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The Biology of the Eastern Pygmy Blue Butterfly, Brephidium pseudofea (Lepidoptera

Permanent Link: http://ufdc.ufl.edu/UFE0024617/00001

Material Information

Title: The Biology of the Eastern Pygmy Blue Butterfly, Brephidium pseudofea (Lepidoptera Lycaenidae): Physiological Adaptations to an Intertidal Environment
Physical Description: 1 online resource (68 p.)
Language: english
Creator: Mcmanus, Valerie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: blue, brephidium, eastern, pseudofea, pygmy, respiration, underwater
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: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE BIOLOGY OF THE EASTERN PYGMY BLUE BUTTERFLY, Brephidium pseudofea (LEPIDOPTERA: LYCAENIDAE): PHYSIOLOGICAL ADAPTATIONS TO AN INTERTIDAL ENVIRONMENT By Valerie C. McManus August 2009 Chair: Jaret C. Daniels Major: Entomology and Nematology The eastern pygmy blue, Brephidium pseudofea (Morrison) (Lepidoptera: Lycaenidae: Polyommatinae), is a small intertidal butterfly (only reaching 8 10 mm) that inhabits salt marshes that are periodically inundated by high tides. This hostile environment poses serious challenges for the egg, larval, and pupal stages of this butterfly, including tidal inundation that possibly blocks respiration and creates stress due to salt exposure. The purpose of this research was to investigate possible adaptations of the larval stages of B. pseudofea to periodic inundation using physiological and morphological analysis. Tidal charts were reviewed for March 2009 to February 2010 for Crystal River (Citrus County) in Florida, a typical habitat, to estimate potential inundation durations. Respiration rates were measured from B. pseudofea while under salt water and compared to five terrestrial lepidopteran species: Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae: Heliothinae), Junonia coenia (Hu umlautbner) (Lepidoptera: Nymphalidae: Nymphalinae), Callophrys irus (Godart) (Lepidoptera: Lycaenidae: Theclinae), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae: Amphipyrinae), and Manduca sexta Linnaeus (Lepidoptera: Sphingidae: Sphinginae). Tidal charts showed that the immature stages of B. pseudofea would be prone to complete inundation two to five times per month during the summer months (May to August) and partial submersion for up to 20 days per month during the rest of the year. Brephidium pseudofea respiration rates were substantially higher than the other five terrestrial lepidopteran species, suggesting adaptation to periodic inundation in these larvae. Preliminary studies suggested that the eastern pygmy blue larvae were also able to survive longer periods of time submerged under salt water than the other species studied. Careful examination of B. pseudofea larvae revealed small air pockets over the spiracles when the organism was exposed to tap water; these air pockets disappeared when exposed to detergent solution. The resulting air pockets may act as a diffusion layer for oxygen to be absorbed from the surrounding water during larval respiration. This diffusion layer may act in conjunction with cuticular gas exchange to meet the larva s respiratory needs. The setae on the larval body were extremely small and were not consistent with a typical plastron structure. Data from the tidal charts confirmed that B. pseudofea larvae would be prone to complete inundation periodically throughout the year. Therefore, larvae must have a means to respire underwater and survive this submersion. Morphological and physiological characteristics suggested strategies of how larvae are able to do this.
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 Valerie Mcmanus.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Daniels, Jaret.
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: UFE0024617:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024617/00001

Material Information

Title: The Biology of the Eastern Pygmy Blue Butterfly, Brephidium pseudofea (Lepidoptera Lycaenidae): Physiological Adaptations to an Intertidal Environment
Physical Description: 1 online resource (68 p.)
Language: english
Creator: Mcmanus, Valerie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: blue, brephidium, eastern, pseudofea, pygmy, respiration, underwater
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: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE BIOLOGY OF THE EASTERN PYGMY BLUE BUTTERFLY, Brephidium pseudofea (LEPIDOPTERA: LYCAENIDAE): PHYSIOLOGICAL ADAPTATIONS TO AN INTERTIDAL ENVIRONMENT By Valerie C. McManus August 2009 Chair: Jaret C. Daniels Major: Entomology and Nematology The eastern pygmy blue, Brephidium pseudofea (Morrison) (Lepidoptera: Lycaenidae: Polyommatinae), is a small intertidal butterfly (only reaching 8 10 mm) that inhabits salt marshes that are periodically inundated by high tides. This hostile environment poses serious challenges for the egg, larval, and pupal stages of this butterfly, including tidal inundation that possibly blocks respiration and creates stress due to salt exposure. The purpose of this research was to investigate possible adaptations of the larval stages of B. pseudofea to periodic inundation using physiological and morphological analysis. Tidal charts were reviewed for March 2009 to February 2010 for Crystal River (Citrus County) in Florida, a typical habitat, to estimate potential inundation durations. Respiration rates were measured from B. pseudofea while under salt water and compared to five terrestrial lepidopteran species: Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae: Heliothinae), Junonia coenia (Hu umlautbner) (Lepidoptera: Nymphalidae: Nymphalinae), Callophrys irus (Godart) (Lepidoptera: Lycaenidae: Theclinae), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae: Amphipyrinae), and Manduca sexta Linnaeus (Lepidoptera: Sphingidae: Sphinginae). Tidal charts showed that the immature stages of B. pseudofea would be prone to complete inundation two to five times per month during the summer months (May to August) and partial submersion for up to 20 days per month during the rest of the year. Brephidium pseudofea respiration rates were substantially higher than the other five terrestrial lepidopteran species, suggesting adaptation to periodic inundation in these larvae. Preliminary studies suggested that the eastern pygmy blue larvae were also able to survive longer periods of time submerged under salt water than the other species studied. Careful examination of B. pseudofea larvae revealed small air pockets over the spiracles when the organism was exposed to tap water; these air pockets disappeared when exposed to detergent solution. The resulting air pockets may act as a diffusion layer for oxygen to be absorbed from the surrounding water during larval respiration. This diffusion layer may act in conjunction with cuticular gas exchange to meet the larva s respiratory needs. The setae on the larval body were extremely small and were not consistent with a typical plastron structure. Data from the tidal charts confirmed that B. pseudofea larvae would be prone to complete inundation periodically throughout the year. Therefore, larvae must have a means to respire underwater and survive this submersion. Morphological and physiological characteristics suggested strategies of how larvae are able to do this.
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 Valerie Mcmanus.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Daniels, Jaret.
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: UFE0024617:00001


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1 THE BIOLOGY OF THE EASTERN PYGMY BLUE BUTTERFLY, Brephidium pseudofea (LEPIDOPTERA: LYCAENIDAE): PHYSIOLOGICAL ADAPTATIONS TO AN INTERTIDAL ENVIRONMENT By VALERIE C. MCMANUS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIE NCE UNIVERSITY OF FLORIDA 2009

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2 2009 Valerie C. McManus

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3 To my future husband, Oren

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. Jaret Daniels as the supervisory chair of my committee for his continuous guidance, support, and encouragement I would also like to thank my committee member, Dr. Daniel Hahn who has provided invaluable a ssistance and a great deal of direction during the course of my research I thank Dr. Jacqueline Miller and Dr. Marc Branham for their guidance and suggestions as members of my committee. I wish to thank Dr. David Julian for assistance with experiments and providing equipment. Lyle Buss deserves great thanks for his time and patience wi th the AutoMontage imaging, as well as Dr. Paul Skelley Division of Plant Industry, with the scanning electron m icroscope. Also, I would like to thank Tom Allen for taking time to collect adults in south Florida and send ing them to Gainesville I would also like to thank Dr. Robert Meagher for donating larvae from his colony at the USDA. I would like to especially thank m y fellow graduate students for their assi stance with the captive colony. I give special thanks to my parents for their continued support. They have shown much encouragement, guidance, and inspiration throughout my college career, which I greatly appreciate. Finally my deepest appreciation and love to my f ianc, Oren Warren. He has put forth hours of laboratory and field assistance, as well as continuous encouragement and support throughout my college career I owe all my success to him.

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5 TABLE OF CONTENTS page ACKNOWLED GMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................................10 CHAPTER 1 LITERATURE REVIEW .......................................................................................................12 Family Lycaenidae ..................................................................................................................12 Life History .............................................................................................................................13 Potential Adaptations to Tidal Inundation ..............................................................................14 Myrmecophily .........................................................................................................................18 Objecti ves ...............................................................................................................................19 2 PHYSIOLOGICAL EXPERIMENTS ....................................................................................27 Introduction .............................................................................................................................27 Material and Methods .............................................................................................................27 Captive Colony ................................................................................................................27 Tidal Activity for 2009 2010 .........................................................................................30 Morphological Analysis ..................................................................................................30 Respirometry Measurements ...........................................................................................31 Spiracular Air Bubble ......................................................................................................33 Early Respirometry Trials ...............................................................................................33 Anoxia Tolerance ............................................................................................................34 Results .....................................................................................................................................35 Tidal Activity for 2009 2010 .........................................................................................35 Morphological Analysis ..................................................................................................36 Respirometry Measurements ...........................................................................................36 Discussion ...............................................................................................................................38 Tidal Activity for 2009 2010 .........................................................................................38 Morphological Analysis ..................................................................................................39 Respirometry Measurements ...........................................................................................40 Spiracular Air Bubble ......................................................................................................41 Early Respirometry Trials ...............................................................................................42 Anoxia Tolerance ............................................................................................................43 Conclusions .............................................................................................................................44

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6 3 CONCLUSIONS AND SUGGE STIONS FOR FURTHER RESEARCH ............................62 LIST OF REFERENCES ...............................................................................................................65 BIOGRAPHICAL SKETCH .........................................................................................................68

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7 LIST OF TABLES Ta ble page 21 Respiration rates (mean SE) showing that Brephidium pseudofea had substantially higher respiratory activity underwater than the other terrestrial species studied and th at boiled larvae consumed only small quantities of oxygen compared to their live counterparts ........................................................................................................................60

