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Cellulolytic and Xylanolytic Gut Enzyme Activity Patterns in Major Subterranean Termite Pests

Permanent Link: http://ufdc.ufl.edu/UFE0021387/00001

Material Information

Title: Cellulolytic and Xylanolytic Gut Enzyme Activity Patterns in Major Subterranean Termite Pests
Physical Description: 1 online resource (126 p.)
Language: english
Creator: Smith, Joseph Anthony
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: cellulase, coptotermes, reticulitermes, subterranean, termite, xylanase
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cellulolytic and xylanolytic termite gut carbohydrolases were assayed for two major subterranean termite pest species; Coptotermes formosanus (Shiraki) and Reticulitermes flavipes (Kollar). Carbohydrolase assays were optimized for buffer and pH. This led to the selection of a 0.1 M pH 5.5 sodium acetate buffer for endoglucanase and xylanase assays and a 0.1M pH 6.5 sodium phosphate buffer for exoglucanase, beta-glucosidase, and beta-xylosidase assays. Endoglucanase activity was found to be mainly localized in the foregut and hindgut of both species, reflecting both endogenous and symbiont enzyme origins. Beta-glucosidase activity was found throughout the gut in both species, but was most prevalent in the midgut or hindgut. Beta-xylosidase activity was mostly restricted to the hindgut and was lowest in the foregut in both species. Exoglucanase and xylanase activities were almost entirely confined to the hindgut in both species. C. formosanus workers were found to have a more active array of gut carbohydrolases, particularly exoglucanase and beta-glucosidase, than R. flavipes. This was consistent with increased metabolic demands from more aggressive foraging and a larger soldier ratio within C. formosanus colonies. Soldier carbohydrolase activities in both of these species were generally lower than worker carbohydrolase activities, consistent with a caste incapable of feeding itself. C. formosanus soldier carbohydrolase activities were especially low, due to a reduced gut size to make room in the soldier abdomen for an enlarged frontal gland. Cellulolytic enzyme activity levels were found to significantly change on differing diets for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to change in a manner consistent with dietary xylan content in both species, being increased on diets containing more xylan. This indicates some adaptability to dietary xylan content in both species. The presence of significant xylanolytic enzyme activities in termites kept on diets without xylan indicates constitutive production of xylanases by symbionts capable of subsisting wholly on cellulose. Alternatively, it is possible that the xylanolytic enzymes seen on xylan-free diets are bifunctional enzymes that also have cellulolytic activities.
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 Joseph Anthony Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Koehler, Philip G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021387:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021387/00001

Material Information

Title: Cellulolytic and Xylanolytic Gut Enzyme Activity Patterns in Major Subterranean Termite Pests
Physical Description: 1 online resource (126 p.)
Language: english
Creator: Smith, Joseph Anthony
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: cellulase, coptotermes, reticulitermes, subterranean, termite, xylanase
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cellulolytic and xylanolytic termite gut carbohydrolases were assayed for two major subterranean termite pest species; Coptotermes formosanus (Shiraki) and Reticulitermes flavipes (Kollar). Carbohydrolase assays were optimized for buffer and pH. This led to the selection of a 0.1 M pH 5.5 sodium acetate buffer for endoglucanase and xylanase assays and a 0.1M pH 6.5 sodium phosphate buffer for exoglucanase, beta-glucosidase, and beta-xylosidase assays. Endoglucanase activity was found to be mainly localized in the foregut and hindgut of both species, reflecting both endogenous and symbiont enzyme origins. Beta-glucosidase activity was found throughout the gut in both species, but was most prevalent in the midgut or hindgut. Beta-xylosidase activity was mostly restricted to the hindgut and was lowest in the foregut in both species. Exoglucanase and xylanase activities were almost entirely confined to the hindgut in both species. C. formosanus workers were found to have a more active array of gut carbohydrolases, particularly exoglucanase and beta-glucosidase, than R. flavipes. This was consistent with increased metabolic demands from more aggressive foraging and a larger soldier ratio within C. formosanus colonies. Soldier carbohydrolase activities in both of these species were generally lower than worker carbohydrolase activities, consistent with a caste incapable of feeding itself. C. formosanus soldier carbohydrolase activities were especially low, due to a reduced gut size to make room in the soldier abdomen for an enlarged frontal gland. Cellulolytic enzyme activity levels were found to significantly change on differing diets for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to change in a manner consistent with dietary xylan content in both species, being increased on diets containing more xylan. This indicates some adaptability to dietary xylan content in both species. The presence of significant xylanolytic enzyme activities in termites kept on diets without xylan indicates constitutive production of xylanases by symbionts capable of subsisting wholly on cellulose. Alternatively, it is possible that the xylanolytic enzymes seen on xylan-free diets are bifunctional enzymes that also have cellulolytic activities.
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 Joseph Anthony Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Koehler, Philip G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021387:00001


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7774bec819e88c985fc0f06733395a87e01a0dff







CELLULOLYTIC AND XYLANOLYTIC GUT ENZYME ACTIVITY PATTERNS IN
MAJOR SUBTERRANEAN TERMITE PESTS




















By

JOSEPH ANTHONY SMITH


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

2007

































2007 Joseph Anthony Smith
































To my family, friends and teachers, without whom this degree would not have been possible









ACKNOWLEDGMENTS

I would like to thank the following people and institutions for their support. I thank

Procter and Gamble for the funding that allowed me to conduct this research. I thank my

committee, Dr. Phil Koehler, Dr. Mike Scharf, Dr. Lonnie Ingram, and Dr. Phil Brode, for their

input and feedback on my research. I thank Bruce Ryser for supplying Formosan subterranean

termites for my research. I thank my fellow students, and Cynthia Tucker in particular, for their

intelligent input and collaboration. I thank my family and friends for their moral support.

Finally, I would like to especially thank Dr. Phil Koehler for his efforts as my advisor and

committee chair.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF TABLES .............. ......... ...................................................8

LIST OF FIGURES .................................. .. .... ..... ................. 10

A B S T R A C T ................................ ............................................................ 1 1

INTRODUCTION ................................. .. ..... .... ................... 13

L ITER A TU R E R E V IE W ....................................................... ............................................. 15

W ood M molecular Structure ........................................................................... ....................15
Termite Taxonomy .............................. .................... ......... 16
Term ite D igestive System A natom y.......................................................................... ... ... 17
Symbiont Taxonomy and Anatomy......................................................... ............... 18
Symbiont Assemblages in Termite Hindguts ........................................ ...... ............... 19
Termite Carbohydrolase Types and Locations............. ... ............................ ............... 20
Termite Carbohydrolase Structural and Functional Characteristics.................. ............21

BUFFER AND PH OPTIMIZATION FOR TERMITE CARBOHYDROLASE ASSAYS.........28

In tro d u ctio n .................................................. ....................... ................ 2 8
M materials and M ethods .................. .... .... ........... ......... ............. ............ 29
Termite Collection, Dissection, and Enzyme Extraction .........................................29
B uffer O ptim ization A says .................................................. .............................. 29
G ut R region A says A cross PH ......... .................. .................................. ............... 31
D ata A n a ly sis ............................................................................................................. 3 2
R e su lts ........... .... .. .... .. ..................33..........
B uffer O ptim ization A says .................................................. .............................. 33
Gut Region pH Optim ization A says ........................................ ......................... 34
D iscu ssion ................ .............................................. ............................34
Activity Magnitudes .................... .................... ................34
A activity C changes w ith pH ....................................................................... ..................35
D differences in G ut R regions ..................................................................... ..................35
C o n c lu sio n s ..............................................................................3 6

CARBOHYDROLASE ACTIVITY PATTERNS IN RETICULITERMES FLA VIPES AND
COPTOTERMES FORMOSANUS WORKERS AND SOLDIERS ............. ...............43

Introduction .............. .. ..... ............ ............. .............. ................. 43
M materials and M ethods .................. .... .... ........... ......... ............. ............ 44
Termite Collection, Dissection, and Enzyme Extraction .........................................44
Term ite Photography ......... .. ......................... ...... ....... .. ........ .... 45









Soluble P protein A ssay s ..................................................................... ....... .. ......45
E ndoglu canase A ssay s............ ... ....................................................................... .. ...... .. 4 5
E x oglu canase A ssay s............ ... ......................................................................... .. ...... .. 46
B eta-G lu cosidase A ssay s......................................................................... .................. 46
X ylanase A says ................................................................................................... ....... 47
B eta-X ylosidase A says ........................................ ................. .... ....... 48
D ata A n a ly sis ............................................................................................................. 4 8
Results ............................. ........................... ................. ........ 49
Termite Gut and Frontal Gland Comparisons ...................................... ............... 49
W orker C ellulolytic A activities ............................................................. .....................49
W orker X ylanolytic A activities ............................................................. .....................50
Soldier C ellulolytic A activities .............................................................. .....................50
Soldier X ylanolytic A activities .............................................................. .....................50
G ut Soluble Protein L evels............................................ .................. ............... 51
D iscu ssio n ................... ...................5...................1..........

