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Survival and Behavior of Coptotermes Formosanus Shiraki (Isoptera

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Title:
Survival and Behavior of Coptotermes Formosanus Shiraki (Isoptera Rhinotermitidae) after Flooding in New Orleans, Louisiana
Creator:
Owens, Carrie B
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (99 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Su, Nan-Yao
Committee Members:
Scheffrahn, Rudolf H
Moore, Kimberly A
Husseneder, Claudia
Riegel, Claudia
Graduation Date:
12/17/2011

Subjects

Subjects / Keywords:
Bodies of water ( jstor )
Boxes ( jstor )
Floods ( jstor )
Foraging ( jstor )
Hurricanes ( jstor )
Mortality ( jstor )
Oxygen ( jstor )
Soldiers ( jstor )
Subterranean termites ( jstor )
Termites ( jstor )
Entomology and Nematology -- Dissertations, Academic -- UF
coptotermes -- flooding -- survival
City of Fort Lauderdale ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Entomology and Nematology thesis, Ph.D.

Notes

Abstract:
Hurricane Katrina made landfall along the Gulf Coast on August 29, 2005, inundating approximately 80% of New Orleans, Louisiana for up to three weeks in some areas. The Formosan subterranean termite, Coptotermes formosanus, has become a well-established structural pest since its introduction to New Orleans. The goal of this study was to determine if and how C. formosanus colonies were able to survive prolonged flooding following Hurricane Katrina. Microsatellite genotyping conducted on termite samples collected from inundated areas pre- and post- flooding yielded that C. formosanus colonies survived prolonged flooding. Several studies were conducted to determine the survival mechanisms of colonies. First, a bioassay was conducted in which groups of termites were maintained at three different temperatures and submerged within containers. Their mortality was recorded at increasing time intervals, and the lethal times were calculated. It was determined that individual termites could not have survived the flooding following Hurricane Katrina by tolerating inundation. A second bioassay was conducted, in which groups of termites were confined to an airtight environment at varying temperatures. Their mortality was recorded at predetermined time intervals, and lethal times were calculated. It was determined that termites could have survived flooding following Hurricane Katrina for up to three weeks if trapped in a large enough pocket of air within their nest, or if the temperature was relatively cool. To determine if C. formosanus colonies survive flooding by evacuating or shifting their foraging areas to escape rising flood waters, a field study was conducted along seasonally inundated areas. It was concluded that termites do not shift their foraging areas, nor do they move vertically within trees, to escape seasonal inundation. A bioassay was conducted to determine if C. formosanus colonies survive inundation by creating a watertight environment within their nest. Termite foragers were added to artificial trees and foraging arenas, then inundated while behavior was observed. It was shown that C. formosanus colonies could have survived the flooding following Hurricane Katrina by creating a sealed nesting system and exploiting oxygen within pockets of air within the carton material or within the gallery systems above the water line. ( en )
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.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Su, Nan-Yao.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31
Statement of Responsibility:
by Carrie B Owens.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Owens, Carrie B. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/31/2013
Classification:
LD1780 2011 ( lcc )

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SURVIVAL AND BEHAVIOR OF Coptotermes formosanus SHIRAKI (ISOPTERA: RHINOTERMITIDAE) AFTER FLOODING IN NEW ORLEANS, LOUISIANA By CARRIE BETH OWENS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1

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2011 Carrie Beth Owens 2

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To my grandfather, Col. Lawr ence G. Brown, who once told me, All persons with effort can attain something worthwhile in lif e, and make contributions to others. 3

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ACK NOWLEDGMENTS I would like to foremost t hank my major advisor, Dr. Nan-Yao Su (University of Florida, Fort Lauderdale, FL). I also t hank the following graduate committee members: Drs. Claudia Riegel (New Orleans Mosquito Termite, and Rodent Control Board, New Orleans, LA), Claudia Husseneder (LSU AgCenter, Baton Rouge, LA), Rudolph Scheffrahn (University of Florida, Fort Lauderdale, FL), and Kimberly Moore (University of Florida, Fort Lauderdale, FL) for their advic e and constructive criticisms. My gratitude also extends to Ed Bordes (NOMTCB, New Orleans, LA), Drs. Mike Carroll (NOMTCB, New Orleans, LA), Alan Lax (USDA ARS, Ne w Orleans, LA), Frank Guillot (USDA ARS, New Orleans, LA), and Kenneth Brown (BASF, St. Louis, MO ) for their involvement. I wish to give a special thanks to Dawn Simms (LSU AgCenter, Baton Rouge, LA) and Jennifer Delatte Donaldson (LSU Ag Center, Baton Rouge, LA) for their assistance and training in microsatellite genotyi ng protocols. I greatly appreciate all the field assistance and encouragement I have received from the following NOMTCB personnel: Ed Freytag, Barry Yokum, Eric Guidry, Barry Lyons L.J. Kabel, Jimmy Jesse, Timmy Madere, Perry Ponseti, Jamie Ward, and Frank DiGiovanni. On a personal note, I want to thank my family for their continued love and support. Without my loved ones, my academic career would not have been possible. 4

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TABL E OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF TABLES............................................................................................................7 LIST OF FI GURES..........................................................................................................8 ABSTRACT .....................................................................................................................9 CHAPTER 1 INTRODUC TION....................................................................................................11 The Formosan Subterranean Termite: Pr oblem and Economic Importance...........12 Distribut ion..............................................................................................................15 Life History..............................................................................................................16 Control Strategies...................................................................................................18 Colony Survival of Inundat ion.................................................................................21 2 MOLECULAR GENETIC EVIDENCE OF THE SURVIVAL OF Coptotermes formosanus COLONIES AFTER FLOODING.........................................................26 Materials and Methods............................................................................................28 Research Sites and Te rmite Samp les..............................................................28 Microsatellite Genotypin g.................................................................................29 Statistical Analys is............................................................................................29 Result s....................................................................................................................30 Discussio n..............................................................................................................31 3 SURVIVAL OF Coptotermes formosanus FORAGERS FOLLOWING INUNDATION IN A LABORATO RY........................................................................36 Materials and Methods............................................................................................37 Termite s...........................................................................................................37 Inundation Bi oassay.........................................................................................37 Statistical Analys is............................................................................................38 Result s....................................................................................................................39 Discussio n..............................................................................................................40 4 EVALUATION OF SURVIVORSHIP OF Coptoterems formosanus IN A HYPOXIC ENVI RONMENT....................................................................................46 Materials and Methods............................................................................................47 Termite s...........................................................................................................47 Termite Survivorship in an Airtight Environm ent...............................................47 5

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Data Collection and Stat istical An alysis ........................................................... 48 Result s....................................................................................................................49 Discussio n..............................................................................................................51 5 SURVIVAL OF Coptotermes formosanus COLONIES IN SEASONALLY INUNDATED LOCATIONS OF NE W ORLEANS, LO UISIANA...............................59 Materials and Methods............................................................................................59 Research Sites.................................................................................................59 Monitoring Termite Foraging Ar eas..................................................................60 Determining Vertical Movement of Termites within Tr ees................................62 Colony Delineation...........................................................................................62 Result s....................................................................................................................63 Discussio n..............................................................................................................64 6 ABILITY OF Coptotermes formosanus COLONIES TO SURVIVE flooDING WITHIN THEIR N ESTING SYST EM.......................................................................74 Materials and Methods............................................................................................75 Termite s...........................................................................................................75 Artificial Tree I nundation Bioas say...................................................................75 Foraging Arena Inundat ion Bioa ssay...............................................................77 Statistical Analys is............................................................................................79 Result s....................................................................................................................80 Discussio n..............................................................................................................82 7 CONCLUS IONS.....................................................................................................90 LIST OF RE FERENCES...............................................................................................92 BIOGRAPHICAL SKETCH ............................................................................................99 6

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LIST OF TABLES Table page 2-1 Summary of termite samples colle cted from in-ground monitoring stations and flood depth and duration at each resear ch site............................................34 2-2 Summary of colony survival and breeding structure before and after inundatio n...........................................................................................................35 3-1 Comparison of lethal times, LT50s and LT90s (hours), with their associated slopes, 95% CLs, and Pearson X2 values from inundated C. formosanus workers and soldiers maintained at three different temperatures.......................44 4-1 Comparison of lethal times, LT50s and LT90s (days), with their associated slopes, 95% CLs, and Pearson X2 values from groups of 20, 40, and 60 C. formosanus workers and soldiers confined to an airtight environment and maintained at three di fferent tem peratur es.........................................................54 5-1 Foraging activity observed at unf looded and seasonally i nundated areas of New Orleans, Louisiana from Nove mber 2008 through Oc tober 2010...............68 7

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LIST OF FIGURES Figure page 3-1 Percent mortality of 100 inundated C. formosanus foragers maintained at three different temperatur es plotted agai nst time...............................................45 4-1 Percent mortality of termites maintained in an ai rtight environment at 10C plotted again st time............................................................................................56 4-2 Percent mortality of termites maintained in an ai rtight environment at 21C plotted again st time............................................................................................57 4-3 Percent mortality of termites maintained in an ai rtight environment at 32C plotted again st time............................................................................................58 5-1 Locations of in-ground monitoring stations established on 3 m centers extending 9 m from infested tr ees at the cont rol site s........................................69 5-2 Foraging activity of a C. formosanus colony at City Park Popps Fountain.........70 5-3 Foraging activity of a C. formosanus colony on Lakesho re Drive.......................71 5-4 Foraging activity of a C. formosanus colony on Down man Road.......................72 5-5 Foraging activity of a C. formosanus colony along the protected side of the Mississippi River batture.....................................................................................73 6-1 Schematic of an artificial tree infested with C. formosanus foragers within the hollowed center and subject ed to periodic inundation........................................85 6-2 Foraging arena containing 1,000 termi tes, added via the termite release chamber, prior to the addition of water into the bottom half of the arena............86 6-3 Images taken using a video borescope system to determine if water entered hollowed sections of artificial tr ees during periods of inundat ion........................87 6-4 The percent atmospheric oxygen observ ed each week in all artificial trees infested with C. formosanus ...............................................................................88 6-5 Observations of termite movement within a foraging arena subjected to inundatio n...........................................................................................................89 8

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Abstract of Dissertation Pr esented to the Graduate School of the University of Fl orida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy SURVIVAL AND BEHAVIOR OF Coptotermes formosanus SHIRAKI (ISOPTERA: RHINOTERMITIDAE) AFTER FLOODING IN NEW ORLEANS, LOUISIANA By Carrie Beth Owens December 2011 Chair: Nan-Yao Su Major: Entomology and Nematology Hurricane Katrina made landfall along the Gulf Coast on August 29, 2005, inundating approximately 80% of New Orleans, Louisiana for up to three weeks in some areas. The Formosan subterranean termite, Coptotermes formosanus has become a well-established structural pest since its introduc tion to New Orleans. The goal of this study was to determine if and how C. formosanus colonies were able to survive prolonged flooding following Hurricane Katrina. Microsatellite genotyping conducted on termite samples collected from inundated areas preand postflooding yielded that C. formosanus colonies survived prolonged flooding. Several studies were conducted to determine the survival mechanisms of colonies. First, a bioassay was conducted in which groups of termites were maintained at three different temperatures and submer ged within containers. Their mortality was recorded at increasing time intervals, and t he lethal times were calculated. It was determined that individual termites could not have survived the flooding following Hurricane Katrina by tolerating inundation. A second bioassay was conducted, in which groups of termites were confined to an airt ight environment at va rying temperatures. 9

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10 Their mortality was recorded at predetermine d time intervals, and lethal times were calculated. It was determined that termi tes could have survived flooding following Hurricane Katrina for up to th ree weeks if trapped in a la rge enough pocket of air within their nest, or if the temperat ure was relatively cool. To determine if C. formosanus colonies survive flooding by evacuating or shifting their foraging areas to escape rising flood wa ters, a field study was conducted along seasonally inundated areas. It was concluded that termites do not shift their foraging areas, nor do they move vertically within trees, to escape seasonal inundation. A bioassay was conducted to determine if C. formosanus colonies survive inundation by creating a watertight environment within their nest. Termite foragers were added to artificial trees and foraging arenas, then inundated while behavior was observed. It was shown that C. formosanus colonies could have survived the flooding following Hurricane Katrina by creating a sealed nesting system and exploiting oxygen within pockets of air within the carton material or within t he gallery systems above the water line.