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8 LIST OF FIGURES Figure page 11 Typical Brephidium pseudofea habitats in Florida ............................................................21 12 A sample of Brephidium pseudofea specimens collected from Cedar Key (Levy County) in Florida. .............................................................................................................22 13 Host plants of Brephidium pseudofea ................................................................................23 14 Newly emerged Brephidium pseudofea larva measuring only 1 mm in length. ................24 15 Brephidium pseudofea eggshell only partially consumed by the newly emerged larva. ...25 16 Brephidium pseudofea showing color variation with pink hues that some larvae are able to attain. ......................................................................................................................25 17 First instar Brephidium pseudofea larva burrowing into the host plant ( Sarcocornia perennis ) and leaving its posterior end exposed ................................................................26 21 Larval rearing set up for eggs and larvae of Brephidium pseudofea showing 2.5 cm stems of Sarcocornia perennis and small larvae. ..............................................................47 22 Experimental set up for respiration rate measurements. ....................................................48 23 Modified microtitre plate showing wells filled with salt water and Brephidium pseudofea larvae. ................................................................................................................49 24 Exp erimental set up for anoxia tolerance studies showing 10% NaOH filled micro pipette at bottom, cotton, and Brephidium pseudofea larva. ..............................................50 25 Series of photos showing Sarcocornia perennis plan ts partially inundated when high tide was at 4.0 ft high at Crystal River, FL. .......................................................................51 26 Data from tidal charts between March 2009 to February 2010 showing partial and complete inundation of Crys tal River habitat ....................................................................52 27 Series of Brephidium pseudofea life stages .......................................................................53 28 Series of scanning electron microscope photogra phs of Brephidium pseudofea egg ........54 29 Scanning electron micrograph of spiracle surrounded by small, clavate setae of Brephidium pseudofea. ......................................................................................................56 210 Ant organs of Brephidium pseudofea ................................................................................57 211 Series of Brephidium pseudofea spiracles .........................................................................58

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9 212 The significantly lower respiration rates (mean SE) of dead Brephidium pseudofea larvae compared to live larvae indicate that live larvae are able to respire under salt water. ..................................................................................................................................59 213 The significantly higher respiration rates (mean SE) of Brephidium pseudofea larvae under salt water compared to other terrestrial lepidopteran larvae suggest respiratory adaptation to the intertidal environment in B. pseudofea ................................61

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE BIOLOGY OF THE EASTERN PYGMY BLUE BUTTERFLY, Breph idium pseudofea (LEPIDOPTERA: LYCAENIDAE): PHYSIOLOGICAL ADAPTATIONS TO AN INTERTIDAL ENVIRONMENT By Valerie C. McManus August 2009 Chair: Jaret C. Daniels Major: Entomology and Nematology The eastern pygmy blue, Brephidium pseudofea (Morrison) ( Lepidoptera: Lycaenidae: Polyommatinae ) is a small intertidal butterfly (only reaching 8 10 mm) that inhabits salt marshes that are periodically inundated by high tides. This hostile environment poses serious challenges for the egg, larval, and pupa l stages o f this butterfly, including tidal inundation that possibly blocks re spi ration and creates stress due to salt exposure The purpose of this research was to investigate possible adaptations of the larval stages of B pseudofea to periodic inundation using physiological and morphological analysis Tidal charts were reviewed for March 2009 to February 2010 for Crystal River (Citrus County) in Florida a typical habitat, to estimate potential inundation duration s R espiration rates were measured from B. pseudof ea while under salt water and compared to five terrestrial lepidopteran species : Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae: Heliothinae) Junonia coenia ( Hbner ) (Lepidoptera: Nymphalidae: Nymphalinae) Callophrys irus (Godart) (Lepidoptera: Lycaeni dae: Theclinae) Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae: Amphipyrinae) and Manduca sexta Linnaeus ( Lepidoptera: Sphingidae: Sphinginae)

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11 Tidal charts showed that the immature stages of B. pseudofea would be prone to complete inundati on two to five times per month during the summer months (May to August) and partial submersion for up to 20 days per month during the rest of the year. Brephidium pseudofea respiration rates were substantially higher than the other five terrestrial lepidopteran species, suggesting adaptation to periodic inundation in these larvae. Preliminary studies suggested that the eastern pygmy blue larvae were also able to survive longer periods of time submerged under salt water than the other species studied. Caref ul examination of B. pseudofea larvae revealed small air pockets over the spiracles when the organism was exposed to tap water ; these air pockets disappeared when exposed to detergent solution. The resulting air pockets may act as a diffusion layer for oxy gen to be absorbed from the surrounding water during larval respiration This diffusion layer may act in conjunction with cuticular gas exchange to meet the larvas respiratory needs The setae on the larval body were extremely small and were not consistent with a typical plastron structure. Data from the tidal charts confirmed that B. pseudofea larvae would be prone to complete inundation periodically throughout the year. Therefore, larvae must have a means to respire under water and survive this submersion. Morphological and physiological characteristics suggested strategies of how larvae are able to do this.

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12 CHAPTER 1 LITERATURE REVIEW Family Lycaenidae The family Lycaenidae, the gossamer winged butterflies makes up about 32% of all butterflies and c ontains almost half of the described species of the superfamily Papilionoidea with roughly 6,000 species ( Shields 1989). Lycaenids are widely distributed and can be found on all continents except Antarctica ( Howe 1975), with the greatest diversity being in the tropics ( Braby 2000). Lycaenids utilize a broad range of habitats, from climax forests, scrublands, grasslands and wetlands to semi arid desert communities. Although the family tolerates a variety of habitats, many species have precise environmental r equirements S ome lycaenids are considered significant environmental indicators and therefore play an important role in conservation studies (New 1993). Of the known life histories within the family, 75% of lycaenids have an associ ation with ants (Pierce e t al. 2002). This family is divided in to seven subfamilies worldwide (Wahlberg et al. 2005) with most falling into the blues, coppers, and hairstreaks ( Eliot, J. N. 1973; Smart 1975). The s ubfamily Polyommatinae or blues means many eyes in Greek (pol y= many; ommat= eye) and refers to the eyespots on the underside of the hindwing (Zirlin 2007). The name blues is derived from the fact that the wings on most adults are blue or purple, with the males typically more vibrant than the females. Blues can be found year round (Howe 1975) and are mostly distr ibuted in temperate regions, with a few species found in the tropics (Howe 1975). Polyommatinae is divided into four tribes (Eliot 1973) and there are over 140 species in North America located north of Mexi co ( Wagner 2005). Polyommatine larvae utilize a broad array of different host plants, but are most commonly associated with the families F abaceae and Sapindaceae. The larvae typically feed on

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13 the flower buds, flowers, and young leaves, while some species even eat developing seeds or pods If food supplies diminish, larvae can become cannibalistic (Braby 2000). Life History The eastern pygmy blue, Brephidium pseudofea (Lepidoptera: Lycaenidae: Polyommatinae ), was originally described in 1873 by Morrison f rom a small series of specimens collected in the Florida Keys (Pavulaan and Gatrelle 1999). The genus name Brephidium means little infant in Greek (brephos= an infant; idium= little) (Zirlin 2007). This genus is small, with only one other member in the New World ( B. exilis ) in the western U.S. and one member in the Old World ( B. metophis ) in Africa Th is butterfly is primarily subtropical in its range and habitat associations. B rephidium pseudofea is found in close association with tidal flats (Figure 1 1) and the margins of coastal salt and brackish water marshes from the Florida Keys north along the Atlantic coastal plain to southeastern South Carolina and along the Gulf Coast to southern Louisiana and Texas (Hall and Butler 2003). Such coastal ecosyst ems especially those of transitional waters are dynamic environments, varying in both time and space and are prone to an array of natural and anthropogenic disturbances. As a result, populations of B pseudofea are common, but tend to be local ized ephem eral and often isolated. When encountered, however, populations may support a very high density of individuals. Despite these factors, relatively little detailed information is available on the butterflys biology, ecology, and behavior. Additionally, ther e remain many unresolved taxonomic questions surrounding the eastern and western U.S. representatives of this small genus. The adults of both sexes are si milar in size with a forewing chord length between 8 to 10 mm (n= 70; Mean= 8.686; Stand ard error= 0 .076) (Figure 1 2) The dorsal surface of the wings is uniformly brown with a marginal row of prominent black spots on the hindwing. The ventral surface of the wings is light brown with numerous white lines and spots and contains no blue

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14 color whatsoever. The hindwing bears a marginal row of equally sized black spots with faint highlights. Gravid females deposit tiny blue green eggs singly reaching only 400 m in diameter, on Sarcocornia perennis (Mill.) A. J. Scott (Amaranthaceae), Salicornia bigelovii To rr. (Amaranthaceae) (Figure 1 3) and possibly Batis maritima L. (Bataceae) In 2008, the peak population density on the west coast of Florida was between May and October, with few sightings through November (personal observation). However, weather plays a n extremely important role as to when these butterflies emerge because in 2009, populations were seen as early as the beginning of March (personal observation). Larvae emerge five to seven days after the eggs are laid a t approximately 1 mm in length (Figur e 1 4) and can reach up to 10 mm when fully mature The larvae consume very little of the eggshell (Figure 1 5) (Rawson 1961; personal observation) and undergo four or five larval instars in 17 to 20 days ( 23 to 25oC ) (personal observation). The slug like larvae are usually uniformly green, but sometimes show color variations with pink hues at the posterior and anterior ends of the larvae ( Figure 1 6) (personal observation). First and second instar larvae chew and burrow into the host plant (usually new gro wth) and feed w ith their posterior end exposed (Figure 1 7 ) Later instars feed externally on the fleshy, jointed branches (personal observation). The pupae are 7 to 8 mm in length uniformly green and are secured to the plant surface by a silken girdle (R awson 1961) and a silken button at the base. The pupal stage lasts seven to eight days (personal observation). The entire life cycle from egg to adult takes approximately 29 to 35 days to complete when temperatures are between 23 and 25oC (personal observa tion). Potential Adaptations to Tidal Inundation Estuary and saltmarsh communities, which are transitions between fresh and marine waters, maintain a group of phytophagous insects within their intertidal zone (Gullan and