CHANGES IN RETICULITERMES FLAVIPES GUT CELLULOLYTIC ACTIVITIES IN
RESPONSE TO DIET ............... .............. ......... ..................... ....... 63

Introduction ................ ...... ........... .............. ...............63
M materials and M methods ...................................... .. .......... ....... ...... 64
T erm ite C collection .......... ......................................................................... ........ ....... 64
T erm ite D iets and Feeding ........................................... .................. ............... 65
Termite Dissection and Enzyme Extraction..... .................... ..............65
Endoglucanase A says ........................................................... ... ..... .... 66
Exoglucanase and Beta-glucosidase Assays ....................................... ............... 67
D ata A n a ly sis ............................................................................................................. 6 7
R e su lts ............... ..... ........... ....................................................................................... ............... 6 8
T erm ite G ut O b servations ....................................................................... ..................68
E ndoglucanase A activities ........................................................................ .................. 69
Exoglucanase A activities ........................................ .... ....... .... ....... 69
B eta-glucosidase A activities ..................................................................... ..................70
D iscu ssio n ................... ...................7...................0..........

CHANGES IN RETICULITERMES FLA VIPES GUT XYLANOLYTIC ACTIVITIES IN
R E SP O N SE T O D IE T .................................................................................... .................. 77

In tro du ctio n ................... ...................7...................7..........
M materials and M methods ...................................... .. .......... ....... ...... 78
T erm ite C o lle ctio n ..................................................................................................... 7 8
T erm ite D iets and Feeding ........................................... .................. ............... 79
Termite Dissection and Enzyme Extraction........................................................80
X y lan ase A ssay s ................................................................................ 8 1
B eta-X ylosidase A says ........................................ .... ....... .... ....... 82
D ata A n a ly sis ............................................................................................................. 8 2
R e su lts ............... ..... ........... ....................................................................................... ............... 8 3
T erm ite G ut O b servations ....................................................................... ..................83


6









X y lan ase A ctiv ities.......... .............. ...................................................... ......... ....... .. 83
B eta-X ylosidase A activities ........................................... ................................. 84
D iscu ssio n ................... ...................8...................5..........

CHANGES IN COPTOTERMESFORMOSANUS GUT CELLULOLYTIC ACTIVITIES IN
RESPONSE TO DIET ............... .............. ......... ..................... ....... 93

Introduction ................ ...... ........... .............. ...............93
M materials and M methods ...................................... .. ......... ......... .....94
T erm ite C collection .......... ....................................................................... ....... ....... 94
T erm ite D iets and Feeding ........................................... .................. ............... 95
Termite Dissection and Enzyme Extraction...... .................... ...............95
Endoglucanase A says .............................................................. .. .... 96
Exoglucanase and Beta-glucosidase Assays ....................................... ............... 97
D ata A n aly sis.........................................................................9 7
R e su lts............... ...... ...... .......................... ............................... ................9 8
T erm ite G ut O b servations ....................................................................... ..................98
E ndoglucanase A activities ........................................................................ .................. 99
Exoglucanase A activities ........................................ .... ....... .... ....... 99
B eta-glucosidase A activities ..................................................................... ..................99
D iscu ssio n ................... ...................9...................9..........

CHANGES IN COPTOTERMESFORMOSANUS GUT XYLANOLYTIC ACTIVITIES IN
R E SP O N SE T O D IE T .............................................................................. ......................105

Introduction ................... ......................................................................... 105
M materials an d M eth od s .............................................................................. ..................... 106
T erm ite C collection .......... ...................................................................... ........ .... 106
Term ite D iets and Feeding .................................................. .............................. 107
Termite Dissection and Enzyme Extraction.................. ........................................108
X y lan a se A ssay s ...................................................................................................... 10 9
B eta-X ylosidase A ssay s ................................................................... ......... ............ 110
D ata A n a ly sis ........................................................................................................... 1 1 0
R results .................... ......... ...................................................... ......... 111
Term ite G ut O observations .................................... .................................. ............... 111
X ylanase A activities .................. ............................. .. ......................... .. 111
B eta-X ylosidase A activities .................................... ................................................ 112
D isc u ssio n ................... ............................................................................1 12

C O N C L U SIO N ................... ............................................................ ................119

L IST O F R E F E R E N C E S .................................................................................... ...................122

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 126









LIST OF TABLES


Table page

4-1 A comparison of endoglucanase activities in Reticulitermesflavipes and Coptotermes
formosanus w workers and soldiers......................................................... .............. 55

4-2 A comparison of exoglucanase activities in Reticulitermesflavipes and Coptotermes
formosanus workers and soldiers......................................................... .............. 56

4-3 A comparison of beta-glucosidase activities in Reticulitermesflavipes and
Coptotermesformosanus workers and soldiers ...................................... ............... 57

4-4 A comparison of xylanase activities in Reticulitermesflavipes and Coptotermes
formosanus workers and soldiers......................................................... .............. 58

4-5 A comparison of beta-xylosidase activities in Reticulitermesflavipes and
Coptotermesformosanus workers and soldiers ...................................... ............... 59

4-6 A comparison of soluble protein concentrations in Reticulitermesflavipes and
Coptotermesformosanus worker and soldier gut extracts...............................................60

5-1 Endoglucanase activities in the three gut regions of Reticulitermesflavipes workers
in response to different field diets ........................................................ ............. 74

5-2 Exoglucanase activities in the three gut regions of Reticulitermesflavipes workers in
response to different field diets............................................................................ ... ... 75

5-3 Beta-glucosidaase activities in the three gut regions of Reticulitermesflavipes
workers in response to different field diets ............................ .................................... 76

6-1 Xylanase activities in the three gut regions of Reticulitermesflavipes workers in
response to different field diets............................................................................ ...... 89

6-2 Beta-xylosidase activities in the three gut regions of Reticulitermesflavipes workers
in response to different field diets ........................................................ ............. 90

7-1 Endoglucanase activities in the three gut regions of Coptotermesformosanus
workers in response to different field diets ..................................................... 102

7-2 Exoglucanase activities in the three gut regions of Coptotermesformosanus workers
in response to different field diets ....................................................... ............... 103

7-3 Beta-glucosidaase activities in the three gut regions of Coptotermesformosanus
workers in response to different field diets ..................................................... 104

8-1 Xylanase activities in the three gut regions of Coptotermesformosanus workers in
response to different field diets.......................................................................... ...... 115









8-2 Beta-xylosidase activities in the three gut regions of Coptotermesformosanus
workers in response to different field diets ..................................................... 116









LIST OF FIGURES


Figure page

2-1 Inverting cellulase m mechanism ............................................................................ ..... ... 26

2-2 R etaining cellulase m echanism ............................................................................ ... .... 27

3-1 Beta-glucosidase activities in Reticulitermesflavipes workers across pH and buffer.......38

3-2 Endoglucanase activities in different gut regions of Reticulitermesflavipes workers
acro ss p H ............ ............................. ................................................ 3 9

3-3 Beta-Glucosidase activities in different gut regions of Reticulitermes flavipes
w workers across pH ................ .... .... .......... .. .. .......... .. ............40

3-4 Hindgut exoglucanase activities in Reticulitermesflavipes workers across pH ..............41

3-5 Hindgut xylanase activities in Reticulitermesflavipes workers across pH.....................42

4-1 Digestive systems of: A) R. flavipes worker, B) C. formosanus worker, C) R. flavipes
soldier, and D) C. formosanus soldier. ........................................ .......................... 61

4-2 Heads and frontal glands of a R. flavipes soldier, and a C. formosanus soldier ..............62

6-1 Xylanase activities in different gut regions of Reticulitermesflavipes in response to
dietary xylan content .................. .................. .................. ......... .. ............ 91

6-2 Beta-Xylosidase activities in different gut regions of Reticulitermesflavipes in
response to dietary xylan content.......................................................................... ... .... 92

8-1 Xylanase activities in different gut regions of Coptotermesformosanus in response to
dietary xylan content .................. ........................................ .. ........ .. .. 117

8-2 Beta-xylosidase activities in different gut regions of Coptotermesformosanus in
response to dietary xylan content........................................................................ ....... 118









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


CELLULOLYTIC AND XYLANOLYTIC GUT ENZYME ACTIVITY PATTERNS IN
MAJOR SUBTERRANEAN TERMITE PESTS

By

Joseph Anthony Smith

August 2007

Chair: Philip G. Koehler
Major: Entomology and Nematology

Cellulolytic and xylanolytic termite gut carbohydrolases were assayed for two major

subterranean termite pest species; Coptotermesformosanus (Shiraki) and Reticulitermesflavipes

(Kollar). Carbohydrolase assays were optimized for buffer and pH. This led to the selection of a

0.1 M pH 5.5 sodium acetate buffer for endoglucanase and xylanase assays and a 0.1M pH 6.5

sodium phosphate buffer for exoglucanase, beta-glucosidase, and beta-xylosidase assays.

