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Behavioral and Histological Observations of the Molting Process of the Formosan Subterranean Termites (Isoptera

Permanent Link: http://ufdc.ufl.edu/UFE0045101/00001

Material Information

Title: Behavioral and Histological Observations of the Molting Process of the Formosan Subterranean Termites (Isoptera Rhinotermitidae) and Changes Induced by the Chitin Synthesis Inhibitor, Noviflumuron
Physical Description: 1 online resource (58 p.)
Language: english
Creator: Xing, Lin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

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

Notes

Abstract: Molting process of Formosan subterranean termites is described by histological study. The ecdysisial behaviors and effects of nestmates during ecdysis are also studied in this thesis. After the normal molting process is described, noviflumron is applied to termites to observe both the histological and behavioral changes. Chapter 1 is the general introduction about termite biology, distribution, taxonomy and most frequently used control methods. Ecdysisal behaviors and effects of nestmates on ecdysis are studied in Chapter 2. The presence of termite nestmates is not required for molting individual to finish the ecdysis, which leads us to reject a speculation of Raina et al. (2007). In Chapter 3, molting process of termites is described at the histological level. After noviflumuron is applied to termites, the changes induced by it are described at both behavioral and histological levels. Termites affected by noviflumuron begin the ecdysis as usual, but no breach is formed on the dorsal abdomen. From the histology study, new cuticles are either missing or incompletely formed, which may explain why the ecdysisial inhibition appears in termites treated with noviflumuron.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Lin Xing.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Su, Nan-Yao.
Local: Co-adviser: Kern, William H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045101:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045101/00001

Material Information

Title: Behavioral and Histological Observations of the Molting Process of the Formosan Subterranean Termites (Isoptera Rhinotermitidae) and Changes Induced by the Chitin Synthesis Inhibitor, Noviflumuron
Physical Description: 1 online resource (58 p.)
Language: english
Creator: Xing, Lin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

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

Notes

Abstract: Molting process of Formosan subterranean termites is described by histological study. The ecdysisial behaviors and effects of nestmates during ecdysis are also studied in this thesis. After the normal molting process is described, noviflumron is applied to termites to observe both the histological and behavioral changes. Chapter 1 is the general introduction about termite biology, distribution, taxonomy and most frequently used control methods. Ecdysisal behaviors and effects of nestmates on ecdysis are studied in Chapter 2. The presence of termite nestmates is not required for molting individual to finish the ecdysis, which leads us to reject a speculation of Raina et al. (2007). In Chapter 3, molting process of termites is described at the histological level. After noviflumuron is applied to termites, the changes induced by it are described at both behavioral and histological levels. Termites affected by noviflumuron begin the ecdysis as usual, but no breach is formed on the dorsal abdomen. From the histology study, new cuticles are either missing or incompletely formed, which may explain why the ecdysisial inhibition appears in termites treated with noviflumuron.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Lin Xing.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Su, Nan-Yao.
Local: Co-adviser: Kern, William H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045101:00001


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1 BEHAVIORAL AND HISTOLOGICAL OBSERVATIONS OF THE MOLTING PROCESS OF THE FORMOSAN SUBTERRANEAN TERMITES (ISOPTERA: RHINOTERMITIDAE) AND CHANGES INDUCED BY THE CHITIN SYNTHESIS INHIBITOR, NOVIFLUMURON By LIN XING A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Lin Xing

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3 To my family

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4 ACK NOWLEDGMENTS First and foremost, I would like to thank my major advisor, Dr. NanYao Su, for keep giving me opportunities until I finished my master thesis project despite lots of trouble I brought to him in my course studies. Not only does he provides me with helpful advices and guidance, but also teaches me how to read the papers critically and define the question clearly. Without his forgiveness for my mistakes and financial support, I would not be able to finish my Master degree. I would like to thank D r.Thomas Chouvenc for teaching me the histology techniques and editing my thesis and powerpoint in past two years. I am equally thankful to Dr. William H. Kern for taking care of my academic progress. Instead of online videos, some of the local Lectures al low me to have more time getting adapted to American education system. I also would like to acknowledge my committee member, Dr. GiblinDavis, for keeping tracking of my experimental progress and giving me valuable suggestions about thesis writing. I woul d like to thank Dr. Houfeng Li, who taught me to wisely give up things that do not belong to me. Joanne Korvick and Nancy taught me to use English in proper ways. Finally, I would like to thank my parents for their support of my daily life. I am grateful to Yu Wang, who always understands me and support my professional career. Looking back to my memories, she is always there and accompanies me when I am doing experiments at weekends.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIG URES .......................................................................................................... 7 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 2 DESCRIPTION OF THE MOLTING PROCESS OF THE FORMOSAN SUBTERRANEAN TERMITE (ISOPTERA: RHINOTERMITIDAE) ......................... 19 Materials and Methods ............................................................................................ 19 Termite collection ............................................................................................. 19 Identification of preecdysis individuals and description of molting process ..... 19 Effects of nestmates on molting individual in ecdysis ....................................... 20 Results .................................................................................................................... 21 Description of molting process ......................................................................... 21 Phase 1: From peristalsis to appearance of abdominal breach (Fig. 21) .. 21 Phase 2: From breach forming to leg pulling (Fig. 22) .............................. 22 P hase 3: From the initiation of leg pulling to the separation of appendage cuticles (Fig. 23) ................................................................. 22 Phase 4: From the separation of appendage cuticles to exuvia separation (Fig. 24) ............................................................................... 23 Effects of nestmates to molting individual in ecdysis ........................................ 24 Discussion .............................................................................................................. 24 3 CHARACTERIZING THE MOLTING PROCESS IN COPTOTERMES FORMOSANUS SHIRAKI (ISOPTERA: RHINOTERMITIDAE) WITH HISTOLOGICAL STUDY ........................................................................................ 28 Materials and Methods ............................................................................................ 28 Sample individuals at different molting phases ................................................. 28 Histological preparation .................................................................................... 29 Results .................................................................................................................... 29 Termites in intermolt period (1) ......................................................................... 29 Termites in premolting fast period (2) .............................................................. 30 Termites in pre ecdysis and ecdysis (3,4) ........................................................ 30 Termites in newly molted period (5) ................................................................. 30 Discussion .............................................................................................................. 30

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6 4 HISTOLOGICAL OBSERVATION OF MOLTING ALTERATION OF FORMOSAN SUBTERRANEAN TERMITES (ISOPTERA: RHINOTERMITIDAE) CAUSED BY NOVIFLUMURON .......................................... 42 Materials and Methods ............................................................................................ 42 Results .................................................................................................................... 43 Discussion .............................................................................................................. 43 5 CONCLUSIONS ..................................................................................................... 51 LIST OF REFERENCES ............................................................................................... 53 BIOGRAPHICAL SKETCH ............................................................................................ 58