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15 Cranston 1994). Only a few hundred species of insects are associated with coastal habitats (including saltmarshes, mangroves, decaying seaweed on the seashore, and estuaries), while several million species are found in terrestrial environments and more than 30,000 species are found in fres hwater environments (Williams and Williams 1998). Stress keeps diversity in transitional habitats low; a mong the se stresses that intertidal insects must conquer, respiration is possibly the most limiting. Other boundaries that such insects must overcome ar e buoyancy, surface tension, and osmoregulation (Cheng 1976). As components of transitional coastal environments, the herbaceous larval host plants of B. pseudofea, as well as the eggs, larvae, and pupae they support are periodically inundated by tidal wa ters. The resulting complete or partial submersion, salt exposure and temporal dynamics pose serious challenges for survival. All animals require a source of oxygen to live, and insects that deal with inundation have evolved a variety of specialized morphological and physiological adaptations including : direct diffusion through the cuticle taking oxygen from the water s surface by a spiracular tube taking oxygen directly from the water using a submerged plastron, breathing f rom enclosed air bubbles, switc hing to anaerobic respiration and/ or reducing metabolic rate s (Zerm and Adis 2003). Direct diffusion, which is the most primitive type of respiratory exchange (Kirsch and Nonnotte 1977), is an adaptation that very small insects are able to use to respir e Instead of a tracheal system, these insects diffuse gases directly through their cuticle (Daly et al. 1998) Cutaneous oxygen uptake is important for organisms living in water or mois t habitats (Kirsch and Nonnotte 1977). I nsect s may also use cutaneous respiration in combination with other adaptations.

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16 Some insects may respire using a spiracular tube. This tube is located at the posterior end of the body attached to the terminal spiracles and extended to the surface. Such insects can essentially hang upside down by the tension of the water via this spiracular tube (Daly et al. 1998). Other insects utilize air spaces in aquatic plants by piercing the plant with specialized structures. These insects have spiracles located in modified spines at their poste rior end that they insert into the air spaces of inundated plants (Borror et al. 1989). Other insects rely on air bubbles that they carry beneath the surface of the water on their body. These air bubbles are held by special hairs and are located on top of the spiracles; the spiracles will then be able to open into the bubble to retrieve oxygen. When air stores begin to diminish, the insect may break the surface film of the water to re plenish the bubble (Daly et al. 1998). The air bubble can also act as a p hysical gill, diffusing oxygen from the water into the bubble when the content of oxygen in the bubble falls below that of the wate r surrounding it (Borror et al. 1989). A plastron is a modification of the air bubble observed in some insects to respire wh ile submerged. A plastron is a permanent layer of air surrounding the body or region of the body (Borror et al. 1989) that is held in place by numerous dense hairs or by a fine cuticular meshwork (Daly et al. 1998). As long as the water is well aerated, th e insect can remain submerged indefinitely because w hen there is a drop in gas pressure within the plastron due to oxygen consumption by the insect, oxygen from the surrounding water diffuse s in to replenish it. Compared to the air bubble, plastron gas exc hange is faster because the surface area of the plastron is typically larger, and the air space is never lost because the hairs are s tiff and do not collapse (Evans 1984), a s long as the area is not disrupted by a wetting agent and the surface tension is not broken. Most insect eggs have plastron structures used to respire in case they are

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17 flooded by rains. They may either contain plastron bearing horns or the plastron may be extended over the entire shell or over a certain area of the shell (Hinton 1969). In addition to respiratory adaptations that facilitate aquatic gas exchange, insects may also have other metabolic strategies to deal with inundation, such as switching to anaerobic respiration and reducing metabolic rates S witch ing between aerobic and an aerobic respiration help s maintain energy in the organisms tissues when oxygen is limited (Hoback et al. 2000). Aerobic respiration is when oxygen is used by the cells for adenosine triphosphate (ATP) production through aerobic catabolic pathways. These p athways can be subdivided into glycolysis, the Krebs cycle, the electron transport chain, and oxidative phosphorylation. Aerobic catabolic pathways use oxygen to oxidize food molecules to form carbon dioxide and water, therefore retrieving ATP (Hill et al. 2004). Anaerobic respiration functions without oxygen. Devoid of oxygen, oxidative phosphorylation can not take place and ATP molecules will not be produced in this stage Also without oxygen, the electrontransport chain will become a dead end because el ectrons will build up with no place to go and ATP (or energy) will not be produced. The alternative to using oxygen is through anaerobic glycolysis, where glucose is converted to pyruvic acid that is then used as the electron acceptor; pyruvic acid will th en be reduced to lactic acid with the help of various forms of lactate dehydrogenase (LDH). As long as LDH is effectively converting pyruvic acid to lactic acid, a redox balance will be sust ained and ATP will be produced. Insects will store the lactic acid that they produce until they switch to aerobic respiration. Once oxygen is available, lactic acid will be converted back to pyruvic acid by the reversal of the reaction that produced it and it can be used as a catabolic substrate to fuel oxidative phosphorylation ( Hill et al. 2004).

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18 Another adaptation to inundation is for insects to reduce their metabolic rate The metabol ic rate is the speed that an organism converts chemical energy into external work (Hill et al. 2004); because oxygen consumption tends to be directly proportional to energy use it is a common measure for metabolic rate. When an insect is introduced to an environment where oxygen is limited it will suspend some physiological functions until it returns to a state of adequate oxygen conce ntration. By doing this, the insect will not expend as much energy and will limit the amount of anaerobic end products it produces (Wegener and Moratzky 1995). When insects lower their metabolic rate s they force their bodies into an oxygen debt because of the lack of oxygen surrounding them When the insects return to normal conditions, the oxygen debt must be balanced and the buildup of anaerobic by products must be catabolized. They do this by raising their metabolic rate about 50% above the normal ra te (Kolsch et al. 2002); once the oxygen debt is corrected, the insects metabolic rate will slow to baseline levels (Hoback et al. 2000). Myrmecophily The association that lycaenid larvae have with ants is a well studied aspect of the family. Full life h istories have been studied for 20% of the species in the family Lycaenidae. Of the known life histories, 75% of the species associate with ants. Myrmecophily, which means ant loving, can be parasitic, facultative, obligate or mutualistic (Pierce et al. 20 02). Lycaenid larvae that associate with ants are morphologically and behaviorally distinct from those that do not by their extra thick cuticle, specialized organs, and the lack of a thrashing response to disturbance by ants (Eastwood and Fraser 1999). Mos t ant interactions are mutualistic with the ants presumably providing protection to the larvae from parasitoids and other various predators in return for nutritive droplets that the larvae secrete from specialized organs (Cottrell 1984). These specialized organs that receive attentio n from attendant ants are: the N ewcomers organ, the

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19 tentacular org ans, and the pore cupolas. The N ewcomers organ secretes liquid for the ants to retrieve. The tentacular organs are a pair of eversible organs that attract the a nts. The pore cupolas are small organs located over much of the dorsal surface of the larvae and secrete an additional food source of amino acids for the ants (Kitching 1983). It has been reported by Harvey and Longino (1989) that Brephidium pseudofea lar vae and pupae associ ate with ants, specifically with the species Tapinoma sessile ( Say ) ( Formicidae ) in the Florida Keys. They even suggest that the adult females of B pseudofea may only oviposit on plants that bear patrolling ants, although personal obse rvation has shown this unlikely from witnessing several gravid females ovipositing eggs on host plants bearing no ants. However, little detailed information is available on this myrmecophilous relationship or on the potential involvement of this or other a nt taxa. Objectives The purpose of this research was to investigate possible physiological adaptations of the larval stages of Brephidium pseudofea to periodic tidal inundation, focusing on respiratory capabilities of the larvae in salt water. Tidal chart s were reviewed for March 2009 to February 2010 at Crystal River (Citrus County) Florida a typical habitat, to estimate the monthly inundations that immature stages would endure. Respiration rates were measured under salt water with a fiber optic oxygen sensing probe for B. pseudofea, and five other terrestrial lepidopteran species for comparison, including : Helicoverpa zea, Junonia coenia, Callophrys irus, Spodoptera frugiperda, and Manduca sext a. Comparative studies were included to distinguish whether B. pseudofea larvae have different underwater respiration rates than other lepidopteran species that are not periodically inundated by salt water. Careful examination of B. pseudofea larvae revealed spiracular air pockets under water and further assessmen t showed that

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20 these air pockets disappear ed with exposure to detergent Additional morphological characteristics of larval ant organs and setae were also examined and described in B. pseudofea.

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21 A B Figure 1 1. Typical Brephidium pseudofe a habitat s in Florida A) Cedar Key (Levy County) showing Sarcocornia perennis along the left side, B) Crystal River (Citrus County) showing S. perennis disperse d i n clumps throughout the habitat, along with Batis maritima

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22 Figure 1 2. A sample of Brephidium pseudofea specimens collected from Cedar Key (Levy County) in Florida

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23 A B Figure 1 3. Host plants of Brephidium pseudofea. A ) Salicornia bigelovii B) Sarcocornia perennis

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24 Figure 1 4. Newly emerged Brephidium pseudofea larva measuring only 1 mm in length.

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25 Figure 1 5. Brephidium pseudofea eggshell only partially consumed by the newly emerged larva. Figure 1 6. Brephidium pseudofea showing color variation with pink hues that some larva e are able to attain.

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26 Figure 1 7. First instar Brephidi um pseudofea larva burrowing into the host plant ( Sarcocornia perennis ) and leaving its posterior end exposed; a typical way to feed during early instars.