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CHA PTER 1 INTRODUCTION It is unknown how the flooding that occurred in New Orleans, Louisiana after Hurricane Katrina in 2005 affect ed Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), colonies. This is primarily because termite populations within the city hav e never experienced such wide scale and prolonged inundation. According to Federal Emergency Management Agency (FEMA) data, approximately 80% of New Orleans was inundated following Hurricane Katrina due to levee breaches (www.fema.gov). Some areas of the city were inundated for one to three days, and other areas for two to three weeks. Between two to four months after the fl ood waters receded, members of the New Orleans Mosquito and Termite Control Boar d staff began checking field research sites for termite activity in flooded areas. Many of t he in-ground monitoring stations contained C. formosanus foragers. These observations indicated that at least some C. formosanus colonies survived heavy flooding for sustained periods of time. However, it has not yet been determined if the termite colonies main tained their foraging areas, or if they originated from higher elevations and inv aded foraging areas of flooded colonies. The mechanisms that enable the colonies to survive prolonged inundation are not understood. The objectives of the studies following this introduction are: 1. Determine if C. formosanus colonies survived prolonged flooding following Hurricane Katrina 2. Determine if C. formosanus colonies survive flooding by tolerating inundation for extended periods 11

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3. Determine if C. formosanus colonies survive flooding by exploiting air trapped in the soil during inundation 4. Determine if C. formosanus colonies survive flooding by evacuating or shifting their foraging areas to escape rising flood waters 5. Determine if C. formosanus colonies survive flooding by creating a watertight environment within their nesting system The Formosan Subterranean Termite: Problem and Economic Importance Coptotermes formosanus is a pest of major economic importance in New Orleans, Louisiana and other areas of the southeastern United St ates. This exotic pest was introduced into United States port cities such as New Orleans and Lake Charles, Louisiana, from eastern Asia after World Wa r II via infested wooden cargo crates and pallets (La Fage 1987). By definition, an ex otic pest is one that not only survives migration, but also becomes successfully est ablished in its new location (Su 2003a). There has never been a documented case of C. formosanus being eradicated from an area once it has been established (Su 2003a). In the continental United States, the first specimen of C. formosanus was collected in Charleston, South Carolina in 1957 (Su 2003a), but it was not posit ively identified until 1969. Coptotermes formosanus was first identified in Houston, Texas in 1966 (B eal 1987). Documentation of infestations followed in New Orleans and Lake Charles, Louisiana and Galveston, Texas (La Fage 1987). In New Orleans, C. formosanus populations are well-esta blished and ubiquitous in the metro area. It has supplanted the native subterranean termite species, Reticulitermes spp. (Isoptera: Rhinotermitidae), as the primary structural pest in the greater New Orleans area. This ability to outcompete native species has been noted in other areas of the Un ited States (Su 2003a). It has been suggested that the city of New 12

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Orleans has one of the highest termite pressu res in North America (Lax and Osbrink 2003). In general, termites are an important and nec essary component of desert, prairie, and tropical and temperature forest ecosystems. They are un ique in their ability to consume wood produc ts, allowing stored nutrients in cellulose polymers to be liberated and released back into the ecosystem. As s ubterranean termites construct their gallery system while foraging, they play an important role within the soil profile by facilitating soil turnover, aerating the soil, and alte ring the carbon:nitrogen ratio within the soil (Lobry de Bruyn and Conacher 1990). However, termites become pests when they cause damage to buildings, railroad cross ties, boats, and live trees. Throughout its distribution, C. formosanus is the most economically important structural pest species. It has been estimated that C. formosanus is responsible for $66 million in structural repairs and $37.5 million in insecticide treat ments per year in the city of New Orleans (Osbrink et al. 1999). A mo re recent estimate claims that in New Orleans alone, consumers spend approximately $300 million per year in preventative treatments, remedial treatments, and repair of damaged st ructures (Lax and Osbrink 2003). In response to the growing problem of C. formosanus infestations in New Orleans, a management initiative was passed by the United States legislature in 1998. The resulting program, dubbed Operation Full Stop, is an area-wide treatment program that utilizes monitoring and baiting te chnology and non-repellent termiticides to reduce or eliminate subterranean termite popul ations. When the program was initiated and coordinated by the USDA ARS Southe rn Regional Research Center, this technology was implemented withi n the original fifteen squar e blocks of the historic 13

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French Quarter (Lax and Os brink 2003). The treatment area has since expanded to encompass the entire 85 square bl ocks of the French Quarter. An initial quarantine was put into effect by t he Louisiana Department of Agriculture in 1967 to prevent the spread of C. formosanus from areas where it had become established to areas where it had not yet been introduced (La Fage 1987). Quarantined materials included wood products poles, sawdust, contaminated soil, and pilings, but it has been largely ineffective. On October 3, 2005, t he commissioner of the Louisiana Department of Agriculture and Fore stry implemented an additional quarantine in response to Hurricane Katrina, which made landfall along the Gulf coast on August 29 earlier that year, leaving behind millions of tons of wood debris from felled trees and hurricane-damaged structures within Louisiana. This recent quarantine was imposed to prevent the spread of C. formosanus through movement of infested materials to uninfested locations. This quarantine is applic able to the following parishes within the state of Louisiana: Calcasie u, Cameron, Jefferson, Je fferson Davis, Orleans, Plaquemines, St. Bernard, St Charles, St. John the Bapt ist, St. Tammany, Tangipahoa, and Washington. There are four main requirements of the current quarantine. First, newly constructed or reconstructed structures must be treated for C. formosanus Second, movement of wood or cellulose materials out of implicated parishes is prohibited unless the materials are fumigated or written authorization is granted by the commissioner. Third, it is prohibited to relocate temporary housing unless written authorization is granted by the commissioner. Finally, any architectural components used for building material must be treated for C. formosanus before being sold or placed in any structure (www.usda.gov). 14

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The extens ive damage caused by C. formosanus colonies can not be attributed to individuals within the colony consuming a greater amount of cellulose than those of native subterranean termite species (Su and La Fage 1984). The destructive nature is due, instead, to the fact that C. formosanus colonies are more aggressive and contain more individuals than endemic U.S. species. From mark-recapture data, it has been estimated that colonies of C. formosanus can consist of up to one to four million foraging termites (Su et al. 1984), nearly 10 fold the size of a native subterranean termite colony. Colonies are capable of pr oducing foraging galleries that extend 100 m in length and connect multiple feeding site s (King and Spink 1969 ). Therefore, C. formosanus colonies readily locate and feed on mo re cellulose material in a given period than colonies of native s pecies (Lax and Osbrink 2003). Distribution Worldwide, C. formosanus is located in temperate a nd subtropical regions (Su 2003a). This species distribution is limited by its temp erature and humidity requirements. Populations of C. formosanus are typically located within the global area 35 north and south of the Equator (Su and Tamashiro 1987). Worldwide, C. formosanus populations are established throughout China, Taiwan, Japan, Midway, Hawaii, South Africa, and in mu ltiple locations within the continental United States. Currently, established populations of C. formosanus have been reported in the states of Texas, Florida, Mississippi, Alabama, Tenne ssee, North Carolina, South Carolina, Georgia, and Louisiana (Su and Tamashiro 1987, Scheffrahn et al. 2001, Woodson et al. 2001). Although C. formosanus disperses naturally via seasonal reproductive flights, the primary method of range ex pansion is unwitting anthrop ogenic transport of infested 15

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materials (La Fage 1987, Scheffrahn et al 2001, Messenger et al. 2002, Su 2003a). Coptoter mes formosanus was most likely introduced into Japan before the 1600s by trading ships transporting goods from southern China to Nagasaki (Mo ri 1987). During the 19th century, sandalwood trade ships from China may have introduced C. formosanus into Hawaii (Su 2003a). After initial introduction into shipping ports, C. formosanus populations are often moved fu rther inland by human transport. One well-documented example of this is Louisiana, where only two established infestations within the state were known in 1966: one in Lak e Charles, the other in New Orleans (La Fage 1987). A survey thr oughout Louisiana from 1999 to 2001 revealed that C. formosanus had been successfully established in the following parishes within the state: Orleans, Calcasieu, Jefferson, La Fourche, St. Tammany, Lafayette, East Baton Rouge, Ascension, St. Charles, Assumption, Terrebonne, St. Bernard, Plaquemines, Iberia, Vermilio n, St. Landry, Sabine, Ouachita, Acadia, and St. Mary (Messenger et al. 2002). As recently as 2006, C. formosanus distribution in Louisiana had further increased with est ablished populations identified in five parishes where C. formosanus has not been previously documented: Allen, Beauregard, Iberville, Pointe Coupee, and St. John the Baptist (B rown et al. 2007). Yet ongoing survey research has yielded confirmed C. formosanus infestations occurring in the following Louisiana parishes: Avoyelles, Cameron, C oncordia, Jefferson Davis, Livingston, Rapides, St. James, Tangipahoa, Vernon, Washington, and West Baton Rouge (K. Brown, personal communication). Life History Subterranean termites are eusocial insects with a complex life cycle. They are eusocial in that they exhibit parental care there are overlapping generations within the 16

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colony, and there is a divis ion of labor, or ca ste system, within the colony. Individuals of multiple castes serve a different function nec essary for the survival of the colony. Subterranean termite castes include pr imary reproductives, replacement and supplementary reproductives, workers, soldiers, nymphs, and larvae (Imms 1931, Edwards and Mill 1986, Lain and Wright 2003) Each caste contains both male and female individuals (Imms 1931). The pair of primary reproductives, queen and king, will mate throughout their lifetimes (Imms 1931). The kings primary function is to mate with the queen, and the queens primary function is to produce new members of the colony (Edwards and Mill 1986, Imms 1931). Replacement reproductive s are produced if one or both of the primary reproductives die or are isolated from the colony. A colony may contain a single or multiple reproducin g pair(s). Simple family colonies are headed by the founding queen and king. During the life cycle of subterranean termite colonies, the breeding system shifts from a simple fam ily to an extended family, in which secondary reproductives produce offspring, as the primar y reproductives die or as their fecundity decreases (Thorne et al. 1999). The worker caste comprises the highes t proportion of i ndividuals within the colony. Their functions are to forage, feed members of dependent castes, and tend to eggs, newly hatched larvae, and reproductive s (Imms 1931, Edwards and Mill 1986). Approximately 10% of individuals within a C. formosanus colony are soldiers (Lax and Osbrink 2003). The soldier castes primary function is to defend the colony against invaders and predators. Their defense me chanisms include large protruding mandibles and a white glue-like defensive secretion ex uded from the fontanel le located on the 17

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anterior frons. Some i ndivi duals will molt to form nymphs in a mature colony. This caste can differentiate into winged reproductives, or alates, replacement reproductives, or supplemental reproductive s (Imms 1931). A mature C. formosanus colony may contain millions of individua l termites. To accommodate th e ever increasing population, the nest chambers and galle ry systems are expanded. Control Strategies The primary goal of termite control is to protect structures (Su and Scheffrahn 1998) by managing termite populations (Lax and Osbrink 2003), eliminating termite infestations, and suppressing termite populati ons in close proximity of structures (Thorne and Forschler 2000). Control of su bterranean termites begins with detection, proper identification of the termite species, and an underst anding of the extent of the problem (i.e. foraging area, aerial vs. ground infestation) (La Fage 1987). Subterranean termites are cryptic insects that reside underground, inside trees, and remain hidden within walls of buildings (Lax and Osbrink 2003). Within structures, it is difficult to detect a C. formosanus infestation until mud tubes or r eproductive dispersal flights are observed. Preventative and rem edial control of C. formosanus infestations may include the use of liquid termiticides applied to the soil, physical barriers, or monitoring and baiting strategies. Liquid termiticides applied to the soil as a barrier has been the primary method of protecting structur es from termite damage for t he past 60 years (Su 2003a). In 1952, highly effective organochlorides chlordane and heptac hlor, became widely used in preventative and remedial treat ments of subterranean termites (Su and Scheffrahn 1990a, Lax and Osbrin k 2003). Organochlorides per sist in the environment, allowing them to create an effective soil barri er for long periods of time when applied 18

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correctly. They have a residual life of over 25 years, and this raised concerns regarding bioaccumulation of this termi ticide within the environment and its effects on non-target organisms. Because of these environmental concerns and potential health ris ks, organochlorides were no longer registered with the United States Environmental Protection Agency (EPA) after 1988 (Lax and Osbrink 2003, Su and Scheffrahn 1998). Liquid termiticides currently registered with the EPA include pyrethroids, pyrroles, and chloronicotinyls. These are used as soil barriers, and can either be repellent or non-repellent (Lax and Osbrink 2003). Soil termiticides used today are less persistent in the environment than chlordane (Su and Scheffrahn 1998). These termiticides are applied beneath the soil surface around buildi ngs to protect them from soil-borne subterranean termites (Su and Scheffrahn 1990a). Physical barriers may be used as preventative measures. One example of this is stainless steel mesh barriers that can be in stalled prior to construction and serve as a horizontal barrier against term ite infestation (Lenz and Runk o 1994). Another barrier that can be applied prior to construction is an insecticide-impregnat ed polymer film that is installed on top of treated soil before the structural foundation is poured (Su et al. 2004). Uniform sized soil particles are another type of physical barrier. These soil particles are too large to be displaced by te rmites but are also small enough to prevent termites from traveling betw een them (Ebeling and Pence 1957). Though liquid termiticides applied to the soil are still used as a primary method for protecting individual structures, they are not designed for ar ea-wide management of termite populations. Soil te rmiticides are able to kill individual termites; however, surviving termites within the colony can redirect their foragi ng efforts to nearby 19

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structures or live trees that remain unprotec ted. The colony will still exhibit continued growth and continue to thrive. In New Orleans, termite pressure is high enough that neighboring termite colonies readily reinvade areas where existi ng termite colonies have been eliminated (Husseneder et al. 2007). In areas of high termite pressure there is a need to focus on controlling entire populations of C. formosanus New products that use baiting strategies have been developed in response to the publics concer n for the environment and the effects soil termiticides may have on it. These products also address the growing desire to control entire populations of subterranean termites (Lax and Osbrink 2003). Baiting strategies not only involv e controlling populations of subterranean termites, but also involve monitoring them (Su 1994). The high termite pressure in New Orleans illustrates the need for constant monitoring due to the risk of reinvading colonies (Husseneder et al. 2007, Messenger et al. 2005). Baiting methods ideally use active ingredi ents that are slow-a cting, non-repellent, and whose lethal time is not dose-dependent so that they successfully control termite populations when ingested by termites (Su et al. 1994). Active ingredients must be slow-acting to prevent termite s from dying near the site of feeding on the toxicant. Dead termites act as a repellent for other termit es in the colony, and may prevent subsequent feeding on the bait (Su et al. 1991). Insect growth regulators, such as hex aflumeron and noviflumeron, have been shown to be effective active ingredients in baiting systems (Forschler and Ryder 1996, Su et al. 1991, Su et al. 2000, Su 2003b). They are used in baiting technologies because they are slow-acting and readily transferred throughout the colony via 20

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trophallaxis One insect growth regulator, a chitin synthesis inhibitor, prevents individual termites from molting and its lethal ti me is non dose-dependent (Su 2003b). Baiting technology involves placing in-gr ound monitoring stations that contain a cellulose material in the soil at regular inte rvals along the perimeter of the structure. These stations are checked at regular time intervals for foragi ng termites. Once termites are observed in a monitoring station, the cellulose resource is replaced with a substrate that contains active ingredient. However, there are commercially available baits that eliminate the pre-bai ting period by initially inst alling in-ground stations that contain active ingredient. Regardless of the exact protocol designed by the bait manufacturers, the method by which the bait is distributed remains the same. That is, individual termites consume the bait and share it with other individuals within the colony through feeding and grooming behaviors, distributing the bait and eliminating the colony. Colony Survival of Inundation Areas of southern China, from which C. formosanus originated, experience regular droughts and floods related to monsoons (Lau and Li 1984). Because C. formosanus populations continue to thrive in their native areas, despite flooding conditions, it is understandable that populat ions can survive in periodically flooded environments in other areas of the world in which C. formosanus has become established. Survival mechanisms of C. formosanus during inundation, however, have not been widely studied. Ants, another highly successful group of social insects, use a variety of mechanisms to survive inundation. For example, Crematogaster cerasi (Fitch) (Hymenoptera: Formicidae) colonies survive i nundation by vertically evacuating to trees 21