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7 LIST OF FIGURES Figure page 2 1 Phase 1: a--> b -> c ............................................................................................ 21 2 2 Phase 2: a--> b -> c ............................................................................................ 22 2 3 Phase 3: a--> b ................................................................................................... 23 2 4 Phase 4: a--> b ................................................................................................... 23 2 5 Mottled headcapsule during preecdysis indicated that termites were to start the ecdysis within 12 h. ...................................................................................... 26 2 6 Termites without blue color were collected. ........................................................ 26 2 7 The confined tube was designed to restrict the termite activity range ................ 27 2 8 The time required for termites to complete an interval in ecdysis. Significant difference was marked by asterisk (Student t test, =0.05). .............................. 27 3 1 Termites were sampled at five phases before and after the molting period.. ...... 28 3 2 Cuticles of termites during intermolt period are relatively smooth. hg=hindgut, cu=cuticle. .......................................................................................................... 32 3 3 Gut fauna were inside hindgut during intermolt period. hg=hindgut, pro= protozoa. ............................................................................................................ 33 3 4 No trachea shedding from the tracheal system during intermolt period. tra=trachea. ........................................................................................................ 33 3 5 Muscles were tightly connected to the cuticle during the intermolt period. cu= cuticle, mu= muscle. ........................................................................................... 34 3 6 Muscles were tightly connected to the cuticle in the intermolt period. cu= cuticle, mu= muscle. ........................................................................................... 34 3 7 Epidermal cells were in inactive status and they were not easily observed underneath the cuticle during the intermolt period. ep= epidermal cells. ............ 35 3 8 Epidermal cells were not easily observed underneath the cuticle during intermolt period. cu= c uticle ................................................................................ 35 3 9 Gut fauna of hindgut were voided during the premolting fast period. hg= hindgut, mal= malpighian tubule. ........................................................................ 36

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8 3 10 Cuticles inside tracheal system were about to separate. mu= muscle, tra=trachea. ........................................................................................................ 36 3 11 Abdominal muscles began to reattach to new cuticle during premolting fasting phase. nc= new cuticle, oc= old cuticle, mu= muscle. ............................. 37 3 12 Abdominal muscles reattachment progress during premolting fasting phase. nc= new cuticle, oc= old cuticle, mu= muscle, ep=epidermal cells. .................... 37 3 13 Abdominal muscles reattachment was in progress during premolting fasting phase. mu=muscle, nc= new cuticle, oc= old cuticle. ......................................... 38 3 14 Abdominal muscles almost finished reattachment during premolting fasting phase. ep= epidermal cells, mu= muscle, nc= new cuticle, oc= old cuticle. ....... 38 3 15 Wrinkled cuticle structure appeared during preecdysis phase. cut= cuticle, ma= malpighian tubule. ...................................................................................... 39 3 16 New cuticles were already completely formed underneath the old cuticle during preecdysis phase. nc= new cuticle, oc= old cuticle ................................ 39 3 17 Cuticles of old trachea were completely separated from the tracheal system during preecdysis. nc= new cuticle, oc= old cuticle. .......................................... 40 3 18 Cuticles of old trachea were completely separated from tracheal system during preecdysis. nc= new cuticle, oc= old cuticle. .......................................... 40 3 19 Cuticles of newly molted termit es retained the wrinkled feature. cut= cuticle. .... 41 4 1 Chemical structure of noviflumuron .................................................................... 45 4 2 Ziploc plastic box with moist t hin sand layer and soaked bait pieces ................. 45 4 3 Bait pieces that were consumed by termites. ..................................................... 46 4 4 Protozoa were still voided with noviflumuron treatment. cut= cuticle, hg= hindgut. ............................................................................................................... 46 4 5 No new cuticle was formed underneath the old cuticle. ep=epidermal cells, mu= muscle, oc= old cuticle, nc= new cuticle. .................................................... 47 4 6 Thin new cuticle layer showed up underneath the old cuticle. nc= new cuticle, oc= old cuticle. .................................................................................................... 47 4 7 Incomplete new cuticle was formed underneath old cuticle. nc= new cuticle, oc= old cuticle. .................................................................................................... 48 4 8 Old cuticles inside tracheal system were partially detached. oc= old cuticle, tr= trachea. ......................................................................................................... 48

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9 4 9 Muscles were reattached to the incompletely formed new cuticle. mu= muscle, nc= new cuticle, oc= old cuticle. ............................................................ 49 4 10 Termite that has body fluid leaking out ............................................................... 49 4 11 White spots were jammed on the abdomen of termite. ....................................... 50

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10 Abstract of Thesis Presented to the Graduate School of the Univer sity of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BEHAVIORAL AND HISTOLOGICAL OBSERVATIONS OF THE MOLTING PROCESS OF THE FORMOSAN SUBTERRANEAN TERMITES (ISOPTERA: RHINOTERMITIDAE) AND CHANGES INDUCED BY THE CHITIN SYNTHESIS INHIBITOR NOVIFLUMURON By Lin Xing Dec ember 2012 Chair: Nan Yao Su Co chair: William Kern Major: Entomology and Nematology Molting process of Formosan subterranean termites is described by histological study. The ecdysisial behaviors and effects of nestmates during ecdysis are also studied in this thesis. After the normal molting process is described, noviflumron is applied to termites to observe both the histological and behavioral changes. Chapter 1 is the general introduction about termite biology, distribution, taxonomy and most frequently used control methods. Ecdysisal behaviors and effects of nestmates on ecdysis are studied in Chapter 2. The presence of termite nestmates is not required for molting individual to finish the ecdysis which leads us to reject a speculation of Raina et al. (2007). In Chapter 3, molting process of termites is described at the histological level. After noviflumuron is applied to termites, the changes induced by it are described at both behavioral and his tological levels. Termites affected by noviflumuron begin the ecdysis as usual, but no breach is formed on the dorsal abdomen. From the histology study, new cuticles are either

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11 missing or incompletely formed, which may explain why the ecdysisial inhibition appears in termites treated with noviflumuron.