Endoglucanase activity was found to be mainly localized in the foregut and hindgut of both

species, reflecting both endogenous and symbiont enzyme origins. Beta-glucosidase activity was

found throughout the gut in both species, but was most prevalent in the midgut or hindgut. Beta-

xylosidase activity was mostly restricted to the hindgut and was lowest in the foregut in both

species. Exoglucanase and xylanase activities were almost entirely confined to the hindgut in

both species.

C. formosanus workers were found to have a more active array of gut carbohydrolases,

particularly exoglucanase and beta-glucosidase, than R. flavipes. This was consistent with

increased metabolic demands from more aggressive foraging and a larger soldier ratio within C.

formosanus colonies. Soldier carbohydrolase activities in both of these species were generally









lower than worker carbohydrolase activities, consistent with a caste incapable of feeding itself.

C. formosanus soldier carbohydrolase activities were especially low, due to a reduced gut size to

make room in the soldier abdomen for an enlarged frontal gland.

Cellulolytic enzyme activity levels were found to significantly change on differing diets

for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to

change in a manner consistent with dietary xylan content in both species, being increased on

diets containing more xylan. This indicates some adaptability to dietary xylan content in both

species. The presence of significant xylanolytic enzyme activities in termites kept on diets

without xylan indicates constitutive production of xylanases by symbionts capable of subsisting

wholly on cellulose. Alternatively, it is possible that the xylanolytic enzymes seen on xylan-free

diets are bifunctional enzymes that also have cellulolytic activities.









CHAPTER 1
INTRODUCTION

Two of the most economically significant pest termites in North America are the eastern

subterranean termite, Reticulitermesflavipes (Kollar), and the Formosan subterranean termite,

Coptotermesformosanus (Shiraki). One thing that distinguishes termites from the majority of

other insect pests is their ability to digest wood, and their consequent ability to cause significant

structural damage to most types of buildings. This capacity for wood digestion is based upon a

complex array of enzymes, mainly carbohydrolases, which allow termites to digest cellulose and

hemicelluloses such as xylan. In all lower termites, including subterranean termites, some of

these enzymes are endogenously produced, while others are produced by symbiotic flagellates

found in the physiologically specialized, expanded hindgut.

Perhaps more important than their status as structural pests is the enormous impact that

termites have on terrestrial ecological processes. Termites are among the major terrestrial

recyclers of cellulose, one of the most abundant substances in nature. Finding a way to disrupt

or harness the enzymatic mechanisms that allow subterranean termites to digest cellulose and

other polysaccharides may be a key to discovering new approaches in termite control or

industrial methods to generate alternative fuels from cellulosic materials. As such, an

understanding of the termite digestive system has significant applications.

The first objective in this dissertation is the optimization of the chosen carbohydrolase

assays, which will be dealt with in Chapter 3. The next objectives concern the characterization

of the activity levels of the various cellulolytic and xylanolytic gut enzymes, which will be dealt

with in Chapter 4. The patterns of activity for each carbohydrolase along the gut must be

determined, as well as the differences seen between the worker and soldier castes for R. flavipes

and C. formosanus. This will provide insight into the mechanisms of cellulose and xylan









digestion for these two species of termites, allowing a better understanding of the interaction

between the termites and their symbionts, as well as the interaction between the termite workers

and soldiers. This understanding may facilitate the development of successful termite control

strategies, or the discovery of novel enzymes for industrial polysaccharide degradation.

Once these basic patterns of activity have been determined, the next main objective is the

exploration of how these patterns are affected by changes in termite diet, and whether the

termites can adapt to their diets. Changes in patterns of R. flavipes worker cellulolytic and

xylanolytic enzyme activities will be addressed in Chapter 5 and Chapter 6, respectively.

Changes in patterns of C. formosanus worker cellulolytic and xylanolytic enzyme activities will

be addressed in Chapter 7 and Chapter 8, respectively. In particular, the changes in xylanolytic

enzyme activities across diets of varying xylan content will provide insight into the adaptive

abilities of these termites with respect to diet. In turn, this will provide some idea of how

termites may adapt to attempts to inhibit their digestive processes. Furthermore, any

understanding of termite adaptation to diet would be useful if whole, living termites were to be

incorporated into industrial processes involving cellulose and xylan degradation.

The final aim of this dissertation is to facilitate an understanding of lower termite digestive

processes, both in terms of enzyme activity patterns and adaptation to different diets. This

understanding may be used for development of control strategies, or for the harnessing of these

termites and their enzymes for the removal of cellulosic waste and the generation of alternative

fuels.









CHAPTER 2
LITERATURE REVIEW

Wood Molecular Structure

The majority of wood consists of polysaccharides and lignin. The dominant

polysaccharide is cellulose, which exists within the plant cell walls as microfibers roughly 5 nm

in diameter (Astley et al. 1997). Each fiber consists of multiple chains of cellulose, which are

largely aligned in tight, crystalline configurations. These configurations make this molecule

particularly difficult to digest. There are amorphous regions as well, where the configuration is

disrupted. It is generally thought that these amorphous regions are caused by interactions with

other molecules in the cell walls. Each cell wall has several layers, called lamellae, roughly one

microfiber thick. The microfibers in each layer are roughly parallel, but their orientation changes

between layers.

The surrounding matrix of the cell wall contains other polysaccharides, predominantly

hemicelluloses, and lignin, a non-repeating aromatic polymer (Whistler and Chen 1991). Unlike

cellulose, which is made up of beta-glucose units, hemicelluloses contain a variety of sugar

subunits. In addition, hemicellulases are much shorter, only a few hundred units long, with short

side chains (Timell 1964). These traits mean that they do not exist as microfibers, but instead

form part of the matrix surrounding the cellulose microfibers. In some models, the ends of the

hemicellulose chains are aligned with the cellulose microfibers while the bulk of the chains form

cross-bridges between the microfibers (Whistler and Chen 1991). The majority of

hemicelluloses in hardwoods consist of modified polymers of xylose, commonly called xylans.

Softwoods contain other hemicelluloses in addition to xylans, which occur in roughly

comparable amounts. These are known as glucomannans and galactoglucomannans, and they are

mainly polymers of mannose and glucose.









Lignin is a highly complex, cross-linked polymer. It is fundamentally different from the

polysaccharides, as it is a polymer of various hydroxycinnamyl alcohols. In addition, the overall

structure is far more amorphous and less predictable than that of polysaccharides. A limited

degree of degradation of aromatic monomers has been demonstrated in the hindgut of lower

termites (Brune et al. 1995). However, it has been difficult to obtain evidence of degradation of

polymerized lignin in termites. Based on the high lignin content of fecal material and nest carton

in multiple termite genera (Mishra and Sen-Sarma 1979), it may be assumed that lignin

degradation is limited in most termites, and certainly in lower termites. As a result, the relevance

of lignin to termite digestion is fairly limited and this paper will focus on the degradation of

cellulose and hemicelluloses.

Termite Taxonomy

The order Isoptera is generally divided into six families. These are the Hodotermitidae,

Termopsidae, Mastotermitidae, Kalotermitidae, Rhinotermitidae and Termitidae. Termitidae

contains the so-called "higher" termites, and none of the termites in this family have symbiotic

flagellates in their hindguts. The remaining five families of termites are commonly called

"lower" termites, and they are all symbiotically associated with flagellates in their hindguts in

addition to the more ubiquitous bacteria and fungi found in termite guts.

The Hodotermitidae and Termopsidae are most often found in the tropics and are often

regarded as relatively primitive termites (Eggleton 2001). The Mastotermitidae, generally

considered to be the basal group, consists of the Australian species Mastotermes darwiniensis.

