult male and female Aedes aegypti when reared separately and together on three different diets. Ann. Entomol. MacVicker, J. A. K., J. S. Moore, D. H. Molyneux, and M. Maroli. 1990. Honeydew sugars Res. 80: 339-344. iptera: Magnarelli, L. A. ing, F. 1973. The world distribution of Wuchereria bancrofti and of Brugia malayi 9thIntern. Congr. Trop. Med. Mal., Abstr. Inv. Heimpel, G. E., J. C. Lee, Z. Wu, L. We iser, F. Wackers, and M. A. Jervis. 2004. Gut su analysis in field-caught parasitoids: adapting obler, B., and E. O. Wilson. 1990. The ants. Harvard Universi ty Press, Cambridge, MA. Beier, A. Hassanali, and B. G. J. Knols. 2004. Feeding and survival of them malaria vector Anopheles gambiae on plants growing on Kenya. Med. Vet. Entomol. 18: 108-115. Jacob, H. S., and E. W. Evans. 2000. Influence of carbohydrate food on mating and longevity of the paras S. C., J. Morris, G. Hill, M. Alderman, and R. C. Ratard. 2002. St. Louis encephalitis outbreak in Louisiana in 2 Joseph, S. R. 1970. Fruit feeding of mosquitoes in na ture. Proc. Annu. Meeting N.J. Mosq. Exter Lee, J. C., G. E. Heimpel, and G. L. Leibee. 2004. Comparing floral nectar and aphid honeydew diet on the longevity and nutrient leve ls of a parasitoid wasp. Entomol. Exp. Soc. Am. 53: 277-280. in wild-caught Italian phlebotomine sandflies (Diptera: Psychodidae) as detected by high performance liquid chromatography. Bull. Entomol. Magnarelli, L. A. 1979. Diurnal nectar feeding of Aedes cantator and Ae. sollicitans (D Culicidae). Environ. Entomol. 8: 949-955. 1983. Nectar sugars and caloric reserv es in natura l populations of Aedes canadensis and Aedes stimulans (Diptera: Culicidae). Environ. Entomol 12: 328-332. 87

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Marinotti, O., and A. A. James. 1990. An -glucosidase in the sa livar y glands of the vec mosquito, Aedes aegypti. Insect Biochem. 20: 619-623. tor Martin, O. Y., and D. A. Hosken. 2004. Copulation reduces male but not female longevity in McCann, S. 2006. Senescence and ot her factors aff ect fecundity in two species of Culex McCrae, A. W. R., Y. Ssenkubuge, P. Manuma, C. Mawejje, and A. Kitama. 1969. Mosquito and tabanid activity at plant sugar sources. E. Afr. Virus Res. Inst. Rep. 1968: Mevi-Schutz, J., and A. Erhardt. 2003a. Effects of nectar amino acids on fecundity of the wall Mevi-Shutz, J., and A. Erhardt. 2005. Amino acids in nectar e nhance butterfly fecundity: a Mola M. Armstron g, J. F. Anderson, and C. R. Vossbrinck. 2004. Host feeding patterns of Culex mosquitoes and West Nile vi rus transmission, Northeastern Molaei, G., T. G. Andreadis, P. M. Armstrong, R. Bueno Jr., J. A. Dennett, S. V. Real, C. l, feeding pattern of Culex quinquefasciatus (D iptera: Culicidae) and its role in transmission of West Nile virus in Harris Coun ty, Texas. Am. J. Trop. Med. Molleman, F., J. Ding, J. -L. Wang, P. M. Br akefield, J. R. Carey, and B. J. Zwann. 2008. Amino acid sources in the adult diet do not affect lifespan and fecundity in the fruitMort Red Sage ( Lantana camara L., [Verbenaceae]), Notorious weed and popular garden flower; some cases of poi soning in Florida. Econ. Bot. 48: 259-270. Muir 7-282. 2. Saltella sphondylli (Diptera: Sepsidae). J. Evol. Biol. 17: 357-362. mosquitoes. M.S. Thesis. University of Florida, Gainesville. 96-102. brown butterfly ( Lasiommata megera L.). Basic Appl. Ecol. 4: 413. Mevi-Schutz, J., and A. Erhardt. 2003b. Larval nutrition affects female nectar amino acid preference in the map butterfly ( Araschnia levana ). Ecology 84: 2788. long-awaited link. Am. Nat. 165: 411-419. ei, G., T. G. Andreadis, P United States. Emerging In fectious Diseases 12: 468-474. Sargent, A. Bala, Y. Randle, H. Guzman, A. Travassos da Rosa, T.Wuithiranyagoo and R. B. Tesh. 2007. Host Hyg. 77: 73-81. feeding butterfly Bicyclus anynana Ecol. Entomol. 33: 429-438. on, J. 1994. Lantana or L. E., and B. H. Kay. 1998. Aedes aegypti survival and dispersa l estimated by markrelease recapture in Northern Aust ralia. Am. J. Trop. Med Hyg. 58: 27 Mller, G., and Y. Schlein. 2005. Plant tissues: the frugal diet of mosquitoes in adverse conditions. Med. Vet. Entomol. 19: 413 Murphey, F. J. 1961. The binomics of Culex salin arius Coquillett. Ph.D. dissertation, University of Delaware, Newark. 88

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BIOGRAPHICAL SKE TCH Erin Vrzal was born in 1980 in El mira, New York and grew up in Elmira Heights, NY. In scholarships, but transferred to State University of New Yo rk, Environmental Science and Fores took a ted as a b of Florid 1998, she attended Barton College in Wilson, No rth Carolina on cross-country and academic try (SUNY-ESF) where she graduated in 2002 with a Bachel or of Science in Environmental studies with a focus in biological sciences. During her time at SUNY-ESF, she n introductory entomology class and completed an internship at the Rosamond-Gifford Zoo at Burnet Park. Her interests in biology a nd entomology led her to accept a job at the Uni States Department of Agriculture (USDA), Agricultural Research Service in Gainesville, Florida iological science technician working on mos quito behavior. Erin continued to work as a technician there while pursuing he r Master of Science degree in Entomology at the University a, and will continue post-graduation at the USDA. 93