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27 CHAPTER 2 PHYSIOLOGICAL EXPERI MENTS Introduction The eastern py gmy blue Brephidium pseudofea, inhabits intertidal environments that are periodically inundated with salt water During inundation, the egg s, larvae, and pupae must survive salt water submersion. Insects that live in habitats that are subject to water inundation have evolved a variety of specialized morphological and physiological adaptations including : direct diffusion by cutaneous respiration taking oxygen from the water by a spiracular tube or a plastron, breathing f rom enclosed air bubbles, switching to anaerobic respiration and/ or reducing metabolic rate (Zerm and Adis 2003) The objective of this study was to examine the mechanisms used by B. pseudofea larvae to endure salt water inundation by measuring respiration rates, determining anoxia toler ance, and disrupting potential respiratory air bubbles using detergent solutions. Tidal activity was also studied in an attempt to discover precisely when immature stages would be subjected to tidal inundation. To determine whether the respiration rates found in B. pseudofea while under salt water were unu sual for lepidopteran larvae, respiration rates of B. pseudofea were compared to rates of five terrestrial lepidopteran species under salt water inundation. Specifically, B. pseudofea was expected to consu me more oxygen during inundation than the terrestrial species. Not all methods were successful; therefore adjustments were made throughout the course of this research project Material and Methods Captive Colony Wild stock and host plant material of Breph idium pseudofea was obtained from salt marshes in Cedar Key (Levy County) Crystal River (Citrus County), Cape Coral (Lee County)

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28 and St. Augustine (St. Johns County) in Florida. The adults were brought back to Gainesville (Alachua County), Florida where t hey were maintained under controlled laboratory conditions during development at the Department of Entomology and Nematology. To induce oviposition, gravid females (2 to 4 ) were placed in KaratTM 16 ounce clea r plastic cups containing fresh, new growth cu ttings of Sarcocornia perennis (Mill.) A. J. Scott (Amaranthaceae) and a cotton swab soak ed in Fierce Melon Gatorade as an artificial nectar source. Cups were placed 30 cm below a 40watt incandescent light (1 h on, 1 h off for a 12 h period from 8:00 AM to 8:00 PM ) to stimulate oviposition activity. Within 48 h of oviposition, all eggs were removed with a wet artist brush and transferred to 24well cell culture cluster flat bottom plates with lids (Costar) (Figure 21) A 2.5 cm stem of S. perennis and fi ve eggs were placed in each well A paper towel cut out was placed on the lid of the plate and lightly misted with tap water daily to ensure high moisture and relative humidity within the wells. After seven days, larvae were transferred to clean plates and two were placed in each well All adults and larvae were kept at temperatures ranging from 23 to 25oC during their life cycles. Host plant material was replaced on a regular basis and plates cleaned out every other day, or when needed. All experiments use d larvae from this captive colony. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae: Heliothinae) larvae were obtained from a colony at the Department of Entomology and Nematology University of Florida in Gainesville (Alachua County) The larvae were reared on a wheat germ based diet ( Bio S erv New Jersey) and kept in 5 ounce cups until needed for experiments. H elicoverpa zea the corn earworm, is strictly terrestrial and a significant pest of a variety of crops, particularly corn and tomatoes.

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29 Junonia c oenia ( Hbner ) (Lepidoptera: Nymphalidae: Nymphalinae) wild stock was collected from Alachua County, Florida in March 2009. Gravid females were placed in similar oviposition cups as B. pseudofea and eggs were placed in vials along with the host plant Larv ae were fed Linaria canadensis ( L. ) D. A. Sutton (Scrophulariaceae) until needed for experiments. J unonia coenia the common buckeye, is strictly terres trial and is found throughout the year in Florida. Callophrys irus (Godart ) (Lepidoptera: Lycaenidae: T heclinae ) larvae were obtained from Duval County Florida in March 2009. Larvae were placed in 16 ounce clear plastic cups and fed Lupinus perennis ( L. ) (Fabaceae) until large enough to use in experiments. C allophrys irus, the frosted elfin hairstreak is strictly terrestrial. Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae: Amphipyrinae) larvae were taken from a colony at the USDA Center for Medical, Agricultural, and Veterinary Entomology ( CMAVE ) in Gainesville Larvae were reared on a pinto bean diet modified from Guy et al. (1985) and kept in large plastic bins until needed for experiments Spodoptera frugiperda, the fall army worm, is a serious pest of many grasses, including corn. It is strictly terrestrial, although it can get extremely hu mid and moist within the whorls of the corn, wher e the larvae periodically feed Manduca sexta Linnaeus (Lepidoptera: Sphingidae: Sphinginae) larvae were obtained from a colony at the Department of Entomology and Nematology University of Florida in Gaines ville. The larvae were reared on a wheat germ based diet modified from Bell and Joachim (1976) and kept in plastic bins until needed for experiments. M anduca sexta the tobacco hornworm, is strictly terrestrial. The larvae are also a pest of various plants in the family Solanaceae.

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30 Salt water from a salt marsh in Crystal River a typical B. pseudofea habitat, was brought back to the la boratory in Gainesville The salinity of the water from a high tide of 4.0 ft Mean Lower Low Water (MLLW) was found to be 2.6% with a digital refractometer (Model 10480, Reichert Jung, Mark Abbe II Refractometer, Depew, NY). The water was sterilized by boiling for 20 min and then stored at 20 oTidal Activity for 2009 2010 C ; aliquots were thawed as needed. Tide charts wer e obtained from www.saltwatertides.com for March 2009 to February 2010 in Crystal River (Citrus County) Florida, a typical B rephidium pseudofea habitat From these data, observations in the field were made based on the height at high tide on March 21st, 25th, and 29thMorphologic al Analysis where the heights in feet (in relation to the Mean Lower Low Water [MLLW]) were 2.5, 3.4, and 4.0, respectively. While in the field, visual estimations of the habitat under water were made by looking at plants completely out of the water, completely underwater, and partially under water. Given the tidal heights from these three days, estimations wer e made from the tide charts for how many times per month B. pseudofea immature stages would be subjected to inundation at this location Five eggs, five larvae, and two pupae from the captive colony of Brephidium pseudofea were examined and photographed in a tungsten low vacuum scanning electron microscope (SEM) (JEOL JSM 5510LV; Tousimis Samdri 780A) at the Division of Plant In dustry Florida Department of Agriculture, in Gainesville (Alachua County) FL. The eggs, larvae, and pupae were dried by the critical point drying method and coated with gold/ palladium by an ion sputter device (Critical Point Drier Denton Vacuum Desk III Sputter Coater) The alcohol dehydration steps were not needed because the life stages of B. pseudofea are so small. Images of abdominal

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31 segments VII and VIII on the larvae were specifically taken in order to observe whether ant organs were present in B. pseudofea. Several adults from the captive colony were examined and photographed using Auto M ontage Pro software (version 5.02, Syncroscopy, Frederick, MD) and a Leica MZ12.5 stereomicroscope at the Department of Entomology and Nematology at the University of Florida in Gainesville, FL Ten lateinstar larvae from the captive colony were also examined and photographed using Auto Montage while in air, under tap water, and under a 0.6% Triton X 100 detergent ( Sigma ) solution a wetting agent Pictures were specifically taken of abdominal spiracles VII and VIII to show differences within and between the various media. Respirometry Measurements The respiration rate of B rephidium pseudofea larvae was quantified using a ruthenium based fiber optic oxygensensin g probe (FOXY R probe; Ocean Optics Inc., Dunedin, FL) and software (001Sensor v.4) A decrease in oxygen levels in the respirometry chamber were evaluated as consumption of oxygen by the larvae and decreases through time were used to calculate respiration rates. The oxygen probe was calibrated with nitrogen gas (0% oxygen) and room air (20.95% oxygen) before each separate experiment. A respiromet ry chamber was created with a 2 ml vial containing a micro stir bar and a piece of plastic mesh (to keep the lar va and stir bar from coming into contact), and was placed on a stir plate to promote circulation of the water in the vial (Figure 2 2A ). A single 3rd, 4th, or 5th instar larva weighing between 17.5 and 26.1 mg was placed in the chamber and salt water poure d in to overfill the vial. A septa cap was crimped on top of the vial that had a small hole for the probe to go through. After the probe was placed into the respirometry chamber a 3 x 4 cm piece of parafilm was wrapped tightly around the septa cap to seal the vial (Figure 2 2B) The oxygen probe and respirometry chamber were held in

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32 place by clamps over the stir plate Initial studies with this respirometry chamber containing nitrogen gas and deoxygenated water showed no diffusion of oxygen into the vial. After a 10 min equilibration period, oxygen consumption was measured for 1 to 6 h with fluorescence readings taken at 1 min intervals. As a control, B. pseudofea larvae were boiled for 10 min to ensure death and their respiration rates taken and compared t o that of the live B. pseudofea larvae. Additional controls of boil ed seawater in the respirometry chamber containing no larva showed no detectable indications of oxygen consumption. Respiration rates of Helicoverpa zea ( between 9.8 and 25.7 m g), Junonia c oenia ( between 12.4 and 25.8 m g), Callophrys irus ( between 12.2 and 33.0 m g) Spodoptera frugiperda ( between 12.3 and 36.0 m g) and Manduca sexta ( between 11.4 and 26.0 m g ) were also taken with the same methods. Subsets of these larvae were also boiled for 10 min as a control. Behavioral observations of each species during inundations were recorded. ANOVA, T tests, unequal variance tests and Tukeys HSD test s were performed in JMP, Version 7 (SAS Institute). Respiration rates corrected for body mass were f ound by using the following equation T mg vv O O Y 000 102 1 where Y= respiration rate O1= ppm of initial oxygen reading after 10 min equilibration period O2= ppm of final oxygen reading vv= void volume of 1770 l in the respiromet ry chamber mg= mass of larva in mg T= number of min experiment lasted