Kalotermitidae and Rhinotermitidae are the two termite families most commonly

encountered in the temperate zones. The Kalotermitidae include the dampwood termites and the

drywood termites. They typically live in close association with their food source, often nesting









within the wood they consume (Noirot and Darlington 2002). They are also considered to be

more primitive in their behavior and morphology than the Rhinotermitidae.

Rhinotermitidae are commonly called subterranean termites. They are aptly named, as

most species live within the soil and seek out dead wood and other similar food sources. These

termites are typically the worst structural pests in buildings (Potter 2004). A large number of

studies on termite digestion and termite symbiosis have been carried out on genera from this

family. The two genera of particular interest are Coptotermes and Reticulitermes.

Termite Digestive System Anatomy

As with other insects, the termite gut may be divided into three major regions: the

foregut, the midgut and the hindgut. The foregut is composed of the salivary glands, the crop

and the proventriculus. The crop and proventriculus are lined with cuticle, which is shed at each

molt along with the exoskeleton. The crop in termites is relatively small, but the proventriculus

is fairly well-muscled and armed with scraping teeth and ridges on its inner surface (Noirot and

Noirot-Timothee 1969).

The second region is the midgut. This structure is not lined with cuticle, but instead has

epithelial cells on the inner surface responsible for enzyme secretion and nutrient absorption. As

with many insects, there is a peritrophic membrane present in the midgut, though it does not

persist into the hindgut. The midgut of termites is fairly small, often without gastric caecae.

This is mainly due to the degree of digestion that takes place in the hindgut.

At the junction between the midgut and the hindgut, there are several malpighian tubules

which serve to excrete nitrogenous waste. It is thought by some that this waste may be recycled

based on the presence of uricolytic bacteria (Potrikus and Breznak 1980).









Symbiont Taxonomy and Anatomy

The cellulolytic system of lower termites has been extensively studied since Cleveland's

early work on the hindgut symbionts (Cleveland 1924). The hindgut contains both protozoa and

bacteria. These protozoa are basal flagellates. They lack mitochondria and so are anaerobic

organisms, but some possess hydrogenosomes (Cavalier-Smith 1993). Many have vacuoles for

engulfing the wood fragments that enter the hindgut (Cleveland 1925). Cellulolytic bacteria

have been documented in Zootermopsis angusticolis (Wenzel et al. 2002). However, the bulk of

the evidence obtained thus far suggests that the protozoa are the major sources of cellulolytic

enzymes. Removal of protozoa from Reticulitermes speratus has resulted in a significant loss of

both cellulolytic and xylanolytic activity (Inoue et al. 1997).

The taxonomy of these flagellates is continually being revised as more powerful

molecular techniques become available. As it is beyond the scope of this paper to provide a

detailed phylogenetic analysis, only a basic overview of current groupings will be provided.

The symbiotic flagellates may be broadly divided into three major classes. The first,

class Anaeromonadea, consists of organisms often referred to as Oxymonads. Most of these

protozoa have some form of structure which allows them to attach to surfaces, most often the

wall of the termite hindgut (Moriya et al. 2003). They tend to have a long, thin morphology and

few flagellae. They are sometimes grouped within the Kingdom Archezoa, and possess neither

mitochondria nor hydrogenosomes (Cavalier-Smith 1993).

The second and third major classes may be grouped within the phylum Parabasalia, a

group which possesses hydrogenosomes, but not mitochondria. The first of these two classes is

the class Trichomonadea. This is a very diverse group, containing families which include

Monocercomonadidae, Trichomonadidae, Devescovinidae and Calonymphidae. The

Monocercomonadidae and Trichomonadidae are generally small flagellates, and it is doubtful









that they participate in cellulose digestion. The two families can be separated by the presence of

a recumbent flagellum in the Trichomonadidae.

The Devescovinidae and Calonymphidae appear to sort out as one group in recent

phylogenetic studies (Delgado-Viscogliosi et al. 2000, Ohkuma et al. 2000). The flagellates in

these two families tend to be larger, often with many flagella, and in the case of Calonymphidae,

many nuclei as well.

In addition to these families, the class Trichomonadea also contains the orders

Lophomonadida and the Spirotrichonymphida. In terms of overall structure, the

Spirotrichonymphida are fairly similar to the flagellates in the third class, and have been grouped

with them in some earlier taxonomies.

The third class is the class Trichonymphea. These flagellates are typically large and

complex, with many flagella. They are widely distributed among the termites, with

Trichonympha being one of the more well-known genera.

Symbiont Assemblages in Termite Hindguts

The species of flagellates vary with each species of termites. A particularly thorough

documentation of these associations was undertaken by Yamin (1979). This study included

several species of termites from the families Kalotermitidae and Rhinotermitidae.

Upon examination, certain patterns emerge. Most clearly evident is the presence of the

families Devescovinidae and Calonymphidae in the Kalotermitidae, but not in either of the other

termite families. Also noteworthy is the presence of Oxymonad flagellates in the family

Pyrsonymphidae being restricted to the Rhinotermitidae. The order Spirotrichonymphida

appears to be far more prevalent in the Rhinotermitidae than in the Kalotermitidae. However,

the genus Trichonympha is present in at least one species from all three of the termite families.









In summary, each family of termites appears to have its own set of flagellate families

associated with it. Some flagellate groups are more widespread among the termites. The overall

pattern appears to be consistent with the coevolution of termite and flagellate lineages, with the

possibility of occasional horizontal transfer.

Termite Carbohydrolase Types and Locations

Before comparing these enzymes between termites and other creatures, it is necessary to

define certain aspects of their structure and function. First of all, cellulases may be divided into

three varieties, based on their mode of action (Breznak and Brune 1994). Any highly cellulolytic

organism requires all three of these enzymes from one source or another.

The first class of cellulases consists of exoglucanases (EC 3.2.1.91). These enzymes are

processive, binding to the end of a cellulose chain and moving along its length, breaking it down

along the way. Most exoglucanases are cellobiohydrolases, meaning that they reduce cellulose

to cellobiose, a disaccharide of beta-glucose. These enzymes are most active against crystalline

cellulose, where the cellulose chains are arranged in a tightly bound parallel configuration and

held in place by hydrogen bonding.

The second class of cellulases consists of the endoglucanases (EC 3.2.1.4). These

enzymes bind anywhere along the cellulose chain and break it up randomly, eventually reducing

it to cellodextrins, oligosaccharides of beta-glucose. The endoglucanases are most active against

amorphous cellulose, where the chains are not arranged in any particular configuration.

The third class of cellulases is the beta-glucosidases (EC 3.2.1.21). These enzymes break

cellodextrins down into glucose. They are the final enzymes in the process of glucose liberation

from cellulose.

Aside from cellulose, the major polysaccharides in wood are hemicelluloses. The most

well known and thoroughly studied of these hemicelluloses is xylan. It is a polymer of xylose, a









pentose sugar, with side molecules of 4-O-methylglucuronic acid. The xylose molecules may

have a varied degree of acetylation. There is also the possibility of arabinose being incorporated

into the polymer as an additional variety of side group. Since xylan is far more complex than

cellulose, there are several more enzymes required for its complete digestion. However, the

major enzymes of concern are often simply known as xylanases (EC 3.2.1.8). They hydrolyze

the bonds between xylose units in the polymer, much in the same fashion as endoglucanases

hydrolyze cellulose. In addition, there are beta-xylosidases (EC 3.2.1.37) which break down the

resulting oligosaccharides.

Functionally, the termite appears to have its enzymes spatially segregated. The

amorphous cellulose is attacked first in the foregut and midgut. The hindgut symbionts then

appear to digest the crystalline cellulose and the bulk of the hemicelluloses (Hogan et al. 1988,

Mishra 1991). The byproducts of this digestion, mainly acetate, then diffuse out into the termite

tissues.

The xylanases of lower termites have not been specifically characterized. However, the

bulk of xylanase and beta-xylosidase activity has been shown to be located in the hindgut in

multiple cases (Azuma et al. 1993, Inoue et al. 1997). Mannanase activity appears to also be

mainly located in the hindgut.

Termite Carbohydrolase Structural and Functional Characteristics

A vital structural aspect, in the case of cellulases at least, is cleft vs. tunnel geometry. The

catalytic site in exoglucanses is located within a tunnel in the enzyme. This allows the cellulose

chain to travel through the enzyme much as a thread may travel through the eye of a needle. Of

course, this particular needle has a guillotine incorporated into its structure. The cleft geometry,

found in endoglucanases, has the catalytic site located in a groove along the enzyme surface.









This allows the enzyme to effectively clamp onto any available location along the cellulose

chain.