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when water s rise (Ellis et. al 2001). Camponotus anderseni McArthur and Shattuck (Hymenoptera: Formicidae) colonies prevent their nests from being inundated by blocking entrance holes to t he nest itself. However, this also prevents gas exchange between the nest and external environment (Nielsen et. al 2006). This prevention of gas exchange is not detriment al to the colony because C. anderseni is able to respire anaerobically if needed (Niels on and Christian 2007). Anot her ant species, the mangrove mud-nesting ant, Polyrhachis sokolova Forel (Hymenoptera: Formicidae), remains within the nest during inundation, and survives inundation by locating air pockets within the nest chambers until flood waters recede (Nielsen 1997). Red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), colonies survive flood conditions by linking their bodies together to form a raft. In this way, colony members are able to float on t he surface of the water until t he water level recedes or the ants contact surfaces above the water line (Anderson et. al 2002). Some termite species other than C. formosanus are also established in seasonally flooded areas. The results of a survey conducted in Darwin, northern Australia, which encompasses areas that are seasonally flooded, determined that Nasutitermes graveolus (Hill) (Isoptera: Termitidae) was able to inhabit mangrove swamps partially due to their arboreal nes ting behavior (Dawes-G ramadzki 2005). Another termite survey conducted by Constant ino (1992) in the rain forest of Brazilian Amazonia determined that al though termite species were able to inhabit periodically inundated swamp forests, termite species dive rsity in flooded areas was not as great as in forested areas that did not experience inundation. This survey documented the following termite species as inhabiting the periodically inundated forest: Rhinotermes 22

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margin alis (L.) (Isoptera: Rhinotermitidae), Anoplotermes spp., Cavitermes tuberosus Emerson, Termes hispaniolae (Banks), Termes medioculatus (Emerson), Coatitermes clevelandi (Snyder), Ereymatermes rotundiceps Constantino, Nasutitermes spp., and Rotunditermes bragantinus (Roonwal and Rathore) (Isoptera: Termitidae). The mechanisms by which these termite species survive inundation are unknown. In flood-prone areas such as New Orl eans, it is important to understand the impact of inundation on termite populations. Studying the survival and behavior of termite colonies following inundation may yi eld insight to the need of retreating flooded structures for termites, as well as improved management stra tegies. In areas disturbed by landscape, construction, or treatment, C. formosanus colonies are known to shift their foraging areas (Aluko and Husseneder 2007). Though prol onged inundation would also be a form of disturbance for termite col onies, a survey of operational research areas of City Park, New Orleans prior to and following Hurricane Katrina revealed that the overall distribution of C. formosanus within City Park was unchanged (Cornelius et al. 2007). In New Orleans, C. formosanus colonies commonly create carton nests in the trunks of trees (Osbrink et al. 1999). T he hard carton nest material is primarily composed of soil, saliva, and excrement, and provides protection and structure to the nest (Edwards and Mill 1986). The presence of carton material may be a key factor in C. formosanus survival during inundation. Follo wing the flooding caused by levee breaches after Hurricane Katrina in 2005, term ites remained active in the in-ground monitoring stations in New Orleans City Park These areas were inundated with 1 to 2.5 m of brackish water for two to three w eeks (Cornelius et al. 2007). This indicates 23

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that subterranean termites may be capable of maintaining their gallery systems even during considerable inundation for several months. Because the time which would be required for neighboring colonies to repopulat e all flooded areas of the city is greater than the time in which in-ground monitoring st ations were inspected and termite activity was documented, the possib ility of attributing all post-Katrina termite activity to colonies originating from higher el evations is unlikely. A bioassay conducted by Forschler and Henderson (1995) showed that C. formosanus survived up to 16 hours while subm erged under water. They also concluded that saturated soil conditions due to heavy rainfall can cause a significant reduction in foraging populations of subterranean termites, though termite colonies are not eliminated. Carton material may pr ovide protection against water-saturated soil because of its hydrophobic proper ties that naturally provi de structure to the nest (Cornelius et al. 2007). A study by Corneliu s et al (2007) which tested the effect of carton material on termite survival during inundation revealed that termites that were given longer periods of time to construct carton nest within containers in a laboratory setting had a higher survival rate following i nundation. During the study, termites that survived had remained above the water line, or were within the carton material. No survivors were found in the inundated foraging galleries themselves. Coptotermes formosanus colonies have even been observed infesting naturally inundated trees. A study by Delaplane et al. (1991) of fo raging behavior of C. formosanus isolated in cypress trees in the Calcasieu River near Lake Charles, Louisiana revealed that foraging activity va ried seasonally, and the number of foraging workers decreased as precipitation increased. It was also determined that 24

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25 C. formosanus colonies isolated in these cypress trees were smaller than nearby colonies established in the soil (Su and La Fage 1999). It was hypothesized that this was due to restricted populations having limi ted food resources and space. Because C. formosanus colonies are able to infest naturally flooded trees, it follows that colonies may be capable of surviving inundati on caused by natural disasters.

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CHA PTER 2 MOLECULAR GENETIC EVIDENCE OF THE SURVIVAL OF Coptotermes formosanus COLONIES AFTER FLOODING Immediately after the flood followi ng Hurricane Katrina, the question of subterranean termite colony survivorship wa s raised. It was observed that in-ground termite monitoring stations located in previo usly established research sites which were flooded for up to two weeks still contained acti ve foraging termites months after the flood waters receded (Cornelius et al. 2007). T hese observations indicate that at least some C. formosanus colonies survived. The monitoring of established research sites that experienced inundation has shown the distribution of C. formosanus colonies has not altered (Cornelius 2007, Osbrink et al. 2008). However, it has not yet been determined if the termite activity in these ar eas could solely be attributed to colonies which survived inundation and maintained their foraging territories or to surrounding colonies located at higher elevations within trees and structures which assumed foraging territories of flooded colonies. Determining spatial and social organizati on of subterranean termite colonies is difficult due to their complex behavior. The spatial organiza tion can consist of multiple foraging sites and multiple nests. Direct observations of colony behavior are not possible because of the cryptic lifestyle of termites. However, molecular methods can be used to evaluate movement and survival of termite colonies. The field of termite genetics has expande d and has become a valuable tool in understanding termite biology. A be tter understanding of the biology of C. formosanus may lead to better management practices. For example, delineating colonies can elucidate the extent of an in festation that can be attributed to a single colony and allow 26

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us to evaluate remediation efficacy. If a colony infesting a given area following treatment is determined to be a different co lony than that delin eated before treatment using genetic methods, it would be conclude d that the treatment was effective and foraging termites observed following treatment may have originated from a neighboring colony. These methods were employed and colony elimination and reinvasion by neighboring colonies was observ ed by Husseneder et al. (2007). Microsatellites have sufficient genetic variability within single colonies of termites to delineat e colonies (Vargo et al. 2003) determine colony spatial and breeding structure (Husseneder et al 2005; Aluko and Husseneder 2007), and determine survival or elimination of colonies after treatm ent (Husseneder et al. 2007). Microsatellite genotyping can also determine whether new infestations have originated from neighboring colonies, are remain ing individuals from a colony that was not completely eliminated, or are originati ng from individuals outside the area of study (Husseneder et al. 2007). The objectives of this study were to dete rmine colony survival after flooding and evaluate any variation in breeding structure of surviving col onies by using microsatellite genotyping. Evaluating the breeding structur es of termite colonies subjected to inundation is important to det ermine if breeding structure influences survival success. A termite colony may be a simple family co lony, headed by a single reproducing pair, or an extended family colony, headed by multip le reproducing pairs (Vargo et al. 2003, Vargo and Husseneder 2009). Ex tended family colonies ma y have a higher probability of surviving inundation due to reproducing pairs being located in multiple satellite nests, which may be better protected from flood waters. 27

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Materials and Methods Research Sites and Termite Samples Samples of C. form osanus were collected from in-ground monitoring stations located at eight study sites in New Orleans, Louisiana. Each study site was established as a New Orleans Mosquito and Termite Cont rol Board (NOMTCB) research site before Hurricane Katrina. As such, foraging termites were collect ed regularly from Sentricon monitoring stations before t he storm. Pre-flood sample s collected within one year before Hurricane Katrina were compared to post-flood samples from all study sites collected from the same in-ground stations within one year after the flood event. Voucher specimens for all samples were deposited in the NOMTCBs arthropod collection. The French Market (N 29.96072, W -90.05884) was not inundated (Table 2-1). Research sites that experienced inundation fo r a minimum of five days with at least 0.3 m of water were the Parks and Parkwa ys administration building (N 29.99535, W -90.06297), Sewerage and Water Boar d Pump Station #7 (N 29.97677, W -90.09128), Louis Armstrong Park (N 29. 96222, W -90.06701), as well as four research sites in City Park. These City Park sites were Pan American Stadium N 29.99404, W -90.08823), the islands of Old Bayou Metairie (N 29.98445, W -90.09974), Tad Gormley Stadium (N 29.98943, W -90.09971), and the South Golf Course (N 30.01312, W -90.09371). After the flood, termi te foraging was observed at Pan American Stadium in one tree and one structure in close proximity ( 10 m) to in-ground monitoring stations from which preand post-flood samples were collect ed. Shelter tubes in these two areas were observed at higher elevations than the fl ood level. If termites collected from these 28

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areas of higher elevat ion belonged to colonies occupying in-ground monitoring stations that experienced flooding, it ma y imply that these colonies survived flooding by vertically evacuating to escape rising water. If te rmites would have been located higher than the flood waters at the onset of rising water or could have retrea ted to higher elevation, they would have been protected from being inundated. Therefore, termite samples taken from these protected areas were also included in the analysis. A total of 29 pre-flood samples and 38 post-flood samples were used for this study. Microsatellite Genotyping For each preand post-flood sample, 20 individual worker termites were subjected to DNA extractions using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Polymerase Chain R eaction (PCR) was performed to amplify microsatellites at six loci, using locus-spec ific primers under conditions stated in Vargo and Henderson (2000). These loci are k nown to assort independently and have been previously used for genetic analysis of C. formosanus populations in New Orleans (Husseneder et al. 2005, Vargo et al. 2006, Husseneder et al. 2007, Aluko and Husseneder 2007). Electrophoresis was conducted using a LI-COR 4300 DNA Analyzer (LI-COR, Lincoln, NE). Statistical Analysis To determine whether individuals in pr e-flood samples were from the same colony as those in post-flood samples, a llelic frequencies were tested for significant differences using log-likelihood G-statisti cs (FSTAT; Goudet 2002). Individuals were considered to belong to the same colony if there were no differences detected at the 5% level of significance and no private alleles were observed. The breeding structure of each colony was determined by analyzing the M endelian distribution (H usseneder et al. 29

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2005, Huss eneder et al. 2002, Vargo et al. 2003) If the observed genotypic frequency of the colony did not differ significantly from expected Mendelian ratios (G-test for goodness-of-fit summed across all loci), the col ony was classified as a simple family colony (Thorne et al. 1999, Bulmer et al. 2001, Vargo et al. 2003). The colony was classified as an extended family colony if the observed genotypic frequencies differed significantly from the expected Mendelian ratios. Results Permutation tests conducted on a total of 67 samples at the 5% level of significance concluded that three Formosan subterranean termite colonies were present at the non-flooded site and four co lonies were present after Hu rricane Katrina. A total of 17 colonies were present at the flooded sites before and after flooding (Table 2-2). Of the 17 colonies, profiles of 14 post-inundation samples matched those from colonies sampled before flooding. It was determined that profiles of three post-inundation colonies (one each at Pan American Stadium, Pump Station #7, and Louis Armstrong Park) were different from those delineated be fore the storm. Termites collected from the tree in close proximity to in-ground monitoring stations at Pan American Stadium did not belong to the same colonies as those ac tually occupying the inundated stations. Those collected from the structure at P an American Stadium belonged to one of the colonies that occupied the inundated stations. Log likelihood G-statistics ( P < 0.05, G-test summed across all loci) showed that eight of the 17 colonies at the flooded si tes before flooding were simple families, headed by a single reproducing pai r, and nine were extended family colonies, headed by multiple reproducing pairs. After floodi ng, there were six simple family and 11 extended family colonies at the flooded sites. The breeding structure of one colony 30

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each at Parks and Parkways and South Cour se changed from a simple family colony before flooding to an extended family colony after flooding. Three previously undetected colonies consisted of one simple family colony and two extended family colonies. Discussion Because profiles of 14 of 17 (82%) inundated colonies were the same as those occupying monitoring stations before the flooding event, it can be concluded that these termite colonies survived prolonged inunda tion (up to 14 days). Saturated soil conditions have been hypothesized to cause signi ficant reductions in termite foraging populations (Forschler and Henderson 1995). Although mortality within the foraging populations of the C. formosanus colonies in the present study is not known, the results indicate a high degree of survivorship after flooding at the colony level. Samples used in this study were collected within one year after the flood. It is unlikely that colonies present in monito ring stations that were undetected before flooding were the result of primary reproduc tives founding incipient colonies, as they would not have yet reached maturity. It has been shown that Formosan subterranean termite colonies shift their fo raging territories when their area is disturbed by treatment or landscaping, readily moving into nei ghboring monitoring stations (Aluko and Husseneder 2007). Furthermore, if termite colonies are eliminated by treatment, neighboring colonies readily move into previously occupied foraging territories (Husseneder et al. 2007). The flood fo llowing Hurricane Katrina may have caused a similar soil disturbance in the form of prolonged saturation. This disturbance may account for the post-flood detection of three colonies not identified before the flood, which may have moved into monitoring stati ons from neighboring areas. Though there 31