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33 Spiracular Air Bubble A 0.6% solution of Triton X 100 detergent (Sigma) a wetting agent, was prepared and Brephidium pseudofea larvae were dipped in the solution and placed in the res pirometry chamber for their respiration rates to be measured Larvae were kept in the chamber for 1 to 2 h with readings taken at 1 min intervals. Different larvae were used for each experiment. To assess mortality in response to detergent exposure larvae were dipped in the detergent solution (0.6% and 0.3% solution), dipped in de ionized water (to rinse them off), lightly dried with a Kimwipe, and then placed in a container with Sarcocornia perennis Survival rates were measured after 24 hours. Early Res pirometry Trials Methods similar to Szela and Marsh (2005) were used. The BioTek Synergy HT microtitre plate reader loaded with a modified plate was located i n the Zoology Department, University of Florida in Gainesville, FL. A single Brephidium pseudofea larva was placed in each of four wells of the modified microtiter plate that contained silicon imbedded with ruthenium at the base of the wells (Figure 2 3 ). Ruthenium fluoresces in the absence of oxygen and this fluorescence was quantified by the plate r eader and compared to known standards of pure nitrogen (0% oxygen) and well mixed room air (20.95% oxygen). The larvae were slowly inundated with salt water and the caps placed on the wells. The plate reader ran for 2 to 12 h with fluorescence measured at 3 to 5 min intervals. Once the experiment was complete, all the data were transferred to Microsoft Excel and converted to a percentage of oxygen depletion within each well over time. A graph was made in Excel to determine whether oxygen concentration was decreasing over time for each larva, which would demonstrate that the larvae are consuming oxygen. As a control, boiled larvae were used in the experiments, a s well as larvae injected with

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34 c yanide, which directly blocks mitochondrial oxygen uptake, to obser ve larval mortality within the well while respiration rates were being taken Anoxia Tolerance Fo urth and f ifth instar ( 12 .7 to 25.5 m g) Brephidium pseudofea larvae were exposed to anoxia for 2 to 6 h in nitrogen atmosphere and in deoxygenated salt water Ten larvae were randomly placed in a control group or a nitrogen treated group. At least 2 replicates were completed for each time interval. Only larvae used for the control treatments were reused in experiments. Each larva was weighed and placed in a p atent lip vial (4 d ram; BioQuip) that contained a 2 cm piece micro pipet at the bottom. A 100 l disposable micropipet (Fisherbrand) was cut with a file into five 2 cm pieces and the pieces were filled with 20 l of a 10% sodium hydroxide (NaOH) solution to absorb respired CO2. A small piece of cotton separated the larva and the NaOH to ensure that they would not come into contact during the experiment (Figure 2 4). Nitrogen gas was pumped into the vials (to purge any oxygen in the vial) at a constant flow for 2 min for the nitrogen treated larvae, while the control group had room air from an aquarium pump that was dried by running it through a Dryrite column. These 2 min intervals were staggered between the nitrogen group and the control group and as soon as the intervals were completed, rubber stoppers were tightly closed on the vials. The ten vials were left for a period of 2 to 6 h. After the respective time interval, the vials were opened and the larvae transferred to another vial with a fresh sprig of Sarcocornia perennis to feed on. After 24 hours, survival was evaluated among the two groups and recorded. The same methods were used for the experiments conducted in salt water. The vials were half filled with salt water so as to not have the water bubble out the top and nitrogen gas pumped into the vial for 2 min for treated groups and room air for the control groups ; the air in the nitrogen treated vials was also made anoxic, and therefore no oxygen diffused into the water.

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35 Another method was attempted during these experiments pertaining to the control individuals in salt water. The salt water was aerated constantly for the entire duration of the experiment, instead of only 2 min, as in the original experiments. This was to ensure that the control indivi duals constantly had a flow of oxygen. The nitrogen treated individuals were only given 2 min of nitrogen gas and the vial was closed with a rubber stopper that would keep the vial anoxic for the whole time. Results Tidal Activity for 2009 2010 In Cryst al River, Florida, 50% of Sarcocornia perennis stems were evaluated as partially inundated when the high tide level was 4.0 ft, and 100% were completely inundated at 4.5 ft high. During the months of January through March, tides would only reach up to 4.0 ft one to six times per month (Figure 25 and Figure 26). In these early months, larvae may be able to avoid most high tides by crawling up the stem of the plant because they only get partially inundated. During the summer months, May through August ( peak breeding months for Brephidium pseudofea) tides reach heig hts of up to 4.8 ft (Figure 26). In these months, immature stages are prone to complete inundation two to five times per month; throughout these inundation periods, the eggs, larvae, and pupae wo uld be subjected to complete inundation because the water level would exceed the height of the majority of plant stems in the habitats at Crystal River. Also during the summer months, immature stages would be prone to partial inundation 13 to 24 times per month. During all other months (April, September, October, November, and December) high tides would reach up to 4.3 ft to partially inundate most plants, but not quite be high enough to completely inundate them. The immature stages would be prone to parti al inundation f or 7 to 20 times each the month (Figure 2 6). From these data, immature stages would have to survive

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36 complete salt water inundation periodically during the summer months, which is also the eastern pygmy blue butterflys peak breeding season. Morphological Analysis Detailed photographs of all life stages of Brephidium pseudofea were taken (Figure 2 7). The scanning electron micrographs showed t he egg of B. pseudofea (Figure 2 8A ) as somewhat flattened on top. The ribs connect enlarged nodules that sometimes contain aeropyles (Figure 2 8B) The fossae contain numerous pores that may function as a plastron to keep a layer of surrounding air around the egg (Figure 2 8B). The micropylar region within this species seems to be variable between eggs (Figure 2 8C ) The setae on the larvae are very short, clavate and held parallel to the body (Figure 2 9) although prominent setae are found on the prothorax and mesothorax and also on the last abdominal segment. The Newcomers organ, inverted tentacular organs, and the pore copula organs were observed in B. pseudofea and photographed (Figure 210). Nothing on the pupae was found to be unusual. The spiracles of B. pseudofea were investigated because they are the entrance to the tracheal system where respir ation occurs Auto montage images of the spiracles in B. pseudofea in room air appeared shiny (Figure 2 11A) When larva e were placed under tap water, air was clearly retained in and around the spiracles and trachea and also appeared shiny (Figure 2 11B) suggesting that the region around the spiracle may act as an air pocket for diffusion of oxygen from the surrounding water. A darkening of the spiracle occurred immediately when the 0.6% Triton X 100 detergent solution, a wetting agent, was added ; in less than 5 min, water had seeped into the spiracl es and trachea and no air was visible (Figure 2 11C ). Respirometry Measurements As a control, live larvae of Brephidium pseudofea were compared to thei r boiled, dead counterparts that had very low respiration r ates (Figure 2 12) (T Test, T13= 7.853, p<0.0001).

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37 Live B. pseudofea larvae clearly consumed much more oxygen under water than dead larvae. Low respiration rates found in boiled, dead larvae was possibly due to decomposition of the larvae in the respiromet ry chamber over time Comparisons of live and dead individuals were made for all five terrestrial species and all the boiled larvae showed only trace levels of oxygen consumption, therefore respiration rates were only compared among live larvae of all species The larvae of B. pseudofea had substantially higher respiration rates when submerged than the other five terrestrial lepidopteran species (Table 21 and Figure 2 13) ( ANOVA, F5,51Behavioral observations were m ade for all six species while water was being poured into the respirometer and slowly flooding the individual. B rephidium pseudofea s reaction to water was calm as compared to other species, with most B. pseudofea larvae defecating as soon as water would touch them followed by quiescence within 10 s after larvae were fully submerged Some B. pseudofea larvae would slowly try to crawl away from the water before defecating but take no longer than 30 s to fall into quiescence once they were fully submerged. H elicoverpa zea, = 15.172, p<0.0001). These results are consistent for possibly having re spiratory adaptations to periodic inundation in B. pseudofea. Preliminary observation s of survival rates showed that B. pseudofea larvae could survive longer periods of salt water inundation than the other five lepidopteran species studied: 83.3% of larva e were able to survive at least 3 h (n= 5) and two larvae were able to survive at least 4 h (n= 2) One larva was inundated for 6.5 h and survive d. It was found that only 20% of Spodoptera frugiperda larvae were able to survive 3 h or less (n= 5) and 66.7 % of Helicoverpa zea larvae were able to survive 3.5 h or less (n=6) J unonia coenia larvae were only able to survive up to 2 h (n= 4), while Manduca sexta and Callophrys irus were not even able to survive 2 h (n= 10; n= 10).

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38 J. coenia, S. frugiperda, and M. sexta tried to avoid the water and some larvae were very frantic, even when fully submerged. Larvae would remain active for about 30 s after defecating and then enter quiescence. C allophrys irus did not r eact to the water at all and immediately enter ed quiescence. Discussion T idal charts were reviewed to estimate the monthly inundation periods that immature stages would have to endure and they demonstrated that Brephidium pseudofea eggs, larvae, and pupae would be subject to salt water inundation periodically during the summer months. The results of this research indicated that eastern pygmy blue larvae are able to respire during salt water emersion Five other terrestrial lepidopteran species including a nother species in family Lycaenidae, w ere also shown to respire under water, but at substantially lower rates Although this study was not done in a phylogenetically correct ed framework to account for relatedness between species within the lepidoptera, it s till gave a broad view of the tolerance for salt water inundation that lepidopteran larvae have. The substantially higher respiration rates found in B. pseudofea larvae when compared to terrestrial lepidopteran species suggest that the eastern pygmy blue m ay have evolved a physiological adaptation to being periodically inundated by salt water Careful examination of the spiracles through automontage showed that the larvae of B. pseudofea can trap small pockets of air and possibly use them as a diffusion la yer to absorb oxygen from the surrounding water during periods of inundation, although this hypothesis was not able to be tested Among the experiments done during this research, many were unsuccessful and adjustments had to be made throughout the course o f this study Tidal Activity for 2009 2010 Tidal charts were reviewed for Crystal River (Citrus County) Florida in an attempt to estimate the monthly inundation periods that Brephidium pseudofea immature stages would have

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39 to endure (Figure 2 6) During the summer months (May to August), eggs, larvae, and pupae would be prone to complete inundation two to five times per month. These inundations are mandatory because the tide is at its highest point during these months and most of the Sarcocornia perennis plants will be completely submerged under salt water. All other months of the year, immature stages are prone to par t i al inun dation for less time per month; larvae may not even get submerged if they are able to avoid the water by crawling up the stem of t he plant (Figure 2 5) However, eggs and pupae would have to endure the submersion year round because they are immobile during the high tides Because B. pseudofea immature stages are prone to complete salt water inundation during a period where populations increase the eggs, larvae, and pupae are expected to have physiological adaptations to be able to tolerate this harsh environment and the stressors associated with it. Morphological Analysis Scanning electron m icroscope (SEM) images were obtained for all life stages of Brephidium pseudofea. The egg showed characteristics consistent with a plastron that could aid the egg in respiring under water durin g periodic inundation (Figure 2 8) However, many insect eggs are very small and have a plastron as a means to survive when they are flooded by rains (Hinton 1969). Hence t his characteristic is unlikely to be a specific adaptation to tidal inundation in B. pseudofea. The presence of ant organs was confirmed in this species by SEM (Figure 2 10 ) The tentacular organs were observed but only while they were still inverted inside the body. Attempts were made to evert the tentacular organs by boiling the larvae and/ or placing the larvae in 95% ethanol for several minutes (methods shown successful in everting the tentacular organs for other lycaenids ). The tentacular organs in B. pseudofea, however, did not evert and pictures were only obtained of inverted tentacular organs.