The next structural aspect concerns retaining enzymes and inverting enzymes. Using

cellulases as an example, an inverting cellulase inserts the hydroxyl group in the alpha position,

opposite to the bond configuration in the cellulose chain. A retaining cellulase inserts a hydroxyl

group in the beta configuration, the same configuration as the bond in the cellulose chain.

Inverting cellulases work in a single step (Fig 1). The catalytic nucleophile removes a

hydrogen from a water molecule. The remaining hydroxide is bonded to the sugar molecule. At

the same time, the proton donor donates its hydrogen, allowing the oxygen bond between sugars

to be broken, separating off as a hydroxyl group on the other sugar. The remaining hydrogen on

the sugar is then brought back to the proton donor.

Retaining cellulases work in two steps, commonly referred to as a double displacement

mechanism (Davies et al. 1998). First, the catalytic nucleophile forms a covalent bond to the

substrate, while the proton donor allows the separation of the leaving group (Fig 2). In the

second step, a water molecule interacts with the bonded substrate, donating a hydroxyl group to

the substrate and effectively breaking the bond between it and the nucleophilic residue. The

remaining proton is accepted by the proton donor, restoring the enzyme to its original

configuration.

Another structural consideration is the nature of the proton donor and the catalytic

nucleophile. Glycosyl hydrolases function by placing the polysaccharide between two acidic

amino acid residues. If these residues are fairly far apart, there is room for a water molecule to

be incorporated into the configuration. This results in a direct trade of bonds, leading to an

inverting mechanism. If the residues are closer together, there is no room for a water molecule









in an occupied site, and so the sugar becomes temporarily bonded to the nucleophilic residue.

After this step, there is room for a water molecule to enter the site, and it is attached in the beta

configuration. This leads to a retaining mechanism.

There are only two amino acids with acidic side chains. These are aspartic acid and

glutamic acid. Each of these has a carboxyl group at the end of a short carbon chain. The

glutamic acid chain is one carbon longer than that of the aspartic acid, and so leaves less space.

As a result, nearly every carbohydrolase with a glutamic acid in each of the two key positions is

a retaining enzyme, while nearly every one with aspartic acid in each position is an inverting

enzyme. In enzymes with one of each residue, the glutamic acid is nearly always the proton

donor.

A final consideration, particularly applicable to the exoglucanases, is the positioning of

aromatic residues. Aromatic residues at certain locations on the enzyme surface allow the

cellulose chain to slide more easily through the catalytic site. This is often noticeable as a row of

tryptophan residues located within the tunnel of exoglucanases (Parsiegla et al. 2000). While

there are other aspects of structure, such as disulfide bonds, alpha helices and beta sheets, these

are generally more significant to enzyme grouping and relatedness.

So far, all of the endogenous cellulases of lower termites have been in glycosyl hydrolase

family 9 (Watanabe et al. 1998, Zhou et al. 2007). The enzymes in this family have aspartic acid

as a catalytic nucleophile and glutamic acid as a proton donor. They are inverting cellulases with

structures consisting mainly of alpha helices. The catalytic site follows the cleft configuration,

allowing the endoglucanases to bind anywhere along the cellulose chain. In addition, there

appears to be a high density of aromatic residues on the enzyme surface near the catalytic

residues.









The second family found in lower termites, family 7, contains both exoglucanases and

endoglucanases. Their structures are dominated by beta sheets and are rich in disulfide linkages.

They have glutamic acid at both positions of the catalytic site, and are retaining cellulases.

Unlike the other cellulases, the exoglucanases in this family have their catalytic sites located in

tunnels. In addition, they have several tryptophan residues located along the length of the tunnel.

The exoglucanases generally have four tryptophan residues in a row, while the endoglucanases

typically have two or three tryptophan residues along the length of the cleft.

So far, these enzymes have been found mainly in the Coptotermes symbionts

Pseudotrichonympha grassii and Holomastigotoides mirabile. The majority of sequences

recovered have indicated an endoglucanase activity (Watanabe et al. 2002). However, there have

been some promising indications of exoglucanases as well (Nakashima et al. 2002).

Glycosyl hydrolase family 45 consists of endoglucanases, which have aspartic acid in

both positions in the catalytic site. As may be expected, these are inverting cellulases. Like the

family 7 enzymes, their structures are dominated by beta sheets and are rich in disulfide linkages.

Like the termite cellulases, they have an open cleft structure. However, they differ significantly

from these enzymes in that they have very few alpha helices, and instead consist mainly of beta

sheets. In addition, they have several disulfide linkages, while family 9 cellulases tend to have

only one or two. Enzymes in family 45 have been found in the protozoan symbionts of

Reticulitermes speratus (Ohtoko et al., 2000). Two of the clones were localized to

Trichonympha agilis and Teranympha mirabilis. Both of these are large parabasalian flagellates.

Glycosyl hydrolase family 5 contains of exoglucanases, endoglucanases, xylanases,

mannanases, and beta-mannosidases. These enzymes have glutamic acid in both positions in the

catalytic site and follow a retaining mechanism. These enzymes consist of a combination of









alpha helices and beta sheets. These enzymes have been characterized from Coptotermes

formosanus symbionts (Inoue et al. 2005).











O0 OH HO


O-H
H
0


Figure 2-1. Inverting cellulase mechanism.


o o


----


HO O
to












HO

0
HO OH


OH HO


HO


o o




3

OH O-HO ,
O-H
0

0 0


OH 0 0 OH


H O OH I HO -
0 H
0 T 0


'I


OH O OH

OO
0
OH
HO

0 0


Figure 2-2. Retaining cellulase mechanism.









CHAPTER 3
BUFFER AND PH OPTIMIZATION FOR TERMITE CARBOHYDROLASE ASSAYS

Introduction

The gut of the typical subterranean termite consists of a foregut with a small crop and a

proventriculus, a fairly simple midgut and a greatly expanded hindgut containing several species

of symbiotic protozoa, fungi, bacteria, and archaea (Yamin 1979, Lewis and Forschler 2004). A

pair of salivary glands is also present, emptying into the foregut anterior to the crop. To

effectively digest wood, a recalcitrant and nutritionally poor substrate, termites like R. flavipes

have evolved an array of enzymes. Some carbohydrolases characterized from the

Rhinotermitidae have been shown to be endogenous (Watanabe et al. 1998), while others are

produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al.

2000).

In most prior studies, the gut carbohydrolases of subterranean termites have been assayed

using 0.1 M sodium acetate buffer, pH 5.5, as a standard buffer. Substrates have either been

based upon the generation of reducing sugars from specific polymers or the hydrolysis of smaller

molecules consisting of a monosaccharide or oligosaccharide completed with p-nitrophenol, a

molecule that is yellow when isolated and clear when completed with a sugar. Prior to using

these substrates for carbohydrolase activity evaluations, it was deemed necessary to evaluate a

range of possible pH values and buffers to determine whether the standard sodium acetate buffer

would produce optimal results.

Our objectives were to determine 1) the optimal buffer types for p-nitrophenol generating

substrate-based assays, 2) the optimal buffer types for reducing sugar-dependent assays, 3) the

optimal pH values for p-nitrophenol generating substrate-based assays for each major gut region,

and 4) the optimal pH values for reducing sugar-dependent assays for each major gut region.









Materials and Methods

Termite Collection, Dissection, and Enzyme Extraction

Reticulitermesflavipes (Kollar) termites were field collected in termite traps consisting of a

polyvinyl chloride bucket (20 cm high by 20 cm diam.; Item # 811192-4, Ventura Packaging

Inc., Monroeville, Ohio) with 11 holes drilled in the sides and base (3 cm diam.) placed

vertically in the ground to a depth of roughly 19 cm and covered with a PVC lid. Three rolls of

single-faced corrugated cardboard (10 cm diam. by 20 cm length) were placed into the tube side

by side as a food source. Termites were collected from the trap by removal of cardboard rolls,

separated from the cardboard, immediately frozen, and kept at -80C until dissection.

Termites were removed from the freezer and kept on ice until use. Termites were either

homogenized intact or dissected before homogenization. In the case of dissections, each

termite's gut was removed intact and then separated into three regions: foregut (and salivary

glands), midgut and hindgut. The three gut regions were placed into separate 1.5-mL

microcentrifuge tubes (Eppendorf, Westbury, NY) containing the appropriate buffer, and kept on

ice.

Enzymes were extracted using a method adapted from Inoue et al. (1997). Whole termites

or gut contents of each microcentrifuge tube were placed in a 2-mL Tenbroeck glass tissue

grinder and manually homogenized on ice. The homogenates were then centrifuged at 14,000

rpm at 4C for 15 min. The supernatants were collected, frozen, and kept at -80C until use in

the enzyme assays.