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12 CHAPTER 1 INTRODUCTION Termites play an important role as decomposers in tropical and subtropical regions and have a significant impact on pedogenesis and the ecosystem balance (Bignell 2006). However, when termites feed on manmade structures, they become economically important pests (Edward and Mill 1986). According to the records, 80 of the approximate 3,000 termite species are considered important pests to wood structures and 38 of them are subterranean termites (Edwards and Mill 1986). Based on insecticide sales figures in 2010, the cost of controlling subterranean termites was estimated at $32 billion worldwide (Rust and Su 2012). Although this sales figure represents all subterranean termite pest s pecies, different genera and species cause significantly different damage to structures ( Rust and Su 2012). The genus Coptotermes has the largest number of pest species (19 species) in the subterranean termites ( Rust and Su 2012). The two species, Coptoter mes formosanus Shiraki and Coptotermes gestroi Wasmann, are considered the most damaging economical pest species because of their wide distribution and relatively large colony size. This study focuses on C. formosanus Coptotermes formosanus was initially identified in Taiwan by Shiraki (1909), but its true origin has been traced back to China, with the discovery of a termitophile species, Sinophilus xiai Kistner (Coleoptera: Staphylinidae) This conclusion has two levels of evidence to support it. First, highly specialized and host specific termitophile species that is recovered only from the nests of C. formosanus can only be found in China. Second, it is difficult to transport, because it requires the entire colony to be transported ( Kistner 1985). World wide cargo transportation helped to introduce this

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13 species to several countries. Presently, C. formosanus has been found in Japan, China, Taiwan, South Africa, and the United States. It is distributed in both subtropical and temperate climate zones but mos t are found in subtropical areas (Li et al. 2009, Su 2003). In the United States, C. fomosanus was first reported from Hawaii (Sweezy 1914). The earliest C. formosanus existence in the continental United States was recorded in Charleston, South Carolina, but the first positive establishment was reported from Lake Charles, Louisiana in 1967. To date, C. formosanus is reported in nine southeastern states: Alabama, Florida, Georgia, Louisiana, Mississippi, North and South Carolina, Tennessee and Texas and has been considered the most economically important termite pest wherever it occurs (Spink 1967, Messenger et al. 2002, Su 2003, Su and Scheffrahn 2010). Coptotermes formosanus can cause severe damage to wooden structures and there are several ways to suppress or eliminate termite populations, including wood treatment, baits and soil barrier treatment (which include soil termiticide barriers and physical barriers) (Su 1998). The application of chemical barriers and termite baiting systems are the most frequentl y used in both preventive and remedial treatment against subterranean termites (Rust and Su 2012). Wood treatments are still used in preventative practice and some of the commonly used preventatives include creosote and inorganic salts. Although pressuret reated wood is required to be used in soil termites still are able to pass through the untreated portions (Su and Scheffrahn 2010). In soil treatment, currently available termiticides can be divided into two types: nonrepellent and repellent. Current non repellent termiticides include fipronil, imidacloprid, chlorfenapyr and chlorantraniliprole. The current repellent termiticides are pyrethroids

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14 that include bifenthrin, cypermethrin, and permethrin (Su 2011). Both nonrepellent and repellent termiticides can be used either as a preconstruction treatment (spray into subslab soil) or a post construction treatment (by drilling holes and applying beneath the building foundation or trenching). In 2002, a survey of the termite control industry showed that 77% of termite control firms used liquid termiticide and 38% used bait products (Rust and Su 2012). These overlapping figures are due to some company using both baits and liquid termiticides. A similar survey in 2009 indicated that usage of liquid termiticide s increased to more than 80% market share (Rust and Su 2012). This sales figure may change as some bait products have become available that require less labor for monitoring. The usage of the large quantity of liquid termiticide is the potential source of environmental concerns, and its inability to eliminate colony( ies) of subterranean termites may leave the colony to recover and cause reinfestation (Su 2011). Unlike liquid termiticides, some bait products aim at achieving colony elimination. The design of baiting systems takes advantage of termite social interaction. When termites forage and consume the bait, they do not die immediately. Through trophallaxis and grooming, the active ingredient is transferred from one individual to another. Eventually, most of the termites within the colony obtain lethal doses of the active ingredient. When a large proportion of termite workers die, the colony collapses due to labor shortages. Commercially, there are two types of chemical compounds used as bait system acti ve ingredients, metabolic inhibitors (sulfluramid) and insect growth regulators (diflubenzuron, hexaflumuron, and noviflumuron) (Su and Scheffrahn 2010). There are three vital factors that a successful bait system needs to fulfill; 1) lethal time is

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15 dosei ndependent, 2) slow acting, and 3) nonrepellent (Su 2012). Lethal time of metabolic inhibitors is dose dependent and individuals that consume higher doses die more quickly, making horizontal transfer of the toxicant more difficult. Some chitin synthesis inhibitors (CSIs), such as hexaflumuron and noviflumuron, are used in bait products. Chitin, an aminopolysaccharide composed of N acetyl D glucosamine units (Cohen 2001), is one of the constituents of insects procuticle, which develops into the exocuticle, mesocuticle and endocuticle, and serves as an important structural component for the integrity of insect cuticle (Andersen 1979). During the development of insects, growth requires additional volume, so the cuticle is periodically shed through apolysis, w hich is the separation of the cuticle from the underlying epidermis, and ecdysis, which is also referred to the shedding of the old exuviae. A new cuticle is formed in the intermolt period and expanded by modulation of chemical compounds to replace the old one (Chapman 1998). CSIs are known to disrupt the molting process of insects and kill the individual when it tried to finish molting. Nishioka et al. (1979) who studied the effect of CSIs on the formation of cuticle in vitro of Chilo suppressalis Walker ( Lepidoptera) showed that the thickness of cuticle decreased as the concentration of CSIs increased. Mommaerts et al. (2006) showed that bumblebee Bombus terrestris L. (Hymenoptera) larvae treated with CSIs exhibited high mortality, and microscopic observat ions showed abnormally formed cuticle and mechanical weaknesses among affected bees. Diflubenzuron can affect the normal lamellate appearance in procuticle and the deposition of epicuticle in the blowfly, Lucilia cuprina Wiedemann (Diptera) (Binnington 1985). Diflubenzuron was also tested on Tenebrio molitor L. (Coleoptera) pupae during cuticle formation and it was shown that the newly -