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was movement of three colonies the overal l foraging areas of C. formosanus colonies did not alter significantly following Hurricane Katrina (Cornelius et al. 2007, Osbrink et al. 2008). Both simple family and extended family colonies survived flooding. The breeding system of a colony did not seem to have an impact on a colonys survival during an inundation event. A similar lack of association between breeding structure and survivorship was noted by Husseneder et al. (2007) following treatment. If extended family colonies exhibited a higher probabili ty of surviving inundation due to multiple reproducing pairs residing in satellite nests, there would have been a decrease in the percentage of simple family colonies observe d at the research sites after flooding. The observed transition from a simple family to an extended family colony is not necessarily considered a result of flooding, but could be if the primary reproductives were killed due to inundation. This transition is a progression of the colony life cycle resulting from the primary reproductive being replaced or supplemented by secondary reproductives within the colony (Thorne et al. 1999). Th is transition has also been observed previously in an urban landsca pe (Aluko and Husseneder 2007). The native areas of C. formosanus in southeast China experience monsoonrelated seasonal droughts and floods (Kripal ani and Kulkarni 1997 ), and they have adapted to these environments of seasonal flooding It is therefore understandable that C. formosanus could survive flood conditions in other areas of the world in which it has become established. However, the survival mechanisms of C. formosanus during flooding are not well understood. 32

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Termites collected from the tree at Pan Americ an Stadium after flooding did not belong to the same colonies as those occupy ing the in-ground monitoring stations. This is not indic ative of termites occupyi ng the surrounding in-ground monitoring stations surviving inundation by retreating to a prot ected area above the water line. However, termites foraging above the water line wit hin the building belonged to one of the colonies foraging within the in-ground monito ring stations before and after flooding. It may be that this colony survived inundation by retreating to higher elevation or by remaining in this protected area during fl ooding. Whether termit es survived prolonged flooding by this behavioral mechanism remains unknown, as termites could not be observed or sampled while in-ground moni toring stations were inundated. Though C. formosanus workers can not survive longer than 16 hours under water (Forschler and Henderson 1995), it has been suggested that C. formosanus can survive prolonged inundation within their nest system due to the hydrophobic proper ties of carton material (Cornelius et al. 2007) and trapped air within vo ids of infested trees (Osbrink et al. 2008). Further research investigating C. formosanus colony survival by physiological or behavioral mechanisms is warranted. Understandi ng termite colony survival facilitates the making of sound pest management decisions after natural disasters such as Hurricane Katrina, as it is important for the property owners to understand that floods do not eliminate termite colonies, and termite m onitoring and remediation efforts should be maintained. 33

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34 Table 2-1. Summary of termite samples colle cted from in-ground monitoring stations and flood depth and duration at each research site No. of samples collected from in-ground monitoring stations Before After Flood Flood Study Site flood flood depth (m)1 duration (days)2 French Market 5 5 0 0 Pan American Stadium 9 11 1.5-1. 8 10-14 Old Bayou Metairie 5 12 0.3-0.6 5-10 Parks & Parkways 1 1 0.6-0.9 5-10 Pump Station #7 1 1 0.6-0.9 5-10 Tad Gormley Stadium 2 2 1.2-1. 5 10-14 Louis Armstrong Park 5 5 0.3-0.6 5-10 South Course 1 1 1.2-1.5 10-14 Total 29 38 1Estimated by National Oceanic and At mospheric Administration (NOAA) 2Estimated by Federal Emergency Management Agency (FEMA)

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Table 2-2. Summary of colony survival and br eeding structure before and after inundation Pre-floo d Post-floo d Study site No. of colonies Simple families Extended families No. of colonies Simple families Extended families Same colonies Different colonies French Market 3 1 2 4 1 3 3 1 Pan American Stadium 3 0 3 3 0 3 2 1 Old Bayou Metairie 5 2 3 5 2 3 5 0 Parks & Parkways 1 1 0 1 0 1 1 0 Pump Station #7 1 0 1 1 0 1 0 1 Tad Gormley Stadium 2 0 2 2 0 2 2 0 Louis Armstrong Park 4 4 0 4 4 0 3 1 South Course 1 1 0 1 0 1 1 0 Total 20 9 11 21 7 14 17 4 35

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CHA PTER 3 SURVIVAL OF Coptotermes formosanus FORAGERS FOLLOWING INUNDATION IN A LABORATORY Microsatellite genotyping confirmed that at least some C. formosanus colonies present at established research sites before inundation were the same as those present following inundation. Though termite colonies survived prolonged inundation, the survival mechanisms of colonies have yet to be fully understood. One hypothesis is that C. formosanus colonies survived the flooding following Hurricane Katrina by tolerating inundation. A study conducted by Forschler and Henderson (1995) concluded the lethal time for 50% of the test population, LT50, for inundated C. formosanus is 11.1 hours. However, th is may not be sufficiently representative of what would have been observ ed for field colonies following Hurricane Katrina for two reasons. Fi rst, only termite workers we re subjected to inundation bioassays, and the soldier proportion within a C. formosanus colony averages 5-10% ( In Haverty 1977). If the LT50 value for inundated soldiers is di fferent from that of workers, the LT50 value for an inundated colony may also be altered. Second, the bioassay was conducted at ambient laborat ory temperature. Field colonies in New Orleans experience soil temperatures that vary throughout the year. A two-year study conducted by Cornelius and Osbrink ( 2011), which included measuring soil temperatures in New Orleans, showed that soil temperatures ma y range from 10 to 20C during winter months, and from 25 to 35C at their peak in summer months. Insects exhibit increased respiration at relati vely warmer temperatures (Cotton 1932). Therefore, it would be expected that termites maintained at lower temperatures before and during inundation w ould have a higher LT50 value than those maintained at a relatively higher temperature. The objective of the current study is to determine if it was 36

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possible for C. form osanus workers and soldiers to survive the flooding following Hurricane Katrina by tolerating in undation for up to three weeks. Materials and Methods Termites Termites were collected from three C. formosanus field colonies located in New Orleans, Louisiana in June 2011 from previously established milk crate traps (Kleinpeter Farms Dairy, Baton Rouge, LA) located below the soil surface and filled with untreated pine ( Pinus sp.) stakes (2 by 4 by 28 cm in lengt h). The first and seco nd field colonies (Colony 1 and Colony 2) were located at Audubon Zoo (N 29.9237 2, W -90.13063) and were separated by at least 100 m. The thir d field colony (Colony 3) was located on Lakeshore Drive (N 30.03189, W -90.07253). Collected termi tes were transported to the laboratory and stored at 24C within corrugated cardboard moistened with distilled water. Within one week, te rmites were bioassayed. Mean worker weight was determined because individual termite mass may affect termite respiration rates, thus affecting the mortality of inundated termites. For each colony, mean termite weight was calculated by weighing five replicates of 100 workers each. Each group consisted of 90 workers (undi fferentiated larvae of at least the third instar) and 10 soldiers. The average weight of the five replicates we re calculated then divided by 100 to determine the mean weight of an individual worker. Inundation Bioassay Each colony was subjected to the same group of tests. For each colony, there were three experimental groups and three c ontrol groups. The first group consisted of termites maintained at 10C in a wine cool er (Danby, Findlay, OH), the second group was maintained at 21C in the laboratory, and the third group was maintained at 32C in 37

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an incubat or (VWR Scientific Products Randor, PA). One hundred termites (90 undifferentiated larvae of at l east the third instar : 10 soldiers) were placed in a 13 ml snap-seal sample container (Corning, Lowell, MA) with 1 g of play sand and a piece of filter paper (1 by 6 cm). The sand and filter paper were both moistened with deionized water. Termites were mainta ined at their respective tem peratures and allowed to feed on moistened filter paper for 24 hours before commencing the bioassay. A 3 by 3 cm piece of Kimwipe (Kimber ly-Clark, Lake Forest, CA) was inserted into each snap-seal container to prevent te rmites from floating on the waters surface while inundated. Deionized water was added to each container so that all termites and the Kimwipe stopper were subm erged. The formation of air bubbles was prevented by tapping the sides of the container as wa ter was added. Groups of termites were inundated for two-hour intervals, up to 60 cons ecutive hours, while being maintained at their respective temperatures. One group per colony for each temperature was not inundated and acted as the contro l. There were three replic ates for each time interval for each colony maintained at three different temperatures. After each time interval, the snap-seal containers were drained and termites were transferred to a 9 cm Petri dish containing filter paper moistened with deionized water. Termites were then returned to their respective temperature at which they had been maintained. Mortality was recorded 24 hours after containers were drained and termites were transferred. Statistical Analysis Termite weights for all three colonies were compared to each other using analysis of variance (ANOVA) (PROC GL M) and Tukeys Honestly Significant Difference (HSD) test. For each temperat ure at which termites were maintained, 38

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mortality data were corrected using Abbott s (1925) formula. These data were pooled and analyz ed by probit analysis to determi ne the lethal time for 50% (LT50) and 90% (LT90) of the test population. Worker and soldie r mortality rates for each experimental group were compar ed using a paired t -test. Analysis of variance and Tukeys HSD test was conducted to compare mort ality between termites maintain ed at the three different temperatures. Mortality data recorded during each time interval for the three colonies maintained at each temperature were pooled and compared to each other using ANOVA and Tukeys HSD test. All statis tical analyses were conducted using SAS 9.1 (SAS Institute 2003, Cary, NC). Results The average weight of individual termite workers collected from Colony 1 was 3.04 mg. The average weights of individual workers from Colonies 2 and 3 were 3.33 and 2.72 mg, respectively. The mean worker weights for all three colonies were significantly different from eac h other (F = 46 .30, df = 2, P < 0.0001), with Colony 2 having the largest biomass and Colony 3 having the smallest biomass. Once termites became inundated, their move ment ceased. Termites maintained at 10C reached 100% mortality after being i nundated for 60 hours. Total mortality for inundated termites maintained at 21C wa s reached at 30 hours. Inundated termites maintained at 32C reached 100% mortality at 20 hours. Mortality fo r the control groups ranged from 0.9 to 2%. T he percent mortality for all three experimental groups was plotted over time (Figure 3-1). Probit analysis conducted on inundated termites maintained at 10, 21, and 32C yielded LT50 values of 32.2, 10.9, and 6.8 hours, respectively (Table 3-1). This probit analysis also yielded LT90 values of 53.4 hours for i nundated termites maintained 39

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at 10C, 18.0 hours for inundated termites maintained a t 21C, and 10.2 hours for inundated termites maintained at 32C. Paired t -tests conducted on worker and soldier mortality showed significant differences for groups of term ites maintained at 10C ( t = 4.13, df = 29, P = 0.0003), 21C ( t = 4.01, df = 14, P = 0.0013), and 32C ( t = 2.58, df = 9, P = 0.0296). At all three temperatures, soldiers exhibited significantly less mortality than workers. Analysis of variance conducted to compare mortality rate s between colonies at each time interval showed significant differences for termites maintained at 10C (F = 7.65, df = 2, P = 0.0011). Termites from Colony 1 exhibited significantly lower mortality than those from Colonies 2 and 3. There were no significant differences in mortality between the three colonies when maintained at 21C (F = 0.35, df = 2, P = 0.7094). Mortality rates for the three colonies were significantly different when maintained at 32C (F = 9.83, df = 2, P = 0.0013). At this temperature, termites from Co lony 1 exhibited lower mortality rates than those from Colonies 2 and 3. Analysi s of variance also showed significant differences in termite morta lity for those inundated and mainta ined at the three different temperatures (F = 44.48, df = 2, P < 0.0001). Termites mainta ined at 10C exhibited significantly lower mortality than those maintained at 21 and 32C. Discussion Probit analysis showed the LT50 value for inundated C. formosanus workers at 21C was 10.5 hours, lower than the LT50 value of 11.1 hours for inundated C. formosanus workers at 24C shown by Forschler and Henderson (1995). The LT90 value derived from our analysis was 15.2 hour s, which is also lower than the LT90 value of 15.8 hours concluded by Forschler and Henderson (1995). These discrepancies can not be explained by the difference in temper ature, as we have shown that termites 40

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survive inundation for longer periods when mainta ined at relatively cooler temperatures, and this would lead to the expect ation that the LT50 value from our analysis would be higher than that previously established by Forschler and Henderson (1995). These discrepancies may be explained by variation between the colonies from which termites were collected and bioassayed. Termites bioassayed by Forschler and Henderson (1995) did not belong to the same colonies fr om which termites were bioassayed for our study. We have shown significant differences in mortality rates of termites from Colony 1 when compared to those fr om Colonies 2 and 3 at 10 and 32C. Therefore, variations of termite mortality between colo nies can be significant, and could lead to differing lethal times. It would be expected that te rmites from the colony wi th the greatest biomass would not survive inundation for the same per iods as those exhibiting the smallest biomass because it has been shown that term ite species with relatively large biomass exhibit greater respiration rates than species with relatively small biomass (Wheeler et al. 1996). However, this was not observed, even though termites from all three colonies showed significant differences in termite biomass when compared to each other. Termites from colonies that exhibited the greatest and smallest biomass did not show significantly different morta lity rates when inundated at any of the three temperatures at which they were maintained. The colony t hat showed significantly lower mortality than the other two colonies had neither t he greatest nor smallest biomass. At all three temperatures at which termi tes were maintained, soldiers survived inundation for significantly longer periods than workers. In contrast to soldiers, workers have to pursue various tasks, including tunn eling and feeding, thus expending more 41

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energy and increasing their oxyg en requirements. The diffe rence in respiration rates may also be due to physiological differences between the two castes. According to Shelton and Appel (2001), soldiers have lowe r respiration rates than workers, and they hypothesiz e that this may be due to the presence of a pronounced fontanelle located on the soldier head capsule, which increases the area of non-metabolizing tissues, thus reducing the oxygen requirem ent of the soldier caste. It was expected that termites maintained at relatively lower temperatures before and during inundation would su rvive being under water for longer periods than those maintained at relatively higher temperatures This is because increased respiration by termites at relatively warmer temperatures reflects an increase of oxygen required by termites for metabolic activity (Cotton 1932). Furthermore, it is known that termite physical activities such as foraging and wo od consumption increase during months of relatively warmer ai r temperature and soil temperature (Fei and Henderson 2004, Messenger and Su 2005, Cornelius and Osbrink 2011, Delaplane et al. 1991). It was observed that termites maintained at 10C ex hibited significantly lower mortality rates and higher LT50 values than those maintained at either 21 or 32C. Termites maintained at 32C exhibited the lowest LT50 values of all three groups, though the termite mortality was not significantly different than that of termites maintained at 21C. According to figures by Cornelius and Os brink (2011), soil tem peratures in New Orleans, Louisiana ranged from 25 to 35 C between July and October for two consecutive years. The temperature of sa turated soil during the flooding that followed Hurricane Katrina in August 2005 is unknown as measurements were not taken during this time, though it is possible that flood conditions reduced the soil temperature. 42

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43 Therefore, termites inundated following the hurricane would most likely be represented by workers and soldiers bioassayed at 21C and 32C. Termites maintained at 21C had an LT50 value of 10.9 hours, an LT90 value of 18.1 hours, and 100% mortality was reached at 30 hours. Termite s maintained at 32C had an LT50 value of 6.8 hours, an LT90 value of 10.2 hours, and 100% mortality was reached at 20 hours. Even if the soil temperature was 10C when field colonies experienced prolonge d inundation following the hurricane, our data show 100% morta lity was reached at 60 hours for workers and soldiers inundated at this tem perature. Field colonies of C. formosanus established before Hurricane Katrina survived two to th ree weeks of flooding, much longer than the survival capabilities observed of termites subjected to the i nundation bioassays. Therefore, the hypothesis that C. formosanus survived flooding following Hurricane Katrina by tolerating prolonged in undation must be rejected. For C. formosanus colonies to survive this prolonged inundati on, they must have had access to an oxygen source.