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40 The SEM images also showed that the setae on the caterpillars are very short (Figure 2 9) and unlike that of other genera in the family Lycaenidae (personal observation). Ballmer and Pratt (1988) studied the last instar of Brephidium exilis a congener of B. pseudofea in California, and showed that they have similar setae that are short, clavat e capitate, and held parallel to the body. While the setae did not exhibit characteristics consistent with a plastron automontage images of the spiracles of B. pseudofea successfully showed that air is trapped inside while larvae are submerged underwater (Figure 2 11) The larvae possibly use this air as a physical gill to absorb oxygen from the water and replenish the air trapped within the spiracle. Larvae of Spodoptera frugiperda were also placed under light microscopy and the spiracles also showed air trapped within. This may be a feature all lepidopteran larvae possess in case they are flooded by rains. Because all of the larvae studied during this research were able to respire underwater, this may be the means that they are able to breathe It is als o possible that the larvae are using direct diffusion as a source of oxygen exchange by absorbing oxygen directly through their cuticle. Respirometry Measurements The respiration rates of Brephidium pseudofea possibly indicate that larvae have evolved ad aptation s to periodic salt water inundation when compared to rates of other terrestrial lepidopteran species under water Preliminary studies showed that most B. pseudofea larvae could survive at least 4 h of inundation in salt water, with a single larva l iving up to 6.5 h of inundation. B rephidium pseudofea larval respiration rates under water were compared to five terrestrial lepidopteran species in an attempt to observe whether B. pseudofeas rates were unusually high. All six species of lar vae were show n to respire under water, although B. pseudofea larvae had substantially higher respiration rates (Table 2 1 and Figure 2 13 ) This may indicate that all lepidopteran larvae possess some mechanisms for gas exchange during submersion in water

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41 allowing them t o survive periodic inundation, but the intertidal B. pseudofea larvae may have additional mechanisms that allow for greater gas exchange. There have been several studies comparing metabolic rates bet ween species and their habitats, and results found for B pseudofea are consistent with observations in other systems. For example, Apodaca and Chapman (2004) compared metabolic rates of gilled and gill less larval damselflies to discover the mea ns by which each survived under water. A fiber optic oxygen sensor similar to the one used for B. pseudofea, was used to measure oxygen consumption (g O2 h1S piracular Air Bubble ) of Proischnura subfurcatum and found that gilled damselfly larvae had higher metabol ic rates than gillless larvae, suggesting that gills were an adopted mechani sm to increase underwater gas exchange Gillless damselflies altered their behavior under water and did not have to breathe as much ; gillless damselflies were able to survive without utilizing gills to breath Heisey and Porter (1977) found differences be tween Daphnia species and the environment that they live in They me a sured respiration rates (l/ mg dry wt/ h) of two different species of Daphnia, whose habitats vary in oxygen concentration, to see whether environment affected metabolism They showed that D. magna, which inhabits ponds with large fluctuations in oxygen concentration, are able to regulate their oxygen consumption much better than D. galeata mendotae which inhabit environments with constant levels of oxygen at near saturation or above The objective of this experiment was to determine whether the air pockets found within and around the spiracles of Brephidium pseudofea larvae were important for respiration and if wetting the air pockets would alter the larvas metabo lic rate The 0.6% and 0.3% Triton X 100 detergent solution wet the air bubble in a matter of minutes ( as observed in Auto montage imaging) (Figure 2 11) Larvae were then placed in the respirometer to measure respiration rates.

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42 Triton dipped l arvae had mu ch lower respiration rates, as compared to other live larvae that were not dipped. However, larvae died soon after soap exposure thus reduced metabolic rates could have been due to a lack of respiration by the larvae because they were dying. A larva that was dipped in the solution and rinsed off with deionized water but never submerged under water, still died due to soap exposure Although using soap exposure to wet the spiracular air bubble in B. pseudofea was not successful because of the toxicity to the larva e other studies have been conducted to wet plastrons or air bubbles by other means such as using low surface tension water (where water no longer bonds to itself) and mild detergents An air bubble will be made dysfunctional with water of low surf ace tension because instead of bonding to itself, water will bond to the wetting agents nonpolar material. For example, Hebets and Chapman (2000) successfully wetted the plastron of Phrynus marginemeculatus ( whip scorpion) by using water of low surface tension. These whip scorpions inhabit environments that are periodically flooded and therefore need a means of res piring under water: in this case, a plastron. After wetting the plastron with low surface tension water, the animals were unresponsive after 14 m in, while control individuals that did not have their pl astrons wetted were responsive. Various surface tensions of the water were created with different amounts of detergent, but naturally produced low surface tension water was also prepared. The naturall y produced low surface tension water was made by adding one drop of NP40 (detergent) dead crickets, dirty paper towels, and dead cabbage looper caterpillars to water that was allowed to sit for one week. This naturally produced dirty water may be less t oxic and perhaps may be useful in testing the function of spiracular air bubbles in gas exchange Early Respirometry Trials Using a modified microtiter plate reader was designed to simplify this project by allowing respiration rates to be quantified for s everal Brephidium pseudofea larvae during a single run

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43 (Figure 2 3) but poor results were obtained. While live larvae were contained within the wells, the oxygen levels decreased, appearing as if the la rvae were respiring Controls of dead larvae (boiled and cyanide injected ) were used to show that dead larvae consume significantly less oxygen than live larvae, but oxygen levels decreased as well, implying that the dead larvae were also respiring at substantial levels Because it was assumed that the resul ts from the dead larvae were flawed, the modified microtiter plate experiments were discontinued; techniques for measuring respiration rates were altered and rates were measured using an oxygen sensing probe where dead individuals showed expectedly low le vels of oxygen consumption. Although not successful for this research, Szela and Marsh (2005) developed a 384chamber, microrespirometer that they used to quantify respiration rates of nauplii of an Artemia sp (brine shrimp) They used 384well plates tha t contained varying numbers of live nauplii in each well, as well as wells that contained no nauplii and only artificial seawater. More than 1000 measurements were taken to provide a vast distribution of metabolic rates within a cohort of larvae. Interesti ngly, t hey also measured respiration rates of dead larvae as a control, but found high respiration rates in their apparatus for dead nauplii. T hese high respiration rates were assumed by Szela and Marsh (2005) to be due to biochemical oxidation reactions a ssociated with decomposition, particularly in small volume s Anoxia Tolerance The objective of these experiments was to determine if Brephidium pseudofea larvae were able to tolerate anoxia in air and water While the larvae are inundated, the water may become a noxic (or hypoxic), therefore determining whether larvae would be able to survive a lack of oxygen is of importance The B. pseudofea control larvae survived all the treatments they were put through in room air. The nitrogen gas treated larvae surv ived up to 5 h of anoxia. At 6 h, some larvae in the group started to die. In water, results were unusual in that control larvae,

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44 which were only subjected to 2 min of room air, did not survive at 2 h, but nitrogen treated larvae (where the water was made anoxic) did survive. It is assumed that the control larvae were running out of oxygen during the 2 h run. The revised knockdown experiment s were created for this reason and results were that half of the control larvae survived in water at 2 h of submersion and half of the nitrogen treated larvae survived. As the time interval increased to 4 h, all the control larvae survived while all the nitrogen treated larvae died. The knockdown experiments were discontinued because of the inconsistent results and limited biological material, so research was focused on measuring metabolic rates. Insects demonstrate a variety of adaptations to overcome hypoxic and anoxic environments including floodprone soils and burrows, intertidal zones, ice encasement and high alti tudes Insects that experience flooding can overcome hypoxia by quiescence, cuticular gas exchange, air bubbles, lowering metabolic rate, and/ or anaerobic respiration (Hoback and Stanley 2001). For example, Bledius spectabilis rove beetles, are able to t olerate anoxic seawater for up to 36 h by unknown mechanisms (Wyatt 1986). Intertidal root aphids, Pemphigus treherni are able to tolerate 40 h of anoxic water and 240 h of aerated water. These aphids are possibly maintaining low metabolic rates, using cu ticular gas exchange, and maybe even trapping small air bubbles (Foster and Treherne 1976). Cicindela togata, a burrowing beetle, is able to survive 4 to 5 d of anoxic water. These beetles were shown to dramatically reduce their metabolic rates while using anaerobic respiration. Experiments were done in aerated water and anoxic water, showing no differences in survival rates, therefore C. togata was assumed to not be able to extract oxygen from the water (Hoback et al. 1998; Hoback et al. 2000) Conclusion s Brephidium pseudofea inhabits salt marshes that are periodically flood ed with salt water. The immature stages are subject to salt water inundation during this time and therefore must