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16 formed cuticles had structural alterations and a thickness reduction (Soltani et al. 2005). Dopperlreiter and Korioth (1981) demonstrated that diflubenzuron caused ecdysis inhibition on Heterotermes indicola Wasmann and Reticulitermes flavipes ( Kollar ) Subsequent tests proved that diflubenzuron efficiently disrupted the ecdysis of R. flavipes but had no significant effect on C. formosanus (Su and Scheffrahn 1993). Hexaflumuron was the first CSI that was shown to induce molting inhibition against a wide range of subterranean termite species, including Heterotermes, Reticulitermes and Coptotermes species (Su and Scheffrahn 1993, 1996; Su 1994; DeMark et al. 1995, Su et al. 1995, 1997; Forschler and Ryder 1996; Grace et al. 1996). Hexaflumuron was the active ingredient of the first termite bait product, Sentricon (Dow Agroscience, Indianapolis, IN) commercialized in 1995, but in the United States a new CSI, noviflumuron, was recently introduced. Noviflumuron (N (((3,5 Dichloro 2 fluoro4 (1,1,2,3,3,3 hexafluoro propoxy)phenyl)amino)carbonyl) 2,6difluorobenzamide) has faster action, greater potency, and longer toxicity clearance t ime than hexaflumuron ( Sheets et al. 2000, Karr et al. 2004). Although several CSIs are used in termite bait products t here is little documentation about the histological effects of CSIs on the termite molting process. Su and Scheffrahn ( 1993) reported th at termites were killed by the hexaflumuron when their ecdysis process was disrupted, but did not describe how the process was inhibited. To understand the CSI effects on termite molting, we first need to understand the normal molting process. Despite its importance in termite development, the molting process is not well understood due to the termites cryptic behaviors and social organization. So in Chapter 2, our first objective was to describe the ecdysis process of

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17 C. formosanus Moreover, Raina et al. (2008) has offered the only report on the termite molting process and they speculated that termites needed nestmates to help complete the molting process. To have a better understanding about molting behavior of social insects and to examine Rainas speculation, our second objective in Chapter 2 was to study the effect of termite nestmates presence on the completion of ecdysis in C. formosanus After we understand the ecdysis behavior of C.formosanus we can make further efforts to understand the entire molting process by histological study Kunkel (1975) used the histology method to describe the molting in Blattellia germanica L. molting and divided it into several events marked with hours after feeding. In addition, Caveney (1969) studied the muscle att achment to the cuticle structure in some species of Apterygota during molting. The only histological study on molting termites was done on a drywood termite species, Kalotermes flavicollis Fabricius (Soltani Mazouni and Bordereau 1987). They described the changes in cuticles, ovaries and colleterial glands during neotenic and pseudergate molting, but information regarding histological and other internal changes in cuticle and other structures for termites during the molting process is lacking. The objective of Chapter 3 was to describe the changes of protozoa, cuticle, muscle and tracheal systems during the molting process of C. formosanus using histological methods. Understanding about the normal molting process leads us to be able to examine the behavioral and histological effects of noviflumuron on the molting process of Formosan subterranean termites. Although it has been proved effectively against subterranean termite species, its mechanism of causing ecdysis inhibition is poorly

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18 documented. So the Chapter 4 aimed to describe the histological changes of the molting process affected by noviflumuron.

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19 CHAPTER 2 DESCRIPTION OF THE MOLTING PROCESS OF THE FORMOSAN SUBTERRANEAN TERMITE (ISOPTERA: RHINOTERMITIDAE) Material s and Methods Termite collection Termites were collected from three colonies in Broward County, FL, using bucket traps (Su and Scheffrahn, 1986). After termites were collected, they were placed in a plastic jar ( diameter: 11.4 cm, height: 9.7 cm ) containin g ten pieces of moist spruce wood slabs ( Picea sp ., each piece was 7.8 cm by 6.6 cm by 0.6 cm ) and were kept in an incubator at 28.8 0.5 C. All termites were kept in the incubator for two weeks before testing. Identification of preecdysis individuals and description of molting process Raina et al (2007) reported that none of the fieldcollected termites, presumably all being foragers, molted for two weeks. Thus, termites were kept in the laboratory for two weeks before they were fed on Nile Blue A filter paper (diameter: 8.8 cm, Nile Blue concentration: 0.5% w/w). According to Raina et al. (2007), termites stopped feeding approximately six days before ecdysis, and individuals that were about to molt could be identified by the lack of Nile Blue A. In addition, individuals within the 12 h of ecdysis (pre ecdysis) may be identified by the mottled feature of their head capsule (Fig. 25). After being exposed to a Nile Blue A stained filter paper for 48 h, workers (undifferentiated larvae of at least the 3rd instar) without blue color were collected and some were selected for observation under a dissecting scope to identify individuals with the characteristic preecdysis feature (Fig. 2 6). Once the individual with preecdysis features was found, it was transf erred to a plastic tube (interior diameter: 2.54 cm, exterior diameter: 2.94 cm, height: 0.9 cm, interior wall is smooth to prevent termites

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20 from crawling out) (Fig. 27). With the confined tube, the movement range of molting termites could be restricted w ithin the viewing field of a dissecting scope (Model SZX12, Olympus Optical Co., Ltd., Tokyo, Japan), allowing the molting process could be recorded by the mounted camera (Model DP70, Olympus Optical Co., Ltd., Tokyo, Japan). The filter paper placed in the tube was painted black with a black marker pen (Sharpie, Sanford ink industry) to offer a color contrast with termites. The dorsal cuticle of the molting termite was painted with a blue marker pen (Sharpie, Sanford ink industry) to differentiate the new f rom old cuticle during the ecdysis. Four workers that consumed Nile Blue A, and thus nonmolting individuals, were transferred to the confined tube to stay with a single preecdysis individual. Ecdysis process was recorded using a timelapse compound microscope camera (Model SZX12 coupled to a DP70 Camera, Olympus Optical Co., Ltd., Tokyo, Japan). The experiment was replicated five times. Effects of nestmates on molting individual in ecdysis In one group, one preecdysis worker was transferred to the confi ned tube without the presence of nestmates. For another group, four workers that ingested Nile Blue A were transferred to the tube to stay with a preecdysis individual. The ecdysis process was recorded using a timelapse compound microscope camera (Model SZX12 coupled to a DP70, Olympus Optical Co., Ltd., Tokyo, Japan). Both experiments were replicated 15 times. The rate of successful completion of ecdysis was computed for both groups with and without nestmates. Time required to complete the ecdysi s process for individuals with (n=11) and without nestmates (n=5) was compared with Student t test at =0.05. Because it was difficult to determine the exact starting point of pre-

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21 ecdysis activity in some replicates, only those with clear evidence of the beginn ing of preecdysis were used for the analysis. Results Description of molting process The molting process was divided into four intervals. Each interval was delimited by two characteristic molting phases. Phase 1: From peristalsis to appearance of abdominal breach (Fig. 21) The first sign of the ecdysis was the abdominal peristaltic contraction. Contraction pulses started from the tip of the abdomen and moved forward to the mesothorax. Molting termites tended to stay in one place during the peristaltic co ntraction with all legs firmly grasping the floor. The contraction occurred in a relatively low frequency at the beginning and its intensity and frequency increased when the termite approached the abdominal breach. The entire contraction process lasted approximately 15 minutes. When nestmates were present, they occasionally groomed the peristaltic individuals. After successive peristalsis, a breach gradually appeared near the first abdominal segment and metathorax. Figure 21. P hase 1: a--> b -> c ( by Lin Xing ) peristaltic contraction abdominal breach a b c