Buffer Optimization Assays

Five buffers were prepared at 0.1M in all cases. The buffers and their pH values were as

follows: sodium acetate (4.0, 4.5, 5.0, 5.5), sodium phosphate (6.0, 6.5, 7.0, 7.5), MES

(methylethyl sulfide; 5.5, 6.0, 6.5), bis-tris (2-[bis(2-hydroxyethyl)imino]-2-(hydroxymethyl)-









1,3-propanediol; 6.0, 6.5, 7.0), and PIPES (piperazine-N,N-bis(2-ethanesulfonic acid); 6.5, 7.0,

7.5). Two substrate solutions were prepared for each of the buffer/pH combinations. The first

was 2% carboxymethylcellulose (CMC; Sigma-Aldrich) and the second was 4 mM pNPG.

Whole termite extracts were prepared at a concentration of 50 termites/mL.

Endoglucanase assays were conducted using a method adapted from Han et al. (1995).

Assays were conducted in clear, flat-bottomed 96-well microplates. In each well, 10 p.L of tissue

extract was combined with 90 p.L of CMC solution. Only the wells more than 2 wells away from

the edge of the microplate were used for these assays. Perimeter wells were filled with deionized

water (200 ptL per well) to add temperature stability and consistency during boiling. Identical

plates were prepared as controls.

The enzyme and substrate solutions were allowed to react for 10 min at 23C. DNSA

solution (100 [tL), consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium hydroxide

and 30% sodium potassium tartrate, was then added to each enzyme/substrate well of the control

plates. After an additional 60 min, the DNSA solution was added in the same manner to the

sample plates. In both cases, immediately after addition of DNSA solution, the microplate was

placed in boiling water for 10 min to induce an oxidation/reduction reaction with the DNSA,

resulting in a color change. All plates were placed on ice immediately after boiling to cool for

15 min. Each microplate was read at 540 nm immediately after cooling using a [tQuant Universal

Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Standards were generated

using dilutions of glucose in sodium acetate buffer (0.1M, pH 5.5) combined with equal volumes

of DNSA solution. Standards were boiled, cooled, and read as described above.

Beta-glucosidase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-3-D-glucopyranoside (pNPG; Sigma-Aldrich) was prepared in









0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In

each well, 10 ptL of tissue extract was combined with 90 p.L of pNPG solution. The reaction was

allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 230C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

Gut Region Assays Across PH

Assays were conducted using a method adapted from Han et al. (1995). For

endoglucanase assays, both sodium acetate and sodium phosphate CMC solutions were prepared

as described above for the buffer optimization assays. Sodium acetate solutions were prepared at

pH values of 4, 4.5, 5, and 5.5. Sodium phosphate solutions were prepared at pH values of 6,

6.5, 7, and 7.5. For xylanase assays, solutions consisted of 0.5% beechwood xylan (>90%

xylose residues; Sigma-Aldrich) prepared in the same sodium acetate and sodium phosphate

buffers. The solutions were boiled approximately 30 min, until xylan particles were no longer

visible. The solutions were then centrifuged at 2,500 rpm for 5 min at 230C and the supernatants

were used as the final xylan substrate solutions. Termite tissues were extracted into the eight pH

buffers described above. Final tissue extracts consisted of foregut, midgut, and hindgut at a

concentration of 50 termite equivalents/mL in all cases.

The assays were conducted as described above for endoglucanase buffer optimization

assays. In each well, 10 ptL of tissue extract was combined with 90 [tl of CMC or xylan solution.

CMC solutions were allowed to react for 70 min at 230C. Xylan solutions were allowed to react

for 35 min at 230C. Control plates were prepared in the same manner, but were allowed to react

10 min for endoglucanase assays or 5 min before xylanase assays before boiling. Standards were









generated using dilutions of glucose for endoglucanase assays and dilutions of xylose for

xylanase assays. In both cases the standards were prepared in sodium acetate buffer (0.1M, pH

5.5), combined with equal volumes of DNSA solution, boiled, cooled, and read at 540 nm.

Exoglucanase, beta-glucosidase, and beta-xylosidase assays were conducted using a

method adapted from Han et al. (1995). Solutions of 4 mM p-nitrophenyl-P-D-cellobioside

(pNPC; Sigma-Aldrich), 4 mM p-nitrophenyl-P-D-glucopyranoside (pNPG; Sigma-Aldrich), and

4 mM p-nitrophenyl-P-D-xylopyranoioside (pNPX; Sigma-Aldrich) were prepared as described

above for the buffer optimization assays. Sodium acetate solutions were prepared at pH values

of 4, 4.5, 5, and 5.5. Sodium phosphate solutions were prepared at pH values of 6, 6.5, 7, and

7.5. Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of pNPC solution. The reaction was allowed to proceed for 10 min

before being placed in a [tQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments,

Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23C. Mean

velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol.

Data Analysis

The buffer optimization assays were set up as one-factor designs with a single homogenate

for each buffer/pH combination and three technical replicates. The pH assays across gut region

were set up as one-factor designs with a single homogenate for each pH and eight technical

replicates for each gut region.

For the endoglucanase and xylanase assays, the following formula was used to calculate

specific activities;

SA = Cs[(A-Ao)/t]/NT









where: SA = specific activity (nmol reducing sugar/termite equivalent/minute), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after

5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.

For the beta-glucosidase and exoglucanase assays, the following formula was used to

calculate specific activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol/termite equivalent/min), VA = mean

velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard (nmol p-

nitrophenol/mOd), and NT = the number of termite equivalents per sample.

For the time/concentration analyses, the mean specific activities were calculated for each

combination of time and concentration. The variance in mean specific activity was then

calculated among all times for each concentration and among all concentrations for each time.

For the remaining assays, means and standard errors were calculated for each enzyme specific

activity at each combination of pH and buffer, pH and gut region, or concentration and

temperature.

Results

Buffer Optimization Assays

Beta-glucosidase activities increased with pH from 5.5 to 7 (Figure 3-1). Activities were

negligible below pH 5.5. MES buffer caused a greater activity level than phosphate or PIPES

buffers at pH 6.5. Bis-Tris caused significantly lower activities across its functional pH range.

Activities on PIPES buffer were slightly lower than those for phosphate buffer across its

functional pH range. With the exception of the bis-tris buffer, the greatest changes in beta-

glucosidase activity over pH were seen between pH 5.5 and 7.









Gut Region pH Optimization Assays

Foregut endoglucanase activity was greatest at a pH value of 6.5, with a smaller activity

peak at 5.5 (Figure 3-2). Midgut and hindgut endoglucanase activities were greatest at a pH of

5.5. Hindgut activity was significantly lower at pH values above 5.5 and below 5. Changes in

foregut endoglucanase activities were high throughout the pH range tested, while the greatest

change in hindgut endoglucanase activities was the decline in activity from a pH of 5.5 to 6.

Foregut and midgut beta-glucosidase activities increased with pH up to pH 6.5, then

declined at higher pH values (Figure 3-3). Beta-glucosidase activities in the hindgut extract

showed a steady increase with pH up to pH 7. All activities were negligible below pH 5.5.

Changes in foregut and midgut beta-glucosidase activities were greatest between pH 6 and 6.5.

Changes in hindgut beta-glucosidase activities were greatest between pH 5.5 and 7.

Hindgut exoglucanase activities were consistently greater with increasing pH value,

following a sigmoid curve (Figure 3-4). The greatest differences in activity were between pH

5.5 and 7. Activity was negligible below a pH of 6.

Hindgut xylanase activity was greatest at pH 4.5 (Figure 3-5). However, there was little

difference in hindgut xylanase activities from pH 4 to 5.5. The greatest change in hindgut

xylanase activity was seen from a pH of 5.5 to 7.5.

Discussion

Activity Magnitudes

Three qualities of enzymatic activity are of particular interest when selecting buffers for

termite carbohydrolase assays. The first is the magnitude of activity yielded by a given buffer.

Experiments with a goal of quantifying enzymatic activity may underestimate actual activities if

the buffer does not maximize in vitro enzyme activity levels. Lower levels of actual activity may









be missed altogether with some buffers. For comparative studies, lower overall activities may

make differences in activity levels more difficult to distinguish.

For pNPG-based beta-glucosidase assays, the highest activities ranged from 6.5 to 7.5 on

MES, PIPES, and sodium phosphate buffers. Sodium acetate and bis-tris buffers yielded far

lower activities. In the past, investigators have used pH 5.5 sodium acetate buffer for pNPG-

based termite beta-glucosidase assays (Inoue et al. 1997). This may have caused beta-

glucosidase activity to be underestimated within the termite.