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Table 3-1. Comparison of lethal times, LT50s and LT90s (hours), with their associated slopes, 95% CLs, and Pearson X2 values from inundated C. formosanus workers and soldiers maintained at three different temperatures Temperature Caste n Slope SE LT50 95% CL LT90 95% CL X2 (df) P 10C workers 24,300 5.74 0.36 30.60 29.04 31.99 51.12 48.70 54.18 319.71 (27) <0.0001 soldiers 2,700 6.60 1.17 45.71 41.71 47.80 70.73 63.45 88.20 63.19 (27) <0.0001 workers and soldiers 27,000 5.82 0.35 32.16 30.68 33.48 53.38 51.01 56.36 286.10 (27) <0.0001 21C workers 9,720 7.86 0.37 10.45 10.05 10.82 15.21 14.70 15.79 28.89 (9) 0.0007 soldiers 1,350 9.90 1.39 21.44 19.91 22.63 28.88 27.01 32.20 25.49 (12) 0.0127 workers and soldiers 13,500 5.83 0.24 10.88 10.38 11.35 18.05 17.36 18.83 50.20 (12) <0.0001 32C workers 6,480 9.37 0.75 6.64 6.21 6.99 9.09 8.64 9.69 23.12 (5) 0.0003 soldiers 900 7.24 0.84 10.18 9.25 10.89 15.30 14.47 16.44 11.87 (7) 0.0105 workers and soldiers 9,000 7.24 0.43 6.76 6.36 7.12 10.16 9.68 10.75 32.24 (7) <0.0001 44

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Hour 051015202530354045505560 Percent Mortality 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Figure 3-1. Percent mortality of 100 inundated C. formosanus foragers maintained at three different temperatures plotted against time. Closed circles represent mortality of termites maintained at 10C. Open circles represent mortality of termites maintained at 21C, and triangl es represent mortality of termites maintained at 32C. 45

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CHA PTER 4 EVALUATION OF SURVIVORSHIP OF Coptoterems formosanus IN A HYPOXIC ENVIRONMENT Coptotermes formosanus is a cryptic species. It creates gallery systems as well as carton nests consisting of soil, wood, sa liva, and frass below the soil surface, within trees, and inside tree stumps (King and Spink 1969). Excavation of C. formosanus foraging galleries showed that branching subterranean galleries were connected to central nests consisting of cavities enclosed by carton material (King and Spink 1969). Coptotermes formosanus readily infests live trees, creat ing voids and building carton material within them (La Fage 1987, Osbrink et al. 1999). According to Osbrink et al. (1999), these nests can be located both under the soil surface or meters above the ground level within tree trunks. In a laboratory bioassay, C. formosanus had a higher survival rate when located in carton materi al during flooding than those located outside of carton material (Cornelius et al. 2007). It has already been determined that C. formosanus colonies infesting research sites that had been flooded survived the prolonged inundation following Hurricane Katrina, and that termites were not able to su rvive this flooding by tolerating inundation. It has been suggested that survival of C. formosanus during prolonged inundation may be due to hydrophobic properties of carton material (Corneliu s et al. 2007) and trapped air within voids of infested trees (Osbrink et al. 2008). The hypothesis tested in the current study is that C. formosanus colonies survived the flooding following Hurricane Katrina by exploiting oxygen trapped within t heir nesting system. The objective was to determine the length of time termite for agers can survive in an environment that contains a limited amount of oxygen. 46

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Materials and Methods Termites Termites were collected from three field colonies in New Orleans, Louis iana. The first field colony (Colony A) was located at Audubon Z oo (N 29.92372, W -90.13063). The second colony (Colony B) was located at the corner of Marcon i Drive and Robert E. Lee Boulevard (N 30.01825, W -9 0.09747). The third colony (C olony C) was located on Lakeshore Drive (N 30.03189, W -90.07253). Termi tes were collected from previously established milk crate traps si milar to those described in C hapter 3. Collected termites were transported and stored within corrugated cardboard in the laboratory at 24C. Within one week, termites were bioassayed. For each colony, the mean worker weight was calculated using methods described in Chapter 3. The mean worker weight was calculated because termite species with a relatively large biomass exhibits greater respirati on rates than those with a relatively small biomass (Wheeler et al. 1996). Therefore, wo rker mass may affect mortality of termites within a hypoxic environment. Termite Survivorship in an Airtight Environment For each colony, there were three experim ental groups and three control groups. The first group consisted of 20 termites per replicate, the second group consisted of 40 termites per replicate, and the third group consisted of 60 termi tes per replicate. Each group consisted of 10% soldiers and 90% undiffe rentiated larvae of at least the third instar. Each bioassay was conducted at thr ee different temperatures. Termites were maintained at 10, 21, and 32C. There were three replicates for each experimental and each control group. 47

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Bioassays were conducted within 9 ml glass vials, each containing 3 ml 3% agar to maintain moisture and two cylindrical piec es (2 mm i n diameter by 20 mm in length) of untreated wood as the food source. When the volumes of agar and wood resource were subtracted from the total volume of the vial, it was determined that the available air within each vial was 5.94 cm3. Termites were added to their re spective vials. The vials containing the experimental groups were topped with a screw cap and wrapped in Parafilm (Pechiney, Chicago, IL) to creat e an airtight environment. The vials containing the control groups were topped with a piece of stainless steel mesh (2 by 2 cm) (Termimesh LLC, Austin, TX), wh ich allowed for air circulation. Data Collection and Statistical Analysis Mortality was evaluated at 12 hour interv als until 100% mortality was reached. Data were pooled and analyzed by probit analysi s to determine the lethal time, in days, for 50% (LT50) and 90% (LT90) of the test population for all groups of termites maintained at the three different temperatures. The resp iration rates of termites maintained at all three temperatures were es timated by dividing the amount of available air by the number of termi tes remaining when the LT50 was reached and the LT50 value. Mortality rates for groups of 20, 40, and 60 termites maintained at each temperature were compared to each other using analysis of variance (ANOVA) (PROC GLM) and Tukeys Honestly Significant Difference (HSD) test. Worker and soldier mortality rates at each temperature were compared using a paired t -test. Analysis of variance (PROC GLM) was conducted to determine if morta lity between colonies was significantly different. Mortality data of all termites maintained at each temperature were pooled and compared to each other using ANOVA (PROC GLM). All statistical analyses were conducted using SAS 9.1 (SAS Institute 2003, Cary, NC). 48

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Results The average weight of indiv idual termite s collected from Colony A was 3.33 mg. The average weights of individual termites from Colonies B and C were 3.46 and 3.73 mg, respectively. The mean termite weights of each colony were significantly different from each other (F = 6.34, df = 2, P = 0.0224). The biomass of termites collected from Colony A were significantly larger than those collected from Colony B, while the biomass of termites collected from Colony C were not significantly different from those from either Colony A or Colony B. At 10C, total mortality for groups of 20 and 40 termites was reached at 89.5 days. At this temperature, groups of 60 termites exhibited 100% mortality at 57.5 days. At 21C, total mortality for groups of 20, 40, and 60 termite s was reached at 52.0, 51.0, and 22.5 days, respectively. At 32C, 100% mortality was reached for groups of 20 and 40 at 3.5, and total mortality for groups of 60 termites was observed at 1.0 day. Percent mortality for all groups of termites maintain ed at three different temperatures was plotted over time (Figures 41, 4-2, and 4-3). The control groups maintained at all three temperatures ranged from zero to 5% mortality. Mortality data were corrected using Ab botts (1925) formula. Probit analysis on groups of 20, 40, and 60 termites maintained at 10C confined within airtight vials containing 5.94 cm3 of available air yielded LT50 values of 23.0, 20.3, and 16.8 days, respectively (Table 4-1). Groups of 20, 40, and 60 termites maintained at 21C yielded LT50 values of 7.0, 4.4, and 1.9 days, respectively. At 32C, groups of 20, 40, and 60 termites yielded LT50 values of 1.1, 0.4, and 0.3 da ys, respectively. From the LT50 values, respiration rates of 20, 40, and 60 te rmites maintained at 10C were estimated to be 0.0258, 0.0146, and 0.0118 cm3 air per termite per day, respectively. At 21C, 49

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respiration rates of groups of 20, 40, and 60 termites were estimated to be 0.0849, 0.0675, and 0.1042 cm3 air per termite per day, respective ly. At 32C, respiration rates of groups of 20, 40, and 60 termites were estimated to be 0.5400, 0.7425, and 0.6600 cm3 air per termite per day, respectively. Mortality rates of groups of 20, 40, and 60 termites were significantly different at 10C (F = 227.95, df = 2, P < 0.0001), 21C (F = 125.57, df = 2, P < 0.0001), and 32C (F = 7.86, df = 2, P = 0.0066). At 10 and 21C, groups of 20 termites yielded the lowest mortality, while groups of 60 termite s yielded the highest mortality. At 32C, groups of 20 termites exhibited si gnificantly lower mortality ra tes than that for groups of 60 termites, though mortality of groups of 40 termites was not significantly different than that of groups of 20 or 60 te rmites. Workers showed signif icantly lower mortality rates than soldiers at 10C ( t = -14.26, df = 178, P <0.0001) and at 21C ( t = -6.61, df = 103, P <0.0001). There was no significant difference in worker and soldier mortality at 32C ( t = -0.70, df = 6, P = 0.5119). At 10C, there was a significant differ ence between mortality rates of the three colonies (F = 70.06, df = 2, P <0.0001). At this temperature, termites from Colony A exhibited the lowest mortality, while termi tes from Colony C exhibited the highest mortality. There was also a significant difference between mortality rates of the three colonies at 21C (F = 105.00, df = 2, P <0.0001). At 21C, te rmites from Colony A exhibited the lowest mortality, while termi tes from Colony B exhibited the highest mortality. Mortality rates of the three colonies were not sign ificantly different from each other at 32C (F = 3.00, df = 2, P = 0.0880). 50

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Analys is of variance conducted on pooled mo rtality of all groups of termites at each temperature showed a significant diffe rence between mortality rates of termites maintained at 10, 21, and 32C (F = 143.61, df = 2, P <0.0001). Termites maintained at 10C exhibited significantly lower mortality than termites maintained at either 21 or 32C. Termites maintained at 32C exhibited significantly higher mo rtality than termites maintained at either 10 or 21C. Discussion Significant differences of mortality ra tes between the experimental groups show that the greater the ratio of available air s pace to the number of te rmites confined to that space, the greater the surv ivorship. At 10 and 21C, groups of 20 termites, when compared to groups of 40 and 60 termites, surv ived for a significantly longer period due to fewer termites consuming the available oxygen. This was also observed for groups of 20 termites when compared to groups of 60 termites at 32C. At 10 and 21C, workers survived significantly longer than sold iers. This is a c ontrast to what was observed in Chapter 3, in which soldiers survived for longer periods than workers when inundated. This may be due to workers cons erving resources in a hypoxic environment, at the expense of soldiers, which rely on workers for food. According to the data presented here, 50% of a group of 20 termites (i.e. 10 termites) can survive being confined to approximately 6 cm3 of air space for 23 days at 10C, seven days at 21C, and one day at 32C. Ten percent of a group of 20 termites (i.e. two termites) can survive in this same space for almost 62 days at 10C, 27 days at 21C, and three days at 32C. As stated in Chapter 3, soil te mperatures in New Orleans, Louisiana typically range from 25 to 35C between July and October (Cornelius and Osbrink 2011). The temperatur e of saturated soil during the flooding 51

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that followed Hurricane Katrina was unknown, but it is possible that flood conditions following Hurricane Katrina reduced the soil temper ature. Therefore, termites inund ated following the hurricane would likely be repr esented by workers and soldiers bioassayed at 21C and 32C. Between the temperatures of 32 and 21C, the LT50s range from one to seven days for 20 termites in a hypoxic environment. At these same temperatures, the LT90s range from three to 27 days. When considering subterranean termite col ony mortality due to treatment, or in this case a flood event which may confine termit es within pockets of ai r, the colony itself must be considered as a unit, rather than placing focus solely on the mortality of individual termites. Even if a portion of a col ony survives, the colony itself will survive, as more foragers and reproductives are produced (Su and Scheffrahn 1990b). A study of field C. formosanus colonies within City Park revealed an initial reduction of C. formosanus populations after being inundated for more than two weeks (Osbrink et al. 2008). However, this study showed popu lations rebounded the following year. This is indicative of a colony that experienced loss of individual termites due to inundation, but continues to produce foragers, t hus increasing its colony size. It was concluded in Chapter 3 that C. formosanus colonies could not have survived the two to three week inundation period after Hurricane Katrina by simply tolerating inundation. The LT90s derived from our data show that 10% of the test population can survive in approximately 6 cm3 of available air up to 27 days (3.8 weeks), which is longer than the two to th ree week inundation duration observed after Hurricane Katrina. During the flooding that followed Hurricane Katrina, termites not succumbed to rising water may have survived by remaining in pockets of air within their 52

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53 hydrophobic carton material. Whether C. formosanus survived inundation by this mechanism remains unknown, as carton materi al within tree voids and structures could not be observed during flooding. However, groups of termites can survive more than three weeks within a hypoxic environment, d epending on temperature and the ratio of available space to the number of termites exploiting oxygen. Survival mechanisms of insects inhabiting hypoxic environments vary. Several species of insects reduce their metabolic rates and become quiescent when exposed to hypoxic environments. For exam ple, intertidal root aphids, Pemphigus trehernei Foster, become quiescent when submerged (Hoback and Stanley 2001). Tiger beetle larvae of the species Cicindela togata Laferte reduce their metabolic rates to survive repeated flooding of their larval habitat alo ng flood plains (Hoback et al. 1998). Based on our findings, the hypothesis that C. formosanus colonies survive flooding by exploiting trapped air is accept ed. It would be possible for inundated colonies to survive the flooding following Hurricane Katrina by remaining within air pockets located in their nesting system until flood waters recede, but only if their nesting system was sealed and provided sufficient pr otection from being filled with water.