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22 Phase 2: From breach forming to leg pulling (Fig. 2 2) The abdominal breach continued to expand and at the same time the exuvia at the thoracic segments split laterally. Part of new abdomen emerged with the exuvia forming a V as the breach expanded. With sufficient expansion on the dorsal abdomen, the termite lay on its side and began to pull its legs out of the exuvia. In interval 2, nestmates occasionally came and groomed the molting individual. An exuviae sac was gradually formed during interval 1 and 2. Figure 22. Phase 2: a--> b -> c ( by Lin Xing ) Phase 3: From the initiation of leg pulling to the separation of appendage cuticles (Fig. 2 3) Coincident with a termite starting to pull its legs out of the exuviae, the old cuticle of the head capsule stretched to the antennal sulcus. During the leg pulling phase, two long white strings of old trachea were pulled out of the spiracular openings at the mesothorax. When the legpulling phase was almost complete, the termite began to extricate its antennae from the old cutlicle. By the time that antennae were completely separated from the exuvia, the mandibles and maxillary palps were also separated from the old cuticle. Each molting termite remained in the jack knife posture until the end of interval 3 (Fig. 23).

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23 Figure 23. Phase 3: a--> b ( by Lin Xing) Phase 4: From the separation of appendage cuticles to exuvia separation (Fig. 2 4) After the antennae were pulled out of the exuvia, the termit e stretched the body straight from the jack knife posture but remained mostly motionless sideways. Nestmates helped the molting individual to finish the ecdysis through grooming and pulling of exuviae. Additional old tracheas were extricated from the molti ng individual during interval 4. Figure 24. Phase 4: a--> b ( by Lin Xing) a b old tra cheal system antennae separation a b

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24 Effects of nestmates to molting individual in ecdysis For both groups with or without nestmates, 14 out of 15 individuals successfully completed ecdysi s. Termites with nestmates completed period 1 and period 4 faster than those left alone ( P < 0.05), while time taken to finish period 2 and period 3 were similar for both groups ( P >0.05) (Fig. 28). Discussion Our definition of preecdysis is based on the m ottled headcapsule, which is different from the definition of Nation (2008) that the beginning of preecdysis is characterized by dorsoventral contractions. We adopted the preecdysis definition from Raina et al. (2007) is because precise beginning of dors oventral contraction is difficult to measure in Formosan subterranean termites. In insects such as Manduca sexta L. (Lepidoptera: Sphingidae), the molting fluid temporarily exists in the apolysial space after apolysis and disappears shortly before ecdys is (Malpighi 1669, Zacharuk 1976, Jungreis 1979). During our observations of termite ecdysis, molting fluid was visible for the entire process. For insects such as Rhodnius prolixus Stal (Hemiptera: Reduviidae) and Hyalophora cecropia L. (Lepidoptera: Saturniidae), the molting fluid was resorbed through the integument. But for others such as M. sexta, it may be absorbed by oral and anal consumption (Wigglesworth 1933, Passonneau and Williams 1953, Jungreis 1979, Cornell and Pan 1983). Although termites are supposed to recycle the molting fluid that contains proteinase and chitinase (Nation 2008), its mechanism remains unknown. Termites with nestmates took significantly less time to finish interval 1 and interval 4 than termites alone (Fig. 28) (P< 0.05). W hereas termites with nestmates took approximately the amount of time to complete interval 2 and interval 3 as those

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25 without nestmates (P>0.05). For interval 1, we suggest that mechanical stimuli such as grooming may help stimulate ecdysis. Molting individuals tend to start the ecdysis earlier when nestmates are present. At interval 4, the long tracheas need to be extricated from the mesothorax (Fig. 24) and the presence of nestmates could facilitate this process. Although in most cases, molting termites could extricate the old tracheas alone, it took longer to finish the task. In all observed cases, two long old tracheas were extricated which may be the longitudinal trunks. From the shed exuvia, some white strand structures were also observed, indicating part of the tracheal system may have broken from longitudinal trunks at nodes and were shed with the exuvia. Besides the tracheal shedding, different insects also have different mechanisms for tracheole shedding. In insects such as Sciara sp. (Diptera: Sciar idae), the tracheoles are shed at each larvalarva molt. However, in other insects such as Rhodnius sp., the tracheoles are never shed. The new trachea can link with the previous existed tracholes (Wigglesworth 1954, Nation 2008). In termites, there is lit tle available information about the shedding or resorption of tracholes during molting. Contraction during abdominal peristalsis in the first time interval helped to push the termite out of the old cuticle, indicating that before the initiation of the ecdysis process, most of the muscles of the abdomen may have completed the deand reattachment processes. Our results showed that, contrary to the speculation of Raina et al. (2007), termites were able to complete the molting process without assistance f rom nestmates. However, nestmates presence made the ecdysis process significantly faster than when termites were isolated.

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26 Figure 25. Mottled headcapsule during preecdysis indicated that termites were to start the ecdysis within 12 h. (by Lin Xing) Figure 26. Termites wi thout blue color were collected. ( By Lin Xing )

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27 Figure 27. The confined tube was designed to restrict the termite activity range ( By Lin Xing ) Figure 28. The time required for termites to complete an interval in ecdysis. Significant difference was marked by asterisk (Student t test, =0.05).

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28 CHAPTER 3 CHARACTERIZING THE MOLTING PROCESS IN COPTOTERMES FORMOSANUS SHIRAKI (ISOPTERA: RHINOTERMITIDAE) WITH HISTOLOGICAL STUDY Materials and Methods Sample i ndividuals at different molting phases Collection of termites and selection of molting individuals followed the same protocols as C hapter 2. After termites were exposed to Nile Blue A filter paper, individuals without blue color were placed in a petridish (radius: 4.6 cm, height: 2.1 cm) provisioned with moist filter paper for daily observation. The asynchronous molting time of termites make it difficult to time the exact molting events. According to the distinguishable features available, samples were coll ected at different phases to help identify the timed sequence of molting events. Samples from different phases were collected, 1) termites in the intermolt period, 2) termites in premolting fasting period (10 individuals per day for 5 days), 3) termites i n preecdysis period, 4) individuals undergoing ecdysis, and 5) newly molted termites with white mandibles and head capsule (Fig. 31). Samples from different periods were used to describe the molting process. Figure 31. Termites were sampled at five phases before and after the molting period.