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45 endure many stressors, including respiratory limitation and salt exposure Tidal charts were reviewed from March 2009 to February 2010 to estimate the monthly inundation periods that the larvae will be subjected to In the summer months (May to August), larvae must survive two to five complete inundations per month, while the rest of the year they are subject to fewer inundations each month. These periodic complete inundations suggest that B. pseudofea larvae must have some means to respire and survive during this time. Larval respiration rates were measured and B. pseudofea larvae consume d oxygen much faster while inundated by salt water than any other terrestrial species studied, including Helicoverpa zea larvae, Junonia coenia larvae, C allophrys irus larvae, Spodoptera frugiperda larvae, and Manduca sexta larvae. All larvae stu died were able to respire under water, but t he substantially higher metabolic rates demonstrate d by B. pseudofea suggest physiological adaptation s to periodic salt water inundation. Light microscopy showed spiracular air pockets in and around the spiracles while under water in B. pseudofea, although this was also seen in S. frugiperda. These spiracular air pocket s may be the means that lepidopteran l arvae are able to respire under water ; although cuticular respiration is also likely to be an important fact or Preliminary s urvival rates under salt water were observed for all the species studied; B rephidium pseudofea larvae were able to survive salt wat er inundation for at least 4 h, while the other terrestrial species studied could not survive longer than 3 h. Scanning electron microscope (SEM) images confirmed the presence of larval ant organs (N ewcomers organ, inverted tentacular organs, and pore copula organs) in B. pseudofea larvae that would be used in the myrmecophilous relationship that larvae may ha ve with ants. SEM images clearly showed that larvae do not have setae substantial enough to support a plastron around the body although the setae are extremely differ ent from other genera within the

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46 lepidoptera ; the setae are similar to the description of Brephidium exilis setae, a congener to B. pseudofea, as was described by Ballma r and Pratt (1988). Scanning electron microscope images of B. pseudofea eggs showed the presence of numerous pores within the fossae, typically used for holding a plastron. Mos t insect eggs are able to hold plastrons, therefore this is unlikely to be a specific adaptation for B. pseudofea to periodic inundations. Though many experiments attempted during this project were unsuccessful, much was learned. Adjustments made to corre ct the experiments led to a new protocol for measuring res piration rates of insects under water.

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47 Figure 2 1. Larval rearing set up for eggs and larvae of Brephidium pseudofea showing 2.5 cm stems of Sarcocornia perennis and small larvae.

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48 A B Figure 2 2. Experimental set up for respiration rate measurements. A) respirometry chamber showing the micro stir bar at bottom, mesh, and Brephidium pseudofea larva, B) showing oxygen probe during experiment inside respirometry chamber wrapped in parafilm.

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49 Figure 2 3. Modified m icrotitre plate showing wells filled with salt water and Brephidium pseudofea larvae.

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50 Figure 2 4. Experimental set up for anoxia tolerance studies showing 10% NaOHfilled micro pipette at bottom, cotton, and Brephidium pseudofea larva.

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51 A B Figure 2 5. Series of photos showing Sarcocornia perennis plants partially inundated when high tide was at 4.0 ft high at Crystal River, FL. A) close view showing water line and extent of plants inundated, B) expanded view of plants being inundated.

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52 0 5 10 15 20 25 Mar-09 Apr-09 May-09 Jun-09 Jul-09 Aug-09 Sep-09 Oct-09 Nov-09 Dec-09 Jan-10 Feb-10 Month Inundation occurance (days) Partial inundation Complete inundation Figure 2 6. Data from tidal charts between March 2009 to February 2010 showing partial and complete inundation of Crystal River habitat. Partial inundation signifies hi gh t ides of >4.0 ft and complete inundation signifies high tides of >4.5 ft.

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53 A B C D Figure 2 7. Series of Brephidium pseudofea life stages. A) adult (ventral view), B) adult (dorsal view), C) 4th instar larva, C ) pupa.

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54 A B Figure 2 8. Series of s canning electron microscope photographs of Brephidium pseudofea egg. A) whole egg, B) close up showing ribs and nodule containing aeropyle and fossae containing pores for holding a plastron, C) micropylar region

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55 C Figure 2 8. Continued

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56 Figure 2 9. Scanning electron micrograph of spiracle surrounded by small, clavate setae of Brephidium pseudofea.

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57 A B Figure 2 10. Ant organs of Brephidium pseudofea. A) Newcomers organ surrounded by pore cu pola organs, B) inverted tentacular organ.

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58 A B C Figure 2 11. Series of Brephidium pseudofea spiracles. A) spiracles in room air, B) spiracles exposed to tap water clearly showing air pocket, C) spiracles exposed to 0.6% Triton X 100 detergent where air pocket has disappeared.

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59 Live Dead Mean Respiration Rates (ul/ mg/ min) 0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 p< 0.0001 Figure 2 12. The significantly lower respiration rates (m ean SE) of dead Brephidium pseudofea larvae compared to live larvae indicate that live larvae are able to respire under salt water. p< 0.0001

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60 Table 2 1. Respiration rates (mean SE) showing that Brephidium pseudofea had substantially higher respiratory activity underwater than the other terrestrial species studied and that boiled larvae consumed only small quantities of oxygen compared to their live counterparts Weights ( m g) are also shown for each species. Species Respiration rate ( l/ mg/ min ) (n= 10) Boiled larva rate ( l/ mg/ min ) (n=5) Live larval weight range ( m g) (n= 10) Boiled larval weight rang e ( m g) (n=5) B. pseudofea 0.0005 3.315 0 e 5 a 0.0001 1.6655e 5 a Z 17 .5 to 26 1 15 .8 to 20 0 H. zea 0.0003 2.8055e 5 b 8.5083e 5 2.4643e 5 a 9 .8 to 25 7 10 .3 to 25 2 J. coenia 0.0002 1.9584e 5 b 7.9462e 5 2.8781e 5 a 12 .4 to 25 8 13 .2 to 20 3 S. frugiperda 0.0002 4.0448e 5 b 0.0001 2.9309e 5 a 12 .3 to 36 0 11 .1 to 26 3 M. sexta 0.0002 1.9054e 5 b 9.0305e 5 1.8396e 5 a 11 .4 to 26.0 11 .7 to 17 8 C. irus 0.0002 3.857 0 e 5 b 7.4342e 5 3.2166e 5 a Y 12 .2 to 33 0 X 8 .3 to 31 3 Y ZValu es followed by different letters are significantly different within a column at p< 0.05, according to Tukey Kramer HSD test for multiple comparisons. YC allophrys irus live larvae had different sample sizes t han the other species with n= 7, but the variance for this group was not different from the others. X XC allophrys irus boiled larvae had different sample sizes th an the other species with n= 3, but the variance for this group was not different from the others.

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61 Species BP HZ JC CI SF MS Mean Respiration Rates (ul/ mg/ min) 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 A B B B B B Figure 2 13. The significantly higher respiration rates (m ean SE) of Brephidium pseudofea larvae under salt water compared to other terrestrial lepidopteran larvae suggest respiratory adaptation to the intertidal environment in B. pseudofea. Values followed by d ifferent letters are significantly different at p< 0.05, according to Tukey Kramer HSD for multiple comparisons. Species are as follows: BP= Brephidium pseudofea, HZ= Helicoverpa zea JC= Junonia coenia, CI= Callophrys irus SF= Spodoptera frugiperda, and MS= Manduca sexta.

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62 CHAPTER 3 CONCLUSIONS AND SUGGESTIONS FOR FURTHER RESEARCH The intertidal eastern pygmy blue butterfly, Brephidium pseudofea, inhabits salt marshes that are periodically inundated by salt water. The objective of this research was to investigate possible physiological adaptations of the larval stages to periodic inundation, focusing on respiration of the larvae under water. Tidal charts for Crystal River (Citrus County) in Florida (a typical B. pseudofea habitat) were reviewed for the p eriod of March 2009 to February 2010 to estimate how many times per month the habitat would be subjected to partial or complete submersion. During the summer months (May to August), plants and larvae will be subjected to complete submersion two to five times per month. During the rest of the year, plants would only be subjected to partial submersion of up to 20 times per month; partial submersion indicates that the larvae may be capable of avoiding the water by crawling up the stem if the plant is not compl etely submerged. Because larvae will be subjected to complete inundation periodically during the summer months, larvae must have a means to respire during this time. Respiration rat es were measured with a fiber optic oxygensensing probe for B. pseudofea. To show whether metabolic rates in B. pseudofea were different from other lepidopteran species that are not prone to periodic inundation, respiration rates of five terrestrial lepidopteran species were measured : Helicoverpa zea, Junonia coenia, Callophrys irus Spodoptera frugiperda, and Manduca sexta. Brephidium pseudofea larvae had significantly higher respiration rates underwater than the other five terrestrial species studied, suggesting adaptation to a periodically inundated habitat. Control treatment s were run using boiled, dead larvae of each species, which had significantly lower respiration rates than each of their live counterparts. B rephidium pseudofea larvae were also observed to survive at least 4 h of complete submersion in salt water, with one larva surviving a 6.5 h submersion.