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Table 4-1. Comparison of lethal times, LT50s and LT90s (days), with their associated sl opes, 95% CLs, and Pearson X2 values from groups of 20, 40, and 60 C. formosanus workers and soldiers confined to an airtight environment and maintained at three di fferent temperatures Temp No. of termites Caste n Slope SE LT50 95% CL LT90 95% CL X2 (df) P 10C 20 workers 28,998 3.11 0.07 24.61 23.75 25.45 63.50 61.99 65.12 274.79 (176) <0.0001 soldiers 2,160 2.24 0.11 8.21 7.00 9.43 30.75 27.83 34.11 45.92 (117) <0.0001 workers and soldiers 32,220 2.98 0.07 23.00 22.01 23.96 61.89 60.22 63.69 372.14 (176) <0.0001 40 workers 57,348 2.76 0.12 23.63 21.61 25.52 68.80 66.32 71.54 803.05 (174) <0.0001 soldiers 5,076 2.00 0.07 5.39 4.68 6.12 23.55 21.55 25.74 72.51 (138) <0.0001 workers and soldiers 64,440 2.51 0.12 20.31 18.06 22.43 65.96 63.41 68.71 921.64 (176) <0.0001 60 workers 55,887 4.23 0.21 18.24 17.02 19.34 36.61 35.26 38.12 1,290.83 (112) <0.0001 soldiers 2,646 2.41 0.18 4.98 4.07 5.88 16.94 14.59 20.11 153.71 (46) <0.0001 workers and soldiers 62,100 3.91 0.21 16.80 15.44 18.02 35.71 34.27 37.32 1,505.76 (112) <0.0001 21C 20 workers 16,848 2.17 0.10 6.91 5.85 7.97 26.92 24.49 29.62 594.89 (101) <0.0001 soldiers 1,134 1.91 0.13 5.73 4.61 6.88 26.85 22.80 32.37 63.55 (60) <0.001 workers and soldiers 18,720 2.19 0.10 6.99 5.92 8.06 26.82 24.44 29.46 650.10 (101) <0.0001 40 workers 31,104 1.86 0.08 4.46 3.72 5.23 21.78 19.64 24.12 692.99 (93) <0.0001 54

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55 Table 4-1. Continued. Temp No. of termites Caste n Slope SE LT50 95% CL LT90 95% CL X2 (df) P 21C 40 soldiers 3,670 1.62 0.07 3.57 2.81 4.40 22.19 19.27 25.50 41.20 (99) <0.001 workers and soldiers 36,720 1.88 0.07 4.41 3.70 5.14 21.18 19.19 23.34 768.66 (99) <0.0001 60 workers 21,384 1.48 0.08 1.92 1.43 2.44 14.05 11.92 16.63 316.58 (41) <0.0001 soldiers 1,566 2.13 0.18 1.91 1.33 2.50 7.59 6.24 9.23 46.78 (26) 0.0074 workers and soldiers 24,300 1.51 0.08 1.92 1.47 2.42 13.56 11.63 15.83 313.02 (42) <0.0001 32C 20 workers 1,134 3.58 0.74 1.14 0.54 1.72 2.59 1.71 6.36 58.09 (4) <0.0001 soldiers 126 3.78 0.61 1.05 0.78 1.31 2.29 1.83 3.14 7.67 (4) 0.0104 workers and soldiers 1,260 3.53 0.70 1.14 0.57 1.71 2.64 1.76 6.11 57.61 (4) <0.0001 40 workers 2,268 2.13 0.64 0.43 0.0006 1.32 1.70 0.11 6.33 77.23 (4) <0.0001 soldiers 252 2.19 0.45 0.45 0.13 0.84 1.72 0.94 2.78 4.27 (4) 0.0371 workers and soldiers 2,520 2.14 0.62 0.43 0.0003 1.28 1.70 0.16 5.71 79.45 (4) <0.0001 60 workers 1,944 4.10 0.88 0.26 0.00 0.52 0.54 0.004 1.02 15.39 (2) 0.0005 soldiers 216 3.79 0.79 0.26 0.11 0.38 0.56 0.37 0.75 3.10 (2) 0.0213 workers and soldiers 2,160 3.94 0.69 0.27 0.07 0.38 0.57 0.39 2.02 29.40 (2) <0.0001

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Day 05101520253035404550556065707580859095100 Percent Mortality 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Figure 4-1. Percent mortality of termites maintained in an airtight environment at 10C plotted against time. Closed circles represent mortalit y of groups of 20 termites. Open circles represent mortalit y of groups of 40 termites. Triangles represent mortality of groups of 60 termites. 56

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Day 0510152025303540455055 Percent Mortality 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Figure 4-2. Percent mortality of termites maintained in an airtight environment at 21C plotted against time. Closed circles represent mortalit y of groups of 20 termites. Open circles represent mortalit y of groups of 40 termites. Triangles represent mortality of groups of 60 termites. 57

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Day 0.00.51.01.52.02.53.03.54.0 Percent Mortality 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Figure 4-3. Percent mortality of termites maintained in an airtight environment at 32C plotted against time. Closed circles represent mortalit y of groups of 20 termites. Open circles represent mortalit y of groups of 40 termites. Triangles represent mortality of groups of 60 termites. 58

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CHA PTER 5 SURVIVAL OF Coptotermes formosanus COLONIES IN SEASO NALLY INUNDATED LOCATIONS OF NEW ORLEANS, LOUISIANA In Chapter 2, it was concluded that field C. formosanus colonies present at established field sites prior to inundation were the same as t hose present following inundation. This indicates that C. formosanus colonies were able to survive inundation for a prolonged period. Chapter 3 showed that termite colonies would not have survived prolonged flooding following Hurri cane Katrina by tolerating inu ndation. It was revealed in Chapter 4 that it would be possible for fl ooded colonies to survive by remaining within air pockets located in their nesting system until flood waters recede, but it is still unknown if their nesting systems were sealed and provided sufficient protection from being filled with water. A study of the behavior of C. formosanus colonies during periodic inundation is warranted because termite colonies within their nesting systems could not be observed during the flooding following Hurricane Ka trina, and because the exact survival mechanisms by which termites survive are still not fully understood. One hypothesis is that termite colonies move away from seasonally rising flood waters to survive inundation. The objectives of this study are to determine if termite colonies shift their foraging areas, or evacuate, during seasonal inundation, and to determine if termite colonies move vertically within trees to escape rising flood waters. Materials and Methods Research Sites Two research sites were established along the river batture in New Orleans, LA. This is an area between the Mississippi Ri ver and the levee, which floods annually between February and June. The flooding at the river batture ranged from 0.3 to 1.8 m 59

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during this study (www.noaa.gov ). The first river batture site was located on the New Orleans west bank, at the foot of the Cr escent City Connection Bridge (N 29.93942, W 90.05397). Five infested black willow trees ( Salix sp.) and two uninfested black willow trees were located at this site. The second river batture site was located at the Jackson Avenue-Gretna Ferry Station in downto wn Gretna, Louisiana (N 29.91834, W 90.06686). One infested black willow tree was located at this site. Three trees that did not sustain seasonal inundation acted as controls. One control was a live oak tree ( Quercus sp.) located in City Park, adjacent to Popps Fountain (N 29.99527, W -90. 09813). The second control was a live oak tree located along the lakefront on Lakeshore Driv e near Beauregard Avenue (N 30.02604, W -90.08444). A third control was a hackberry tree (Celtis sp.) located in New Orleans East, at the corner of Hayne Boul evard and Downman R oad (N 30.03301, W -90.02666). For all research sites, termite activity within the trees was confirmed before and after each flood season. The presence of live termites was documented by drilling into the tree trunk and viewing termites with an Everest XLG3 video borescope system (General Electric, Skaneateles, NY). Monitoring Termite Foraging Areas In-ground monitoring stations were insta lled at all research sites. These monitoring stations were the Pro Series r ound irrigation valve bo xes (Orbit Irrigation Products, Bountiful, UT), 15.24 cm in diameter, buried to lay flush with the ground. Each station contained a wood resource, cons isting of 11 untreated pine slats (18 by 10 by 0.5 cm in thickness) separated by wood tongue depressors and held together by tie wire. 60

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Grids of in-ground monitoring stations were established on 3 m centers, extending 9 m from infested tr ees at all research sites (Figure 5-1). Two in-ground monitoring stations were also installed at the base of each infe sted tree. At the inundated sites along the river batture, rows of 15 in-ground monitoring stations were installed on 3 m centers from the river to 4.5 m from the base of the levee wall. No stations were installed in the levee itself. On the protected side of the levee at t he Crescent City Connection river batture site, two rows of 15 in-ground monitoring stations were installed on 3 m centers, 4.5 m from the base of the levee wa ll. In-ground stations on the protected side of the levee were monitored to determine if termites move into or beyond the levee during flooding. At the Jackson Avenue-Gretna Fe rry Station river batture site a lack of green space on the protected side of the levee prevented the installation of in-ground monitoring stations. The monitoring stations were serviced at monthly intervals for two flood seasons (from November 2008 to October 2010). The onl y exception was the research site on the corner of Downman Road and Hayne Boulevard, which was removed from this study in June 2010 due to construction at an adjacent floodwall. For safety purposes, stations on the flooded side of the levee at th e river batture were not serviced once they became inundated by the Mississippi River. When feeding was observed during monthly inspections, the cellulose was r eplaced and termite samples were taken and preserved in 95% ethyl alcoho l. Voucher specimens were archived and stored at the New Orleans Mosquito and Termite Control Board Biolab facility. 61

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Consumption rates were calculat ed, as wood consumption was used as an indication of relative termite foraging activity. To determi ne consumption rates, the dry weight of each wood resource was re corded before placement within monitoring stations. Once the wood resource was fed upon, it was replaced an d transported to the laboratory, where it was cleaned of debris, dried in a Binder FED720 drying oven (Binder, Tuttlingen, Germany) at 105C fo r 24 hours, and weighed to determine the amount of wood consumed, in dry weight. Determining Vertical Movemen t of Termites within Trees During the course of this study, two infe sted black willow trees located at the Crescent City Connection river batture site h ad fallen due to strong storm winds coupled with termite damage within the trees. These trees were sectioned at 15 cm increments and evaluated for the presenc e of carton material. Colony Delineation To determine if termite colonies were foraging from infested trees to the monitoring stations and not fr om neighboring col onies, microsatellite genotyping was conducted on samples collected from inside the infested trees and those collected from in-ground monitoring stations surrounding the infested trees. For each site, samples were chosen for genotyping during the month in which the highest number of monitoring stations exhibited active foraging termi tes. Samples were chosen based on the proximity of in-ground monitoring stations from which they were collected. Eight samples from the control site on Downman Road, eight samples from the control site in City Park, seven samples from the control site on Lakeshore Drive, and four samples from the protected side of the levee at the river batture were subjected to genotyping. 62

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For each sample, 20 individual worker termites were subjected to DNA extractions using a Qiagen DNeas y Blood and Tissue Kit (Q iagen, Valencia, CA), PCR using locus-specific primers (Vargo and Hen derson 2000) to amplify microsatellites at six loci, and electrophoresis using a LI-COR 4300 DNA Analyzer (LI-COR, Lincoln, NE). Allelic frequencies were tested fo r significant differences using log-likelihood G-statistics (FSTAT; Goudet 2002) to determine if the te rmites collected from infested trees belonged to the same colony as those collected from the surrounding in-ground stations. Samples were considered to be from the same colony if the genotypic frequencies were not significantly different at the 5% level and no private alleles were observed. Results The average number of active stations, average percent wood consumption, maximum foraging distance, and the maximum foraging area observed at each research site were calculated (Table 5-1). At each control site and the protected side of the levee at the river batture, which did not experience seasonal inundation, foraging termites were observed within in-ground moni toring stations. The relative foraging activity, represented by the number of in-gro und monitoring stations containing foraging termites and the respective percent wood c onsumption, varied throughout the course of this study (Figures 5-2, 5-3, 5-4, and 5-5). There was no observed foraging activity in any of the monitoring stations at the seasonally inundated areas alon g the river batture. Live termites were observed inside these seasonally inundated trees between 0. 3 and 1.0 m from gr ound level using a video borescope before and after inundation for both flood seasons. No live termites were observed in the seasonally inundated trees above 1.0 m from ground level. 63

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Permutation tests of termites from 27 samples conduct ed at the 5% level of significance yielded that one colony was pr esent at each of the three c ontrol sites, and two colonies were present along the protected side of the levee at the river batture. After the felled trees on the seasonally i nundated side of the river batture were sectioned, it was observed that foraging galle ries were present within the trees above the water line, 1.8 m from ground level. Ho wever, carton material was only present below the water line, in tree sections that had been inundated. Within the first tree, carton material was observed from ground le vel to 1.4 m above ground level (0.4 m below the water level). Foraging galleri es extended from 1.4 to 7.1 m above ground level (0.4 below water level to 5.3 m above wa ter level). Within t he second tree, carton material was observed in tree sections fr om ground level to 1. 6 m above ground level (0.2 m below water level). Foraging galleries were observed in tree sections from 1.6 to 4.7 m from ground level (0.2 m below wa ter level to 2.9 m above water level). Discussion Foraging was observed within in-ground moni toring stations extending from trees in areas that did not experience flooding, while there was no such foraging observed within in-ground monitoring stations outside the infested trees at the seasonally inundated river batture. However, live termites were observed within seasonally inundated trees prior to and following each flood season. Microsatellite genotyping conducted on samples collected from each contro l site confirmed that termites nesting within infested trees were from the same colony observed foraging within in-ground monitoring stations surrounding the tree, and were not originati ng from neighboring colonies outside the boundaries of the research site. In cont rast, the colonies foraging on the protected side of the lev ee at the river batture site were not the same as those 64