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29 The ecdysis of a termite was defined as 0 day and from 12 h before ecdysis to ecdysis was defined as preecdysis. The period that termites stopped feeding but did not reach preecdysis was defined as premolting fast period. One hour after ecdysis was defined as newly molted period. Head capsules and legs of all the samples were removed before the samples were immersed into Bouins aqueous fixing solution (75% aqueous picric acid, 20% formaldehyde, 5% acetic acid) (Martoja and Martoja Pierson, 1967). Histological preparation Specimens were dehydrated by successively passing them through 75%, 95% and then put into pure nbutanol. After dehydration, they were first placed into a container with 50% paraffin and 50% nbutonal for six hours. Samples were then successively passed through three containers filled with paraffin and were kept in each container for 24 h until the nbutonal was replaced by paraffin. All the specimens were embedded into paraffin blocks. Embedde with the Azan Heidenhaim protocol (Mayer et al. 1979, Chouvenc et al. 2009). Stained slides were observed with a compound microscope (Model BX51 coupled to a DP70 Camera, Olympus Optical Co., Ltd., Tokyo, Japan). Results Termites in intermolt period (1) Cuticles of termites in the inermolt period were relatively smooth (Fig. 32) and the flagellates and spirochetes were present in the termite hindgut (Fig. 33). There was no spiracle cuticle separation during this period (Fig. 34). Muscles were tightly attached to the cuticle (Fig. 35, 6), and epidermal cells underneath cuticles were not easily observed (Fig. 37, 8).

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30 Termites in premolting fast period (2) Termites voided their guts at the beginning of the prem olting fast period, thus no gut fauna was observed from the hindgut of the termites sampled from the late fast period (Fig. 39). Cuticle texture at this period was still smooth and not visibly different from termites in the intermolt period. The lining of the trachea was still properly attached to the new cuticles of the tracheal system (Fig. 310). Before the preecdysis, some of the muscles began to detach from the old cuticles (Fig. 311, 12, 13, 14). Termites in preecdysis and ecdysis (3,4) During the preecdysial phase, cuticular structure appeared heavily wrinkled (Fig. 3 15) and two layers of cuticle were observed (Fig. 316). Similar to pre molting fast period, the protozoa were not observed in the hindgut. Before ecdysis, old tracheae were totall y detached from new tracheae (Fig. 317, 18) and were voided during the ecdysis. Termites in newly molted period (5) New cuticles appeared heavily wrinkled, which were similar to the cuticles of termites in pre ecdysis (Fig. 319). Protozoa were not obser ved in newly molted individuals. After ecdysis, no old tracheae were observed in tracheal system. Discussion Our results showed that at the early stages of molting gut protozoa were voided, which confirmed the finding of Raina et al. (2007). During the pr e ecdysis and ecdysis period, the wrinkled cuticle feature occurred. According to the study of Jenkin and Hinton (1966), a cuticulin layer is formed at the beginning of apolysis before the procuticle is formed underneath it. Therefore, we suggest the wrink led feature of cuticle may have resulted from the deposit of newly

PAGE 31

31 formed procuticle. The fixation and sectioning techniques may have stretched the distance between new cuticle and old cuticle, but it was clearly shown that the new cuticle existed underneath the old cuticle during the preecdysis and ecdysis periods (Fig. 316). Bourguignon et al. (2012) found that when pseudergates of Psammotermes hybostoma Desn. approached the nymphal molt, their new cuticles were completely formed. Old tracheal system c uticles were totally detached from newly formed cuticle during the preecdysis, which allowed the old tracheal lining cuticle to be pulled out during ecdysis. Wigglesworth (1954) who studied molting process of Rhodnius prolixus Stal reported that the old c uticles of tracheae existed inside the newly formed tracheal system, and before the ecdysis, the two layers of cuticle were completely separated. As described in the behavioral observations of Chapter 2, molting termites were mostly motionless, which may be explained by the detachment and reattachment of muscle. Our results showed that not only did termites finish their muscle detachment and reattachment during the ecdysis process, but parts of their body muscle were also detached during preecdysis and even before preecdysis. Our observation on the separation of muscle and cuticle is different from the generally accepted description that muscles detach during the ecdysis and quickly reattach to new cuticle (Nation 2008). We suggest the muscles detachment sequences may occur in two steps, 1) muscles that connect to the abdomen first finish the deand reattachment before ecdysis while part of the leg muscles remain attached to the old cuticle, and 2) in period 2 of ecdysis, the leg muscles begin to deand reattach to the new cuticles.

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32 Behavioral observations of Chapter 2 further confirm our muscle detachment and re attachment hypothesis. The existence of peristaltic abdomen movement may indicate that the muscles may have reattached to new cuticles of the abdomen before ecdysis, because without muscles attachment to the new cuticles, the peristaltic movement could not have happened. We believe that leg muscle deattachment and reattachment may occur in ecdysis because at the beginning of ecdysis, the termite could maintain its standing posture before it lay on the floor sideway to extricate its legs. Newly molted termites maintained the wrinkled feature, which confirms the size enlargement of the new cuticles as compared to old cuticles. After complet ing air intake, the new cuticles eventually expanded. Figure 32. Cuticles of termites during intermolt period are relatively smooth. hg=hindgut, cu=cuticle. ( By Lin Xing )

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33 Figure 33. Gut fauna were inside hindgut during intermolt period. hg=hindgut, pro= protozoa. ( By Lin Xing ) Figure 34. No trachea shedding from the tracheal system during intermolt period. tra=trachea. ( by Lin Xing) hg pro

PAGE 34

34 Figure 35. Muscles were tightly connected to the cuticle during the intermolt period. cu= cuticle, mu= muscle. ( by Lin Xing ) Figure 36. Muscles were tightly connected to the cuticle in the intermolt period. cu= cuticle, mu= muscle. ( by Lin Xing )

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35 Figure 37. Epidermal cells were in inactive status and they were not easily observed underneath the cuticle during the intermolt period. ep= epidermal cells. ( by Lin Xing ) Figure 38. Epidermal cells were not easily observed underneath the cuticle during intermolt period. cu= cuticle ( by Lin Xing )

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36 Figure 39. Gut fauna of hindgut were voided during the premolting fast period. hg= hindgut, mal= malpighian tubule. ( by Lin Xing) Figure 310. Cuticles inside tracheal system were about to separate. mu= muscle, tra=trachea. ( by Lin Xing)

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37 Figure 311. Abdominal muscles began to reattach to new cuticle during premolting fasting phase. nc= new cuticle, oc= old cuticle, mu= muscle. ( by Lin Xing) Figure 312. Abdominal muscles reattachment progress during premolting fasting phase. nc= new cuticle, oc= old cuticle, mu= muscle, ep=epiderm al cells. ( by Lin Xing )