For symbiont exoglucanase from the hindgut, activity was greatest on pH 7.5 sodium

phosphate buffer. The activity of symbiont xylanase from the hindgut was greatest on pH 4.5

sodium acetate buffer, and nearly as high on all sodium acetate buffers tested.

Activity Changes with pH

The second quality of interest is the rate of change in activity with changing pH. If the

selected buffer is in a region of high change, smaller changes in buffer pH will cause greater

changes in activity levels. This can increase variation in experimental results and, in extreme

cases, cause difficulties with experimental repeatability. Therefore, the optimal buffer should

maximize activity within a region of minimal change in activity with change in pH.

Exoglucanase activity declined sharply below pH 6.5 sodium phosphate buffer and was

nearly nonexistent on sodium acetate buffer. However, xylanase activity declined sharply with

increasing pH on sodium phosphate buffer.

Differences in Gut Regions

Different activity maxima were seen for different gut regions in beta-glucosidase and

endoglucanase assays. In addition, pH ranges with the highest changes in activity differed

between gut regions for beta-glucosidase and endoglucanase assays.









In the case of beta-glucosidases, the differences were mainly seen between the endogenous

enzymes from the foregut and midgut, and the symbiont enzymes from the hindgut. In contrast,

the differences in the endoglucanase assays were mainly seen between the foregut and the

hindgut, with midgut activity levels far lower than those seen in the other gut regions. Because

of these differences, the optimal buffers for carbohydrolase assays may not be the same for

endogenous (termite) and symbiont produced enzymes.

Conclusions

Based upon our experimental data, the optimal pH buffer for predicting overall xylanase or

endoglucanase activities is pH 5.5 sodium acetate buffer, which is the standard buffer used in

past termite carbohydrolase assays (Hogan et al. 1988, Inoue et al. 1997). Although

endoglucanase activity shifts considerably from pH 5.5 to pH 6, the proximity of pH 5.5 sodium

acetate buffer to this shift presents no concern. In this case, the observed shift is not only

between two pH values, but also occurs in a shift between sodium acetate and sodium phosphate

buffers.

Previous experiments have generally used pH 5.5 sodium acetate buffer as a universal

buffer for termite carbohydrolase assays. Based on our p-nitrophenol-generating assays, the

optimal pH buffer for predicting overall beta-glucosidase activity using pNPG is pH 6.5 sodium

phosphate buffer. Both beta-glucosidase and exoglucanase activities on pH 6.5 sodium

phosphate buffer are roughly 10-fold higher than on pH 5.5 sodium acetate buffer. The optimal

pH buffer for predicting exoglucanase activity or hindgut beta-glucosidase activity is pH 7.5

sodium phosphate buffer. Hindgut exoglucanase activity is roughly 15-fold higher and beta-

glucosidase activity roughly 20-fold higher on pH 7.5 buffer than on pH 5.5 buffer. We believe

that prior pNPG or pNPC-based assays using pH 5.5 sodium acetate buffer may have

underestimated actual beta-glucosidase or exoglucanase activity levels within the termites.









Based upon these findings, we intend to conduct pNPC, pNPG, and pNPX-based assays

using 0.1 M sodium phosphate buffer, pH 6.5, in the following chapters. We intend to conduct

DNSA-based assays using 0.1 M sodium acetate buffer, pH 5.5.
















1.6 Acetate
"' Phosphate
3 1.2 MES
SBis-Tris
S0.8 PIPES


0.4


0
3.5 4 4.5 5 5.5 6 6.5 7 7.5 8

pH



Figure 3-1. Beta-glucosidase activities in Reticulitermesflavipes workers across pH and buffer.


















Foregut
--Midgut
Hindgut


4 4.5 5 5.5 6 6.5 7 7.5


pH


Figure 3-2. Endoglucanase activities in different gut regions of Reticulitermesflavipes workers
across pH.


EE

S
E

cL E
C-,.










0.9





*D 0.6
c -




E. E 0.3





0


4 4.5 5 5.5 6 6.5 7 7.5


pH


Figure 3-3. Beta-Glucosidase activities in different gut regions of Reticulitermes flavipes
workers across pH.


Foregut
--Midgut
Hindgut










0.5


0.4


> E
0.3


= 0.2


0.1


0
4 4.5 5 5.5 6 6.5 7 7.5

pH


Figure 3-4. Hindgut exoglucanase activities in Reticulitermesflavipes workers across pH.











35

30

S 25

S 20

_E 15
(1) 0 1
SE 10

5

0
4 4.5 5 5.5 6 6.5 7 7.5
pH


Figure 3-5. Hindgut xylanase activities in Reticulitermesflavipes workers across pH.









CHAPTER 4
CARBOHYDROLASE ACTIVITY PATTERNS IN RETICULITERMES FLA VIPES AND
COPTOTERMES FORMOSANUS WORKERS AND SOLDIERS

Introduction

Reticulitermesflavipes (Kollar) and Coptotermesformosanus (Shiraki) are two of the most

economically significant pest termites in North America. Both are subterranean termites, but R.

flavipes is a native termite with relatively small colonies and a low soldier ratio while C.

formosanus is an invasive termite with relatively large colonies and a higher soldier ratio. Such

differences in colony structure may be accompanied by differences in gut carbohydrolase

profiles.

Termites and their symbionts are known to produce three major types of cellulase which

work together to digest cellulose. Exoglucanase (EC 3.2.1.91) cleaves the cellulose chain from

the ends, typically producing cellobiose, and is most active against crystalline cellulose.

Endoglucanase (EC 3.2.1.4) cleaves the cellulose chain randomly along its length and is most

active against amorphous cellulose. Beta-Glucosidase (3.2.1.21) cleaves cellobiose and other

small cellulose fragments, hydrolyzing them to glucose. Xylanolytic enzymes include xylanases

(EC 3.2.1.8) which internally cleave the xylan chain and beta-xylosidases (EC 3.2.1.37) which

cleave xylan oligosaccharides into xylose.

The objectives of this study were 1) determine the patterns of cellulolytic and xylanolytic

enzymes in different termite gut regions, 2) determine the relative cellulolytic and xylanolytic

activities of R. flavipes and C. formosanus workers, 3) determine the relative cellulolytic and

xylanolytic activities of R. flavipes and C. formosanus soldiers, and 4) determine the relative

cellulolytic and xylanolytic activities of the soldiers of each species compared to the workers.









Materials and Methods

Termite Collection, Dissection, and Enzyme Extraction

Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in

Charleston, South Carolina. Termites were collected from the trap by removal of the wood

(southern yellow pine) in the bait stations and brought to the University of Florida. Termites

were then placed in feeding bioassays. Termites were collected, frozen and kept at -800C until

dissection. Collections were restricted to a single colony for each species to eliminate colony as

a source of variation in enzyme activities.

Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5,

and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the

endoglucanase and xylanase assays, while sodium phosphate buffer was used for the

exoglucanase, beta-glucosidase and beta-xylosidase assays.

Termites were removed from the freezer and kept on ice until dissection. Each termite's

gut was removed intact and separated into three regions: foregut (and salivary glands), midgut,

and hindgut. A single enzyme extract was prepared from dissected termites from each feeding

treatment for each assay type using an experimental design similar to the recent previous termite

carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For

the endoglucanase and xylanase assays, 25 termites of each caste and species were dissected in

sodium acetate buffer. For the exoglucanase, beta-glucosidase and beta-xylosidase assay, 25

termites of each caste and species were dissected in sodium phosphate buffer. The three gut

regions were placed into separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the

appropriate buffer, and kept on ice. Final concentrations were 50 termite gut regions per mL in

all cases.









Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of

each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and

manually homogenized on ice. The homogenates were centrifuged at 20,800 g at 40C for 15

min. The supernatants were collected, frozen, and kept at -800C until use in the enzyme assays.

Termite Photography

Guts and frontal glands from both termite species were dissected into 0.1 M sodium

phosphate buffer, pH 6.5, using the methods described above. Termites were then photographed

on an auto-montage system (Syncroscopy, Frederick, MD).

Soluble Protein Assays

Assays for soluble protein content were conducted using the Bradford reagent. Enzyme

extracts (10 [tL) were combined with 250 p.L of Bradford reagent (Bio-Rad, Hercules, CA) in a

96-well microplate. Absorbances were then read at 595 nm using a [tQuant Universal

Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Standards were generated

using BSA standards (Bio-Rad, Hercules, CA) combined with the Bradford reagent in the same

proportion as the enzyme extracts.