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within the trees that experi ence s easonal inundation, as no foraging activity was observed within in-ground monitoring stati ons on the flooded side of the levee. Therefore, these C. formosanus colonies had not originat ed from inundated trees and shifted their foraging area to escape rising flood waters. Termite colonies located along the ri ver batture may be adapted to seasonal inundation by remaining wit hin trees, while those located in areas that do not experience seasonal inundation have larger foragi ng territories. This is similar to what was observed by Su and La Fage (1999), who determined that C. formosanus colonies restricted to cypress trees in the Calcasieu River have smaller foraging populations and foraging range than those obser ved on dry land. Because C. formosanus colonies remained within seasonally inundat ed trees, the assumption that C. formosanus colonies shift their foraging areas to escape rising wate r is incorrect, and our hypothesis that C. formosanus colonies move away from risi ng water to survive inundation is rejected. At least in areas that ex perience seasonal inundation, C. formosanus colonies do not shift their foraging areas to escape rising flood waters. Instead, termites remained confined to their food source throughout this study. This behavior was not observed for C. formosanus colonies which experienced prol onged inundation follo wing Hurricane Katrina, as foraging termites were obser ved within inundated in-ground monitoring stations before to flooding and after the fl ood waters receded (Cornelius et al. 2007, Osbrink et al. 2008). It may be that C. formosanus colonies that experience seasonal inundation have adapted behaviorally to surv ive repeated flood events, while those infesting areas that had not been subjected to inundation prior to Hurricane Katrina do 65

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not exhibit such behav ioral adaptations and readily forage away from protective structures. Whether termites moved from soil into trees during the flooding following Hurricane Katrina is unknown, as carton nest within trees could not be observed during the flooding. This is unlikely, though, as previous studies conducted by Cornelius and Osbrink (2010) have shown that in laboratory tests, C. formosanus does not attempt to evacuate from its gallery system within soil to seek higher elevation during flooding. Carton material was not present above the water line within trees that experienced seasonal inundation. This is not indicative of vertical movement of entire C. formosanus colonies, though foraging galleries located above the water line of seasonally inundated trees c ould provide the means of evacuation for individual termites. Cornelius and Osbrink (2010) have given evidence that C. formosanus when inhabiting hollowed wood, may attempt vertic al evacuation to escape rising waters. However, a relatively small percentage of the colony surviving flooding by vertically evacuating within trees does not explain the survival mechanisms of entire C. formosanus colonies during the four month flood season along the river batture, unless primary reproductives or neotenics vertica lly evacuated to survive and propagate the colony. This is because termites were observed within seasona lly inundated trees immediately after the flood waters receded, and this is not indicative of significant termite mortality. Because C. formosanus colonies nesting within seasonally inundated trees do not evacuate flooded trees, the nesting system within trees may be the mechanism by which C. formosanus survives inundation. Carton material, observed in the bases of seasonally inundated trees, may provide protection against saturated soil because of its 66

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67 hydrophobic properties (Cornelius et al. 2007). If carton material provides a sealed, watertight environment for termites, C. formosanus colonies could survive within inundated trees for extended periods.

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Table 5-1. Foraging activity observed at unfl ooded and seasonally inundated areas of New Orleans, Louisiana from No vember 2008 through October 2010 Research site No. active stations (mean SE) Total no. monitoring stations Percent wood consumption (mean SE) Maximum foraging distance (m)1 Maximum foraging area (m2)2 City Park 12.88 2.45 50 9.21 1.83 25 209 Lakeshore Drive 4.52 0.80 54 20.46 3.83 15 78 Downman Road 22.80 0.99 50 21.68 2.89 18 156 River Batture (protected side of levee) 2.08 0.52 30 5.56 1.38 9 10 River Batture (seasonally inundated areas) 0 150 0 0 0 1 Linear distance between furthest two interconnected stations 2 Area of all interconnected stations; col ony delineated by microsatellite genotyping 68

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69 A. B. C. Figure 5-1. Locations of in-ground monito ring stations established on 3 m centers extending 9 m from infested trees at the following cont rol sites: (A) City Park Popps Fountain, (B) Lakeshore Drive, and (C) corner of Downman Road and Hayne Boulevard.

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Month Nov2008 Dec2008 Jan2009 Feb2009 Mar2009 Apr2009 May2009 Jun2009 Jul2009 Aug2009 Sep2009 Oct2009 Nov2009 Dec2009 Jan2010 Feb2010 Mar2010 Apr2010 May2010 Jun2010 Jul2010 Aug2010 Sep2010 Oct2010 No. Active Stations 0 5 10 15 20 25 30 35 40Average % Consumption 0 5 10 15 20 25 30 35 40 Figure 5-2. Foraging activity of a C. formosa nus colony at City Park Popps Fountain. Bars represent the number of in-ground m onitoring stations in which foraging termites were observed each month. Solid circles represent the average percent of wood consum ed within the aforementi oned in-ground stations. 70

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Month Oct2008 Nov2008 Dec2008 Jan2009 Feb2009 Mar2009 Apr2009 May2009 Jun2009 Jul2009 Aug2009 Sep2009 Oct2009 Nov2009 Dec2009 Jan2010 Feb2010 Mar2010 Apr2010 May2010 Jun2010 Jul2010 Aug2010 Sep2010 Oct2010 No. Active Stations 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Average % Consumption 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Figure 5-3. Foraging activity of a C. formosanus colony on Lakeshore Drive. Bars represent the number of in-ground moni toring stations in which foraging termites were observed each month. Solid circles represent the average percent of wood consum ed within the aforementi oned in-ground stations. 71

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Month Nov2008 Dec2008 Jan2009 Feb2009 Mar2009 Apr2009 May2009 Jun2009 Jul2009 Aug2009 Sep2009 Oct2009 Nov2009 Dec2009 Jan2010 Feb2010 Mar2010 Apr2010 May2010 Jun2010 No. Active Stations 0 5 10 15 20 25 30 35 40 45 50Average % Consumption 0 5 10 15 20 25 30 35 40 45 50 Figure 5-4. Foraging activity of a C. formosanus colony on Downman Road. Bars represent the number of in-ground moni toring stations in which foraging termites were observed each month. Solid circles represent the average percent of wood consum ed within the aforementi oned in-ground stations. 72

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Month Nov2008 Dec2008 Jan2009 Feb2009 Mar2009 Apr2009 May2009 Jun2009 Jul2009 Aug2009 Sep2009 Oct2009 Nov2009 Dec2009 Jan2010 Feb2010 Mar2010 Apr2010 May2010 Jun2010 Jul2010 Aug2010 Sep2010 Oct2010 No. Active Stations 0 5 10 15 20 25 30Average % Consumption 0 5 10 15 20 25 30 Figure 5-5. Foraging activity of a C. formosanus colony along the protected side of the Mississippi River batture. Bars represent the number of in-ground monitoring stations in which foraging termites were observed each month. Solid circles represent the average per cent of wood consumed wit hin the aforementioned in-ground stations. 73

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CHA PTER 6 ABILITY OF Coptotermes formosanus COLONIES TO SURVIVE FLOODING WITHIN THEIR NESTING SYSTEM In Chapter 2, it was conc luded that at least some C. formosanus colonies survived prolonged inundation following Hurricane Katrina. Several hypotheses have been tested to determine the survival mechanisms of C. formosanus colonies during prolonged inundation. In Chapter 3, it was shown that termites did not survive flooding following Hurricane Katrina by tolerating inu ndation. However, Chapter 4 concluded that termites could have survived prolonged flooding inside pockets of trapped air within their nesting and gallery system. This seems most likely, as Chapter 5 demonstrated that termites do not shift their foraging ar ea to escape seasonal inundation. Instead, termite colonies subjected to seasonal inundation remained confined to their food source. There was no evidence of vertical evacuation within seasonally inundated trees to escape rising waters. This contrast s with the previous conclusions made by Cornelius and Osbrink (2010), who observed evidence of vertical movement of C. formosanus within hollowed wood blocks to escape rising waters. It is still unknown whether C. formosanus creates a sealed environment capable of protecting termites from rising waters and trapping air within their nest and gallery system, which would sustain a colony during periods of flooding. This, though, has been speculated by others (Cornelius et al. 2007, Osbrink et al. 2008). Our hypothesis tested here is that C. formosanus colonies survived prolonge d inundation following Hurricane Katrina without evacuating by creating a sealed environment filled with trapped air within their nesting and gallery system and remaining there until flood waters receded. Termites may have also exploit ed air trapped within water-resistant galleries extending above the water line. 74

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Materials and Methods Termites Termites were collected from five C. formosanus field colonies in New Orleans, Louisiana. Two colonies were loca ted along Lakeshore Dr ive (N 30.03189, W -90.06701). One colony was located on the corner of Marconi Drive and Robert E. Lee Boulevard (N 30.01825, W -90.09747). Another colony was located on Audubon Zoo property (N 29.92372, W -90.13063). The fifth colony was located in Brechtel Park (N 29.90533, W -90.00804). Term ites were collected from pr eviously established milk crate traps and stored in the same manner described in Chapter 3. Within one week, termites were bioassayed. Mean worker weights for each colony were calculated as previously described in Chapter 3. The mean worker weight of 100 termites was multiplied by 200 to determine the weight of 20,000 termites for each colony. Artificial Tree Inundation Bioassay To determine if C. formosanus colonies survive inundation by creating a watertight environment within their nest, a bioassay was conducted in which five artificial trees were infested with termite foragers and exposed to periodic inundation. Artificial trees were made by hollowing the centers of 50 cm sections of trees trunks that ranged from 20 to 40 cm in diameter (Figure 6-1). For each tree, the hollowed center had a diameter equal to half that of the tree section. Thr ee tree sections were from cypress trees ( Taxodium sp.) and two were from live oak trees ( Quercus sp.). Tree sections were first heated to 105C in a Binder FED720 drying oven (Binder, Tuttlingen, Germany) for 24 hours to kill any organism s living within the tree sections. 75

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The bottom end of each tree secti on was capped with 1.3 cm thick polyethylene sheeting (United States Plastic Corpor ation, Lima, OH) and sealed with silicone plumbing caulk. Twelve holes that were 0. 3 cm in diameter were drilled into the bottom cap to allow termites to forage outside the tree sections. Each artificial tree was placed within a galvanized steel containe r (69 cm in height by 52 cm in diameter) on top of play sand and gravel to help facilitate drainage. Prio r to placement of artificial trees within the steel containers, a spigot was installed 3 cm from the container bottom for drainage. Play sand was added to the cont ainers and the hollowed centers of tree sections so that the substrate was 18 cm deep in side and surrounding the artificial trees. Play sand was moistened with carbon -filtered water. Twenty thousand termites, by weight, were placed into the hollowed center of each artificial tree. Each tree section contai ned termites from a different colony. After termites were added, the top of each tr ee section was capped with 1.3 cm thick polyethylene sheeting and sealed with silicone pl umbing caulk. The top cap included a 50 cm section of 5 cm polyvinyl chloride (PVC ) pipe protruding upwards. This pipe was filled with corrugated cardboard moistened with ca rbon-filtered water. This was to allow termites to travel vertically, as they would in a non-artificial environment. A 2.5 cm hole was drilled through the top polyethylene shee ting and plugged with a rubber stopper to maintain an airtight environment. This hole was necessary for viewing termites and measuring percent atmospheric oxygen within ar tificial trees. A 28 cm pine stake was inserted into the sand adjacent to each arti ficial tree inside the steel containers to monitor termite foraging out side of the tree sections. 76

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Termites within the arti ficial trees were allowed to forage and tunnel for four weeks. At the end of four weeks, artifi cial trees were inundated with enough carbonfiltered water so that the water level was at 30 cm. The duration of this initial inundation was two hours. At Weeks 6, 8, 10, 12, 14, and 16, artificial trees were inundated for 4, 6, 8, 12, 24, and 48 hours, respectively. One artificial tree was used as a control and was not subjected to inundation. Each week, the pine stakes were checked for evidence of consumption and the presence of active foraging termites. Also at weekly intervals, termites were monitored inside the artificial trees using an Ev erest XLG3 video borescope system (General Electric, Skaneateles, NY). The interiors of inundated artificial trees were monitored for the presence of water during periods of inundation. The percent atmospheric oxygen within each artificial tree was measured each week using an X-am 2000 oxygen monitor (Draeger Safety Inc., Pittsburgh, PA). Vertical movement of termites was m easured by removing corrugated cardboard from the PVC pipe protrudi ng from the tree sections and counting the number of termites located within the cardboard. Followi ng this, termites were returned to their respective artificial trees and new cardboard was inserted into the PVC pipe. During weeks in which artificial trees were inundated, termites within the arti ficial trees, oxygen levels, and termites within cardboard inside the PVC pipe were monitored before and during inundation. Foraging Arena Inundation Bioassay Twelve foraging arenas were construct ed similarly as those described by Su (2005) (Figure 6-2). Arenas c onsisted of two transparent Plex iglas sheets (24 by 24 by 0.6 cm thick). Between these s heets, four Plexiglas laminates (three that were 2 cm in 77

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width, one that was 7 cm in width) that were 0.2 cm thick, were placed along the outer edges. The foraging area within each arena was 20 by 15 cm in size. To maintain structural i ntegrity, an additi onal 2 cm by 2 cm piece of Plexiglas laminate was inserted in the center of the arena. All pieces of Plexiglas and Pl exiglas laminate were bolted into place. An access hole (0.5 cm in diameter) was included on the upper sheet near one of the corners to allow termites to enter the for aging area. A termite release chamber, consisting of a Plexiglas cup (4.5 cm in diameter and 3 cm in height) with a lid, was attached onto the access hole. Additional access holes on the outer edges of the arenas were closed by attaching a piece of stainless steel mesh (2 by 2 cm) (Termimesh LLC, Austin, TX) to the access holes. Approximatel y 60 g of sifted sand (150 500 m sieves) were added to each arena interior. Sand was then moist ened with approximately 15 ml of deionized water. The edges of each arena were made wa tertight by sealin g them with silicone plumbing caulk. Pieces of wood (2.5 by 2.5 by 0.5 cm thick) were saturated with deionized water and placed inside each termite release chamber to serve as a food source. Additional termites collected from three of the five field colonies previously described were included in this bioassay. Thes e three field colonies were located at the corner of Marconi Drive and Robert E. Lee Boulevard, Audubon Zoo, and Lakeshore Drive. One thousand termites (900 worker s and 100 soldiers) were added to the termite release chamber of each foraging arena. Termites were allowed to forage within t he arenas for six weeks. At the end of this period, the stainless steel mesh attached to access holes was removed and 78