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38 Figure 313. Abdominal muscles reattachment was in progress during premolting fasting phase. mu=muscle, nc= new cuticle, oc= old cuticle. ( by Lin Xing) Figure 314. Abdominal muscles almost finished reattachment during prem olting fasting phase. ep= epidermal cells, mu= muscle, nc= new cuticle, oc= old cuticle. ( by Lin Xing )

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39 Figure 315. Wrinkled cuticle structure appeared during preecdysis phase. cut= cuticle, ma= malpighian tubule. ( by Lin Xing) Figure 316. New cuti cles were already completely formed underneath the old cuticle during preecdysis phase. nc= new cuticle, oc= old cuticle. (by Lin Xing)

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40 Figure 317. Cuticles of old trachea were completely separated from the tracheal system during preecdysis. nc= new cuticle, oc= old cuticle. ( by Lin Xing) Figure 318. Cuticles of old trachea were completely separated from tracheal system during preecdysis. nc= new cuticle, oc= old cuticle. ( by Lin Xing )

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41 Figure 319. Cuticles of newly molted termites retained the wrinkled feature. cut= cuticle. ( by Lin Xing )

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42 CHAPTER 4 HISTOLOGICAL OBSERVATION OF MOLTING ALTERATION OF FORMOSAN SUBTERRANEAN TERMITES (ISOPTERA: RHINOTERMITIDAE) CAUSED BY NOVIFLUMURON Materials and Methods Six hundred termites w ere collected from a field colony using bucket traps (Su and Scheffrahn, 1986) in Broward County, Florida. After termites were collected, they were placed into a plastic box (27.5cm by 17cm by 8cm) with a layer of moist sand and soaked Recruit HD pieces (0.5% w/w noviflumuron (Fig. 41) (Karr et al. 2004) Dow Agrosciences, Indianapolis, IN) (Fig. 41). Termites were fed with baits for 3 days immediately after collection. After being forced fed on baits, termites were kept in a plastic jar ( diameter: 11.4 c m, height: 9.7 cm ) containing ten pieces of moist spruce wood slabs ( Picea sp ., 7.8 cm by 6.6 cm by 0.6 cm ) at 28.8 0.5 C. According to Raina et al. (2007), fieldcollected foraging termites did not molt within the first two weeks. Thus, noviflumuronfed termites were kept in the same jar for another 11 days before being placed in a petridish ( diameter: 9.2 cm, height: 2.1 cm ) containing moist Nile Blue A filter papers (0.5% w/w) for 48 h. Based on the study of Raina et al. (2007) termites stopped feeding approximately six days before ecdysis, and individuals that started molting process could be identified by the lack of Nile Blue A. Termites without blue color were placed in a petridish (diameter: 9.2 cm, height: 2.1 cm) provisioned wi th moist filter pa per for daily observation. Freshly dead individuals were collected and fixed in Bouins aqueous fixing solution (75% aqueous picric acid, 20% formaldehyde, 5% acetic acid) (Martoja and Martoja Pierson, 1967). Processing and staining protocols were the same as outlined in C hapter 3. Samples used in Chapter 3 were taken as the untreated control.

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43 Results After 3 days of nochoice feeding, consumption on baits was visible (Fig. 43). Termites fed with noviflumuron were devoid of protozoa in the hindgut. (Fig. 4 4) None of the noviflumuronaffected individuals manifested the wrinkled cuticle feature or mottled headcapsule as described by Raina et al. (2007). Compared with the control group as in Chapter 3, there was no properly formed new cuticle structure under neath the old cuticle. In some cases, no cuticle was formed at all underneath the cuticle (Fig. 45). In other cases, a thin layer of membrane appeared on the surface of epidermal cells (Fig. 46) or an incompletely formed cuticle were observed (Fig. 47). Old cuticles in the tracheal systems for termites treated with noviflumuron appeared to partially separate. However, the separation was not as complete as in the control group (Fig. 48). Muscles were observed to deand reattach to incompletely formed new cuticle (Fig. 49). During our observation, some dying individuals had body fluids leaking out from their bodies (Fig. 410). Discussion Protozoa were not observed in hindgut of termites treated with noviflumuron, (Fig. 4 4) indicating that noviflumuron had no significant effect on gut voiding process. In the normal molting process of termites, the mottled headcapsule feature was observed during the preecdysis. However, we did not observe any wrinkled headcapsule feature from termites treated with nov iflumuron, indicating the lack of the proper foundation of procuticle. The histological observation showed an incompletely formed cuticle underneath old cuticle in noviflumurontreated termites. We suggest that the procuticle formation was interrupted with noviflumuron, resulting in the failure of

PAGE 44

44 ecdysis. Some of the muscles were able to deattach from old cuticles and reattach to new cuticles. The absence of properly formed new cuticle and muscle reattachment may explain the behavioral observations t hat noviflumurontreated termites displayed peristaltic movement without completing the ecdysis. Because some of the muscles were reattached to the new cuticles, the peristaltic contraction movement occurred. However, due to the lack of properly formed new cuticle, the noviflumurontreated termites could not complete the ecdysis. Incomplete separation of the old cuticle in the tracheal systems of termites treated with noviflumuron suggested that even those progressed to period 4 of ecdysis, they were unli kely to finish the process (Fig. 48). In this study, most of the termites that picked up Nile Blue A (and thus were not ready to molt) were alive after 12 weeks, suggesting the clearance of noviflumuron as reported by Sheets et al. (2000) and Karr et al. (2004). After feeding with noviflumuron, we observed leaking of body fluid from some individuals (Fig. 49). These fluids and injuries can induce cannibalism (Castle 1934, Williams 1959), and may further promote the spread of noviflumuron through termite colony. Termites that were sampled for histological study all showed white spots on their abdomen (Fig. 411). Our histological study indicates that those are accumulated crystals in the fat body of termites. Similar crystal structures were recorded in fat bodies of cockroach Periplaneta Americana L. and mosquito larvae during starvation treatment (Wigglesworth 1941, Srivastava and Gupta 1960). We suggest that the crystals

PAGE 45

45 accumulated in fat bodies may be the uric acid. Although it appears in the termites treated with noviflumuron, its accumulation may be not the direct effects of CSI because similar crystal structures were also observed from individuals under stress without CSI treatment. In conclusion, our results confirm statement of Su and Scheffrahn (1993) that the interruption of molting process by CSIs resulted in termite mortality. Figure 41. Chemical structure of noviflumuron Figure 42. Ziploc plastic box with moist thin sand layer and soaked bait pieces ( by Lin Xing )