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63 Careful examination of the spiracles under water revealed spiracular air pockets, and further assessment showed that these air pockets were disrupted when exposed to a detergent solution. The air pockets may act as a d iffusion layer for absorbing oxygen from the surrounding water for the larvae to use while inundated. Interestingly, l arvae of S. frugiperda also had air pockets in their spiracles while submerged under water. Because all six species of larvae stu died were able to respire under water, spiracular air pockets may be one of the mechanisms used for underwater respiration. Lepidopteran larvae may also be using cuticular respirat ion as a means to respire under water by diffusing oxygen through their cuticle. Larval ant organs were confirmed in this species from a s canning electron m icroscope. The N ewcomers organ, tentacular organs, and pore copula organs were seen, but the tentacular organs were inverted and resisted eversion Larval setae was also studied and show n from SEM images to be small, clavate, and held parallel to the body appearing very different from other genera in the lepidoptera. The eggs of B. pseudofea are somewhat flattened and have small pores within the fossae, possibly for holding a plastron. Many experiments were attempted during this research and some proved unsuccessful Soap exposure was used on the spiracles of B. pseudofea larvae to disrupt the spiracular air bubble but the toxicity of the soaps killed the larvae and these experiments were terminated. Anoxia tolerance studies were attempted to observe whether the larvae were able to survive anoxia for various periods of time but these experiments were discontinued due to a lack of consistency between trials Respiration rates were measur ed with a microtiter plate reader (designed to measure rates of several larvae at one time), but dead, boiled larvae were also shown to respire; therefore, these experiments were also discontinued.

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64 More research is necessary to determine the means that B. pseudofea larvae are able to respir e under water. Soap exposure was attempted during this research to break the bubbles surrounding the spiracles, but soap exposure caused mortality; perhaps a different approach to this type of experiment would prove succe ssful. Using various concentrations of low surface tension water may be less toxic to the larvae. P roducing low surface tension naturally would likely be less toxic to the organism, as long as it is able to wet the surface of the spiracle. Further anoxia t olerance experiments are necessary to determine whether the larvae are able to survive without oxygen under water. Larval respiration underwater has been the main focus throughout the course of this research, but there are many other challenges that these l arvae must overcome, including being exposed to salt water and feeding on a plant that is presumably high in salinity. Further studies need to be conducted on these aspect s, as well as the salt water tolerance and aquatic respiratory abilities of the eggs and pupae. Although this research was primarily focused on the physiological processes of larval respiration, further studies on the behavior and life history strate gies of this insect are still required. Further investigation of the myrmecophilous relati onship that the larvae have with ants would prove valuable. There is limited literature on the eastern pygmy blue and there is so much more to be learned about this exceptional intertidal insect

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65 LIST OF REFERENCES Ballmer, G. R., and G. F. Prat t. 1988. A survey of the last instar larvae of the Lycaenidae of California. J. Res. Lepid. 27(1): 181. Bell, R. A., and F. A. Joachim 1976. Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Entomol. Soc. Am. 69: 365373. Borror, D. J, C. A. Triplehorn, and N. F. Johnson. 1989. An introduction to the study of i nsects. Saunders College, Philadelphia PA Braby, M. F. 2000. Butterflies of Australia : T heir identification, biology and distribution, vol. 2. CSIRO, C ollingwood, Australia. Cheng, L. 1976. Marine insects. North Holland, New York, NY Cottrell, C. B. 1984. Aphytophagy in butterflies: I ts relationship t o myrmecophily. Zool. J. Linn. Soc. 79: 157. Daly, H. V., J. T. Doyen, and A. H. Percell III. 1998. Introduction to insect biology and diversity. Oxford University, New York, NY. Eastwood, R. and A. M. Fraser 1999. Associations between lycaenid butterflies and ants in Australia Aust. J. Ecol. 24: 503537. Eliot, J.N. 1973. The higher classificat ion of the Lycaenidae (Lepidoptera): A tentative arrangement Bull. Br Mus. (Nat. Hist.) Ent. 28: 371505. Eva ns, H.E. 1984. Insect biology: A textbook of entomology. Addison Wesley, Reading MA Foster, W. A., and J. E. Treherne. 1976. Insects in m arine saltmarshes: P roblems and adaptations. pp. 542. In L. Cheng, Marine insects. NorthHolland, New York, NY. Gullan, P. J. and P. S. Cranston. 1994. The i nsects: A n outline of entomology Chapman and Hall, London, United Kingdom Guy, R. N., N. C. Leppla, J. R. Rye, C. W. Green, S. L. Barette, and K. A. Hollien. 1985. Trichoplusia ni. Pp. 487494. In P. Sing and R. F. Moore (eds.), Handbook of insect rearing, vol.2, Elsevier, Amsterdam. Hall, D. W. and J. F. Butler. 2003. Eastern Pigmy Blue, Brep hidium isophthalma pseudofoea (Morrison) (Insecta: Lepidoptera: Lycaenidae). http://creatures.ifas.ufl.edu/bfly/eastern_pigmy_blue.htm Harvey, D. J. and J. Longino. 1989. Myrmecophily and larval food plants of Brephidium isophthalma pseudofea (Lycaenida e) in the Florida Keys. J. Lepid Soc. 43(4): 332333.

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66 Hebets, E. A. and R. F. Chapman. 2000. Surviving the flood: P lastron respiration in the nontracheate arthropod Phrynus marginemaculatus (Amblypygi: Arachnida). J. Insect Physiol. 46: 1319. Heisey H., and Porter, K. G. 1977. The effect of ambient oxygen concentration on filtering and respiration rates of Daphnia galeata mendotae and Daphnia magna. Limnol. Oceanogr. 22(5): 839845. Hill, R. W., G. A. Wyse, and M. Anderson. 2004. Animal physiology Sinauer Associates, Sunderland, MA Hinton, H. E. 1969. Respiratory syst ems of insect egg shells. Annu. Rev. Entomol. 14: 343368. Hoback, W. W., J. E. Podrabsky, L. G. Higley, D. W. Stanley, and S. C. Hand. 2000. Anoxia tolerance of con familial tig er beetle larvae is associated with differences in energy flow and anaerobiosis. J. Comp. Physiol. 170: 307314. Hoback, W. W., D. W. Stanley, and L. G. Higley. 1998. Survival of immersion and anoxia by larval tiger beetles, Cicindela togata. Am. Midl. N at. 140(1): 2733. Hoback, W. W., and D. W. Stanley. 2001. Insects in hypoxia. J. Insect Physiol. 47: 533542. Howe W. H. 1975. The butterflies of North America. Doubleday, Garden City, New York JMP, Version 7. 19892007. SAS Institute Inc., Cary, NC. Kirsch, R. and G. Nonnotte. 1977. Cutaneous respiration in three freshwater teleosts. Resp. Phys iol 29: 339354. Kitching, R. L. 1983. Myrmecophilous organs of the larvae and pupae of the lycaenid butterfly Jalmenus evagoras (Donovan). J. Nat. Hi st. 17(3): 471481. Kolsch, G., K. Jakobi, G. Wegener, and H. J. Braune. 2002. Energy metabolism and metabolic rate of the alder leaf beetle Agelastica alni (L.) (Coleoptera, Chrysomelidae) under aerobic and ana erobic conditions: A microcalorimetric stu dy. J. Insect Physiol. 48: 143151. New, T. R. 1993. Conservation biology of the Lycaenidae (butterflies). IUCN, Gland, Switzerland. Pavulaan, H. and R. R. Gatrelle. 1999. A new subspecies of Brephidium isophthalma (Lycaenidae: Polummatinae) from coasta l South Carolina. The Taxonomic Report 1(7): 1 4. Pierce, N. E., M. F. Braby, A. Heath, D. J. Lohman, D. B. Rand, and M. A. Travassos. 2002. The ecology and evolution of ant association in the Lycaenidae (Lepidoptera). Annu. Rev. Entomol. 47: 733771.

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67 R awson, G. W. 1961. The early stages of Brephidium pseudofea (Morrison) (Lepidoptera: Lycaenidae). N. Y. Entomol. Soc. 19: 8891. Shields O. 1989. World numbers of butterflies. J. Lepid. Soc. 43: 178183. Smart, P. 1975. The international butterfly book: A complete guide to butterflies of the world. T. Y. Crowell, New York NY. Strathmann, R. R., L. R. Kendall, and Marsh, A. G. 2006. Embryonic and larval development of a cold adapted Antarctic ascidian. Polar Biol. 29: 495501. Szela, T. L., and A. G. Marsh. 2005. Microtiter plate, optode respirometry, and inter individual variance in metabolic rates among nauplii of Artemia sp. Mar. Ecol. Prog. Ser. 296: 281289. Wahlberg, N., M. F. Braby, A. V. Z. Brower, R. de Jong, M. Lee, S. Nylin, N. E. Pierc e, F. A. H. Sperling, R. Vila, A. D. Warren, and E. Zakharov. 2005. Synergistic effects of combining morphological and molecular data in resolving the phylogeny of butterflies and skippers. Proc. R. Soc. B. 272: 15771586. Wagner, D. L. 2005. Caterpillar s of eastern North America. Princeton University Press, Princeton NJ Wegener, G. and T. Moratzky. 1995. Hypoxia and anoxia in insects: M icrocalorimetric studies on two species ( Locusta migratoria and Manduca sexta) showing different degrees of anoxia tolerance. Thermochimica Acta 251: 209218. Williams, D. D. and N. E. Williams. 1998. Aquatic insects in an estuarine environment: D ensities, distribution an d salinity tolerance. Freshw. Biol. 39: 411421. Wyatt, T. D. 1986. How a subsocial intertidal beetle, Bledius spectabilis prevents flooding and anoxia in its burrow. Behav. Ecol. Sociobiol. 19: 323331. Zerm, M. and J. Adis. 2003. Exceptional anoxia resistance in larval tiger beetle, Phaeoxantha klugii (Coleoptera: Cicindelidae). Phys iol Entomol. 28: 150153. Zirlin, H. 2007. Ta xonomists just wanna have fun: T rue blue, part one. American Butterflies Fall/ Winter: 72 78.

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68 BIOGRAPHICAL SKETCH Valerie McManus was born in 1984, in Hollywood, FL. She grew up with her parents and two older brothers. After high school, she attended Florida Southern College in Lakeland, FL for one year before transferring to the University of Florida. Valerie has had several majors ranging from education, psychology, and animal sciences before finally settling down as an entomology major. She did an internship at the Butterfly Rainforest where her interest in Lepidoptera sparked. After receiving her bachelors degree in en tomology, Valerie continued in entomology to get her Master of Science degree. Valerie recentl y got engaged to her best friend, Oren, and they will be married in November 2009.