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approximately 15 ml of deioniz ed water was added to each arena via these access holes at a rate of 3 ml per minute. Wh ile the top half of each arena, the area which includes the termite release chamber, remained unflooded, the bot tom half of each arena was inundated. There were three replic ates for each colony, and one additional replicate for each colony was not subject ed to inundation and acted as the control. Video photography was employed to reco rd the behavior of termites during inundation using a Canon X L2 camera with a 100 mm macro lens (Canon, Lake Success, NY). Photographs were taken of each arena prior to and following inundation using a Canon EOS 40D with a 100 mm macro lens (Canon, Lake Success, NY). To determine if termites moved away from water of if they remained within their galleries, photographs of each arena were analyzed. Termites in flooded and non-flooded halves of the arenas were counted fo llowing inundation. Termites were then counted in these same areas (those to be flooded and those to remain unflooded) of the arenas in photographs taken before flooding. Statistical Analysis The numbers of termites located in corrugated cardboard in PVC pipe protruding from the artificial trees before inundation were compar ed to those observed during inundation using a paired t -test to determine if termites we re vertically evacuating during flooding. The percent oxygen levels observe d in the inundated artificial trees were compared to those observed in the contro l tree, which was not inundated, using a paired t -test. The percent oxygen le vels in all inundated arti ficial trees observed before flooding were compared to those observed during flooding using a paired t -test to determine if termites within inundated tr ee experience an environment in which less atmospheric oxygen is available. 79

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To determine if termites within the foraging arenas were evenly distributed in the foraging areas before inundati on, the number of termites lo cated in the bottom half of each arena was compared to those loca ted in the top half using a paired t -test. This statistical analysis was also conducted for the control arenas, which were not inundated. Following inundation of the foraging arenas, the number of termites located in the bottom, flooded, half of each arena was compared to the number of termites located in the top half of each arena using a paired t -test to determine if termites in the flooded arenas exhibited an even distribution within the foraging areas dur ing inundation. The number of termites located in the bottom and top halves of arenas before flooding were compared to those located in the same areas during inundation using paired t -tests. All statistical analyses were conducted using SAS 9.1 (SAS Instit ute 2003, Cary, NC). Results During the artificial tree inundation bioassay, termites were observed foraging outside only one artificial tree. However, after the initial inundat ion of artificial trees at Week 4, this foraging was no longer observ ed. Live termites were observed inside each artificial tree using the video borescope every week Three out of four artificial trees did not exhibit water entering into their hollowed interior during periods of inundation (Figure 6-3 A). Water was observed in the interior of the fourth artificial tree during each period of inundation (Figure 6-3 B). As water entered into the interior of the fourth artificial tree, termites were observed fl oating on the water surface. The number of termites located in the corrugated car dboard within the upwardsprotruding PVC pipe prior to flooding was not si gnificantly different from the number of termites in the same cardboard during periods of inundation (t = -1.74, df = 19, 80

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P = 0.0975). The percent oxygen observed in t he control artificial tree ranged from 18.7 to 20.9%, and the percent oxygen observed in the inundated artificial trees ranged from 17.6 to 20.4% (Figure 6-4). The percent oxygen obser ved in the control artificial tree was significantly higher t han that observed in the i nundated artificial trees ( t = 4.40, df = 27, P = 0.0002). Artificial trees subjec ted to periodic inundation exhibited significantly higher oxygen levels within thei r interior before flooding than during flooding ( t = 4.18, df = 27, P = 0.0003). Termites in the foraging arenas were obs erved moving away from water during inundation (Figure 6-5). Thos e that were overcome by ri sing water did not exhibit any movement once they were i nundated. However, termites that became surrounded by water but were located within a pocket of air were observed antennating the waters edge. The foraging tunnels remained intact, though water passed through the galleries. Termites were not observed repairing or constructing any tunnels during inundation to further protect themselves from rising water. The numbers of termites located in t he top halves of the arenas were not significantly different than t he numbers of termites located in the bottom halves of the arenas before flooding (t = -0.40, df = 8, P = 0.349). During inun dation, there were significantly more termites observed in the unflooded areas of arenas than those observed in the flooded areas of arenas ( t = -13.80, df = 8, P <0.0001). In the bottom halves of the arenas, which were inundated, t here were significantly more termites before flooding than during flooding ( t = 6.13, df = 8, P = 0.0003). There were significantly more termites located in t he top, unflooded, halve s of arenas following flooding than before flooding ( t = -6.21, df = 8, P = 0.0003). 81

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Discussio n The lack of foraging outside any of the arti ficial trees following initial inundation was not due to termite morta lity following in undation, as live termites were observed inside artificial trees each week during the artificial tree inundation bioassay. Rather, this behavior is more representative of w hat was observed in Chapter 5, in which C. formosanus colonies infesting seasonally inund ated trees along the Mississippi River batture did not exhibit any foragi ng away from their food source. Because water was not observed within thr ee out of four artificial trees during periods of inundation, the hypothesis that C. formosanus colonies could have survived prolonged inundation following Hurricane Katrina by creating a sealed environment filled with trapped oxygen within their nest and rema ining there until flood waters receded is accepted. This method of protection may not necessarily be solely used for protection against flooding. Creating a sealed environment could also provide protection against predation and other insects that would otherwise exploit the termite nest and resources. However, this adaptation was not observed fo r all colonies, even following the initial inundation period. Ev en without this adaptation, there wa s no evidence of high termite mortality within any artificial tree. Live termites were view ed inside each artificial tree every week during the inundati on bioassay. Termites inhabiting the artificial tree that was not sealed from rising water may have survived by either floating on the water surface until the water receded, as was observed, or by possibly remaining within hidden pockets of air at t he base of the tree section. Coptotermes formosanus is known to readily infest trunks of live trees and create carton material that can ext end meters above ground level (Osb rink et al. 1999, Osbrink et al. 2008). It has been suggested that th ese voids can trap air that can sustain 82

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termites below the water level during in undation (Osbrink et al. 2008) and the C. form osanus colonies may have survived prolonged inundation following Hurricane Katrina by remaining protected within t heir hydrophobic carton nests and gallery systems (Cornelius et al. 2007, Osbrink et al 2008). Galleries located above the water line may also serve to trap air that can be exploited by termites during periods of inundation. This is the most likely survival mechanism of C. formosanus during prolonged inundation for two reasons. First, Chapter 4 concluded that termites can survive within an airtight env ironment for up to a month. Second, it has been shown that termites create a sealed, protective environment within trees that traps air, which can be exploited during periods of inundation. There is no evidence from our data that suggests termites evacuate vertically within trees to escape rising flood waters. A significantly high num ber of termites was not observed moving vertically within the inundated artificial trees, including the artificial tree that was not sufficiently protected against ri sing water. In contra st to this finding, termites occupying foraging galleries within foraging arenas were observed moving away from water during inundation. At t he conclusion of the foraging arena inundation bioassay, significantly more termites occupied nonflooded areas than those which succumbed to inundation. Our findings differ greatly from those di scussed by Cornelius and Osbrink (2010), who concluded that C. formosanus moves vertically within wood in response to rising water, but does not move from their galleries to escape rising water. There was also no evidence of vertical evacuation observed in C hapter 5. This lack of movement from the 83

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gallery system in response to flooding was also observ ed for Reticulitermes flavipes (Forschler and Henderson 1995). Though foraging tunnels within arenas appe ared to remain intact during inundation, water readily passed through t he galleries. There were no observations made of termites attempting to repair tunnels or prevent water from rising further. These observations would indicate that eit her the interphase betwe en the Plexiglas and sand was not watertight, or foraging galle ries may have at least some hydrophobic properties, but would not provide as effect ive protection under t he soil surface during periods of inundation as that provided by ca rton material within the nest inside trees. The significant reduction in atmospheric oxygen experienced by termites within artificial trees that were subjected to per iodic inundation is most likely due to termites occupying a sealed environment. This seal was initially created by capping the top of the tree sections, making them airtight. The percent at mospheric oxygen within the inundated trees was never lower than 17.6%. Even with this reduced oxygen level, termites were able to survive in this hypoxic environment. 84

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Scale: monitoring stake PVC pipe sand gravel cardboard rolls water level during inundation hollowed tree section hole for viewing termites polyethylene sheeting galvanized steel container spigot 10 cm Figure 6-1. Schematic of an artificial tree infested with C. form osanus foragers within the hollowed center and subjec ted to periodic inundation. 85

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Scale: 10 cm Figure 6-2. Foraging arena c ontaining 1,000 termites, added via the termite release chamber, prior to the addition of wate r into the bottom half of the arena 86

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87 A. B. Scale: Scale: 5 mm 10 mm Figure 6-3. Images taken using a video bore scope system to determine if water entered hollowed sections of artificial trees during periods of inundation. (A) Three out of four inundated artificial trees did not exhibit water entering into hollowed tree sections during periods of inundation. (B) One artificial tree filled with water during periods of inundation, and termites were observed floating on the water surface.

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W eek 012345678910111213141516 0 5 10 15 20 25 Percent Oxygen Figure 6-4. The percent atmospheri c oxygen observed each week in a ll artificial trees infested with C. form osanus. The black bars represent oxygen levels wit hin the artificial control tree. Th e light grey bars represent average oxygen levels within inundated artificial trees prior to and between periods of inundation. The dark grey bars represent average oxygen levels within inundated artificial trees during periods of flooding. 88

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A. B. Scale: 10 cm C. Scale: 5 mm Figure 6-5. Observations of termite move ment within a foraging arena subjected to inundation. (A) Prior to flooding, termites are evenly distributed throughout the arena. (B) During inundati on, termites were observed moving away from rising water. (C) At least some term ites within each arena became restricted within a pocket of air surrounded by water. 89

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CHA PTER 7 CONCLUSIONS Genetic analysis was used to compare inundated termite colonies in New Orleans, Louisiana before and after flooding follo wing Hurricane Katrina. The results revealed that termites occ upying inundated in-ground monitori ng stations after flooding belonged to colonies that had occupied the sa me monitoring stations before flooding. Therefore, these termite colonies surviv ed flood conditions for up to 14 days following the storm. Once it was determined that termite colonies survived prolonged flooding, a series of laboratory bioassays and a field study were conducted to elucidate the survival mechanisms of termites during extended periods of inundation. The first hypothesis tested was that C. formosanus colonies survive prolonged inundation by tolerating being under water. This was not accepted, as termi tes forced to remain under water exhibited 100% mortality at 20 hours at a relative ly warm temperature, and 60 hours at a relatively cool temperature. The second hypothesis was that C. formosanus colonies survive prolonged flooding by exploiting air trapped within voids in their nesting system. Based on the LT50 values for groups of termites confin ed to airtight environments, it was concluded that termites could have surviv ed the flooding following Hurricane Katrina by remaining within airtight voids in their nes ts, though this is dependent upon the size of voids and temperature. The third hypothesis was that C. formosanus colonies move away from rising flood waters to avoid satura ted soil conditions. This was not accepted, as termite colonies infesting trees loca ted in seasonally inundat ed locations in New Orleans were not observed moving away from the trees during periods of inundation. Rather, they remained within their nesting syst em in their food source. The fourth 90

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91 hypothesis tested was that C. formosanus colonies create a watertight environment within their nesting system. Bioassays in whic h artificial trees were infested with termites and inundated while termite behavior was observed confirmed that termites can create a sealed environment t hat offers protection fr om rising flood waters. Coptotermes formosanus colonies are known to readily infest trunks of live trees and create carton material that is locat ed below ground level an d can extend meters above ground level (Osbrink et al. 1999, Osbrin k et al. 2008). These colonies may have survived prolonged inundation fo llowing Hurricane Katrina by remaining protected within their hydrophobic carton nests (Osbrink et al. 2008, Cornelius et al. 2007). Galleries located above the water line may also serve to trap air that can be exploited by termites during periods of inundation. Hurricane Katrina gained much notoriety due to its widespread flooding and devastation to an urban area. However, fl ooding of urban areas is not isolated to only New Orleans. Flooding in the United States can occur from tropical storms, hurricanes, and rising rivers due to ice melt in northern st ates. For those whom reside in locations within the United States in which C. formosanus has become established, it is important to understand that a natural disaster, such as a prolonged period of flooding, will not eradicate C. formosanus colonies.

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BIOGRAPHICAL SKETCH Carrie Beth Owens was born in St. Louis, Missouri. She was raised in St. Charles, Missouri, a suburb of St. Louis, and graduated from Francis Howell North High School in 1997. Carrie earned a Bachel or of Science degree in biology ( cum laude ) from the University of Miss ouri St. Louis in 2002. She then moved to Fayetteville, Arkansas, where she was employed full time as a Research Spec ialist for the Department of Entomology at the University of Arkansas. While working full time, she earned her Master of Science degree in cell and molecular biology in 2005, for which her thesis title was, Molecul ar detection and seasonal abundance of Campylobacter and E. coli O157:H7 carried by filth flie s (Diptera: Muscidae). Following graduation, Carrie was offered em ployment at the City of New Orleans Mosquito, Termite, and Rodent Control Boar d (NOMTCB). She moved to New Orleans in June 2005, just two months prior to Hurri cane Katrina making land fall. After safely evacuating, Carrie promptly returned to New Orleans, where she not only assisted with pest control operations following the stor m, but was also assigned the task of establishing a genetics laboratory for NOMTCB from its inception. She is still responsible for research that requires employment of mo lecular methods as well as overall laboratory management. Carrie has been employed full-time while participating in the University of Florida distance educat ion program. Upon completion of her Ph.D. program, Carrie will continue her employment at NOMTCB. 99