PAGE 46

46 Figure 43. Bait pieces that were consumed by termites. ( by Lin Xing) Figure 44. Protozoa were still voided with noviflumuron treatment. cut= cuticle, hg= hindgut. ( by Lin Xing )

PAGE 47

47 Figure 45. No new cuticle was formed underneath the old cuticle. ep= epidermal cells, mu= muscle, oc= old cuticle, nc= new cuticle. ( by Lin Xing) Figure 46. Thin new cuticle layer showed up underneath the old cuticle. nc= new cuticle, oc= old cuticle. ( by Lin Xing) oc nc nc o c nc nc oc ep mu

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48 Figure 47. Incomplete new cuticle was formed underneath old cuticle. nc= new cuticle, oc= old cuticle. ( by Lin Xing) Figure 48. Old cuticles inside tracheal system were partially detached. oc= old cuticle, tr= trachea. ( by Lin Xing) tr oc tr oc

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49 Figure 49. M uscles were reattached to the incompletely formed new cuticle. mu= muscle, nc= new cuticle, oc= old cuticle. ( by Lin Xing ) Figure 410 Termite that has body fluid leaking out ( by Lin Xing )

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50 Figure 411. White spots were jammed on the abdomen of termite. ( by Lin Xing )

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51 CHAPTER 5 CONCLUSIONS Formosan subterranean termite ecdysis process can be divided into four periods, 1) from peristalsis to appearance of abdominal breach, 2) from breach forming to the initiation of legpullin g, 3) from the leg pulling to the separation of antennae cuticle and 4) from antennae separation to exuviae separation. During the ecdysis process, the nestmate presence was not required, which leaded us to reject the speculation of Raina et al. (2008) that termites needed nestmates aid to finish molting. However, with the presence of nestmates, molting individuals took less time to finish the ecdysis than those without nestmate. Histological study of termite undergoing normal molting showed that protozoa were voided at the beginning of premolting fast period as reported by Raina et al. (2007). The deattachment and reattachment of muscles at abdominal segments began to appear in preecdysis, which was earlier than that of leg muscles that occurred in ecdysis. Our results contradicted to the general understanding that muscles quickly deattach from old cuticle and reattach to the new cuticle during ecdysis. During the preecdysis, abdominal cuticles appeared wrinkled and two layers of cuticles were observ ed, indicating the successful formation of new cuticle. In addition, the old treacheas were shed from the tracheal system and were pulled out by nestmates during the ecdysis. Histological study of termites treated with noviflumuron showed that the protozoa were voided as in untreated termites. We observed new cuticles were wrinkled in appearance. Because the surface areas of new cuticles are larger than the old body surface, it is understandable that they have to fold in the old body cavity, thus creating

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52 t he wrinkled feature. Termites treated with noviflumuron initiated the ecdysis as schedul ed and their abdominal peristaltic contractions were observed for a long time until they die. From the histological study, some of the muscles deand reattach to the incompletely formed the new cuticles and it explained why the termites were able to begin the peristaltic contraction. However, because of the lack of new cuticle formation and in some cases the formation of thin layer and incompletely formed cuticle, term ites could not finish the ecdysis. Old tracheas were partially separated from tracheal system, which may not be able to perform a complete deattachment function in ecdysis. In conclusion, our results confirm statement of Su and Scheffrahn (1993) that the interruption of molting process by CSIs resulted in termite mortality.

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53 LIST OF REFERENCES Andersen, S. O. 1979. Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria. J. Insect Physio. 20: 15371552. Bignell, D.E. 2006. Termites as soil engineers and as s oil processors. in: intestinal microorganisms of soil invertebrates. Konig, H. and Varma, A. (Eds). Springer Verlag, Berlin Heidelberg. Binnington, K. C. 1985. Ultrastructural changes in the cuticle of the sheep blowfly Lucilia induced by certain insecticides and bi ological inhibitors. J. Tissue cell. 17: 131140. Castle, G. B. 1934. The dampwood termites of western United States, genus Zootermopsis(for merly Termopsis), pp. 273310. In C. A. Kofoid [ed.], Termites and termite control. University of Califronia Press, Berkeley. Caveney, S. 1969. Muscle attachment related to cuticle architecture in Apterygota. J. Cell Sci. 4: 541 559. Chapman, R. F. 1998. The insects: structure and function, 4th ed. Cambridge university p ress, New York. Chouvenc, T. N. Y. Su, and A. Robert. 2009. Cellula r encapsulation in the eastern subterranean termite, Reticulitermes flavipes (I soptera), against infection by the entomopathogenic fungus Metarhizium ani sopliae. J. Invertebr. Pathol. 101: 234 241. Cohen, E. 2001. Chitin synthesis and inhibition: a revisit. J. Pest Management Sci. 57: 946950 Cornell, J. C., and M. L. Pan. 1983. The disappearance of moulting fluid in the tobacco hornworm, Manduca sexta. J. exp. Biol. 107: 501504. Demark, J. J., E. P. Benson, P. A. Zungoli, and B. M. Kard. 1995. Evaluation of hexaflumuron for termite control in the southeast U.S. Down to Earth 50: 202 6. Dhadialla, T. S., G. R. Carlson, and D. P. Le. 1998. New insecticides with ecdysteroidal and juvenil e hormone activity. 43: 5455 69. Doppelreiter, V. H., and M. Korioth. 1981. Entwicklungshemmung durch diflubenzuron bei den bodentermiten Heterotermes indicola and Reticulitermes flavipes. Z. Angew. Entomol. 91: 131137. Edwards, R., and A. E. Mill. 1986. Termites in buildings: their biology and control. Rentokil Ltd, East Grinstead, England.

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58 B IOGRAPHICAL SKETCH Lin Xing was born in 1988 in Beijing, China. He moved with his family to a small city, Weihai, when he was 4year old. He loved catching bugs and hanging out with his friends when he was a child. Between the years of 2003 and 2 006, he attended a high school, where he studied hard and tried to get a high score from the college entrance examination. After he finished the exam, he began to collect information about all the majors based on his interest. Eventually he chose the China Agricultural University majoring in plant protection. Because he believed that the agricultural development of China had a long way to go and what he studied can benefit the development of Chinese agriculture. The four years in college are the most memorable time in his life. He harvested friendships, love and basic understanding about his major. In 2010, he entered the graduate program at the University of Florida in the Department of Entomology and Nematology. There, he began a journey of study and gradually understood the curiosity and a good attitude is the key for being a good scientist. He learned and changed a lot in the past two years and he believed that there is no best in his world but always to be better. Now he is planned to apply for a graduate school in United States to obtain a doctoral degree in future.