Endoglucanase Assays

The endoglucanase assays were conducted using a method adapted from Han et al. (1995).

A 2% solution of carboxymethylcellulose (CMC; Sigma-Aldrich) was prepared in 0.1 M sodium

acetate buffer, pH 5.5.

Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of CMC solution. The solutions were allowed to react for 70 min at

23C. DNSA solution (100 [tL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was









immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each

microplate was read at 540 nm using a [tQuant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 10 min to

allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A

10 min reaction was used as a control to correct for any differences in initial sample reaction

rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated

using dilutions of glucose. Only the wells in the middle of the microplate were used for these

assays. Perimeter wells were filled with deionized water (200 p.L per well) to add temperature

stability and consistency during boiling.

Exoglucanase Assays

Exoglucanase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-P-D-cellobioside (pNPC; Sigma-Aldrich) was prepared in 0.1 M

sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In each

well, 10 ptL of tissue extract was combined with 90 ptL of pNPC solution. The reaction was

allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 23C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

Beta-Glucosidase Assays

Beta-glucosidase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-P-D-glucopyranoside (pNPG; Sigma-Aldrich) was prepared in

0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In

each well, 10 ptL of tissue extract was combined with 90 p.L of pNPG solution. The reaction was









allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 23C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

Xylanase Assays

The xylanase assays were conducted using a method adapted from Han et al. (1995). A

0.5% solution ofbeechwood xylan (>90% xylose residues; Sigma-Aldrich) was prepared in 0.1

M sodium acetate buffer, pH 5.5. The solution was boiled approximately 30 min, until xylan

particles were no longer visible. The solution was centrifuged at 1250 g for 5 min at 230C and

the supernatant was used as the xylan stock solution.

Assays were conducted in clear 96-well microplates. In each well, 10 pL of tissue extract

was combined with 90 pL of xylan solution. The solutions were allowed to react for 35 min at

23C. DNSA solution (100 [LL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each

microplate was read at 540 nm using a [[Quant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 5 min to allow

for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 5 min

reaction was used as a control to correct for any differences in initial sample reaction rates due to

incomplete mixture of enzyme and substrate solutions. Standards were generated using dilutions

of xylose. Only the wells in the middle of the microplate were used for these assays. Perimeter

wells were filled with deionized water (200 pL per well) to add temperature stability and

consistency during boiling.









Beta-Xylosidase Assays

Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-P-D-xylopyranoside (pNPX; Sigma-Aldrich) was prepared in

0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In

each well, 10 ptL of tissue extract was combined with 90 p.L of pNPX solution. The reaction was

allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 23C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

The experiment with formulated diets was set up as a two-factor design with four

replicates. The two factors were gut region and diet treatments. One microplate well was

assayed for each replicate of each gut region/diet combination. Three gut sections and five diet

treatments were assayed for each replicate, yielding a total of 15 wells per replicate.

Data Analysis

Experiments were set up as one-factor designs with two homogenates for each

caste/species combination; one homogenate for endoglucanase and xylanase assays and another

for exoglucanse, beta-glucosidase, and beta-xylosidase assays. The endoglucanase assays had 6

technical replicates for each gut region while the other assays had 4 technical replicates for each

gut region. The protein assays had 3 technical replicates for each gut region, species and caste.

For the endoglucanase and xylanase assays, the following formula was used to calculate

specific activities;

SA = Cs[(A-Ao)/t]/Nr









where: SA = specific activity (nmol reducing sugar per termite equivalent per min), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after

5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.

For the exoglucanase, beta-glucosidase, and beta-xylosidase assays, the following

formula was used to calculate specific activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA

mean velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard

(nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample.

The data were analyzed using analysis of variance. Student-Neuman-Keuls test, ca = 0.05,

was used to separate the mean activities within each gut region (SAS Institute 2001).

Results

Termite Gut and Frontal Gland Comparisons

Soldiers in both species had smaller, less developed hindguts than workers (Figure 4-1).

Compared to R. flavipes workers, C. formosanus workers had a smaller crop, a larger rectum,

and a small internal sclerotization just behind the malpighian tubules. The frontal gland in C.

formosanus soldiers was far larger and apparently more developed than that in R. flavipes

soldiers (Figure 4-2). Formosan soldier termites had a prolonged pharynx, to accommodate the

enlarged frontal gland.

Worker Cellulolytic Activities

Endoglucanase activities were mainly confined to the foregut and hindgut (Table 4-1).

Activities were significantly greater in the R. flavipes worker foregut than in the C. formosanus

worker foregut, but they were significantly greater in the C. formosanus midgut and hindgut.









Exoglucanase activities were almost exclusive to the hindgut, with C. formosanus worker

activities being significantly greater in all gut regions (Table 4-2). Beta-glucosidase activities

were distributed across the gut, being most prominent in the midgut in C. formosanus workers

and most prominent in the hindgut in R. flavipes workers (Table 4-3). Differences in Beta-

glucosidase activities were significant in all three gut regions among all species-caste

combinations.

Worker Xylanolytic Activities

Xylanase activities were almost exclusive to the hindgut, with no significant differences

between worker hindgut xylanase activities (Table 4-4). Beta-xylosidase activities were mainly

restricted to the hindgut, and were significantly higher in C. formosanus workers in all gut

regions (Table 4-5).

Soldier Cellulolytic Activities

Soldier cellulolytic activities were lower than those seen in workers in most cases.

Endoglucanase activities were mainly confined to the hindgut, with midgut and hindgut activities

significantly greater in R. flavipes soldiers (Table 4-1). Exoglucanase activities were mainly

confined to the hindgut, and hindgut activities were significantly greater in R. flavipes soldiers

(Table 4-2). Beta-glucosidase activities were distributed across the gut, being most prominent in

the midgut and significantly greater in R. flavipes soldier hindguts (Table 4-3).

Soldier Xylanolytic Activities

Soldier xylanolytic activities were lower than those seen in workers in most cases.

Xylanase activities were almost exclusive to the hindgut, and were significantly higher in the R.

flavipes soldier hindgut (Table 4-4). Beta-xylosidase activities were mainly restricted to the

hindgut, and were significantly higher in the R. flavipes soldier hindgut, while midgut activities

were significantly higher in the C. formosanus soldier hindgut (Table 4-5).









Gut Soluble Protein Levels

The hindgut extracts had far more soluble protein than the foregut or midgut extracts in all

cases except for the C. formosanus soldier extract in pH 5.5 buffer (Table 4-6). This difference

was smaller, but the hindgut still contained more protein than the foregut or midgut, even for the

pH 5.5 C. formosanus soldier extract. Soldier protein levels were universally lower than worker

protein levels for each gut region. C. formosanus worker extracts had higher protein levels than

R. flavipes worker extracts in most cases. C. formosanus soldier extracts had lower protein

levels than R. flavipes soldier extracts in all cases. The extracts in pH 5.5 buffer had lower

protein levels than the extracts in pH 6.5 buffer.

Discussion

The patterns of cellulolytic enzymes within the R. flavipes workers appeared to indicate a

processive degradation of amorphous cellulose along the gut. Endoglucanase activity was seen

to be higher in the foregut than in the midgut, whereas beta-glucosidase activity appeared to

increase progressively through the gut.

The majority of exoglucanase and xylanase activities were located in the hindgut of

workers soldiers of both species assayed. Similar exoglucanase patterns have been observed in

Coptotermes lacteus (Hogan et al. 1988). This indicates a large dependence of the termite on its

hindgut symbionts for the digestion of crystalline cellulose and xylan. Our findings are

consistent with prior characterizations of symbiont exoglucanases in C. formosanus and R.

flavipes (Watanabe et al. 2002, Nakashima et al. 2002a, Zhou et al. 2007). This localization of

exoglucanase and xylanase activities to the hindgut is significant, as up to 70% of wood cellulose

may be crystalline in nature and xylan may make up as much as 25% of wood dry weight

(Biermann 1996). This could explain past observations of reliance on hindgut symbionts

(Cleveland 1924) in spite of the characterization of endogenous cellulases in Reticulitermes and









Coptotermes termites (Watanabe et al. 1998, Nakashima et al. 2002b, Zhou et al. 2007). It

should be noted that, thus far, no endogenous cellulases in characterized lower termites have

been exoglucanases.

Formosan subterranean termites are widely regarded as more aggressive foragers than

eastern subterranean termites, with far larger colonies. Their soldiers have a larger frontal gland

than the soldiers of the eastern subterranean termite, producing a significant amount of chemical

exudate in defense of the colony (need to take photos of the frontal glands). This increased

metabolic demand, combined with the larger ratio of soldiers to workers, roughly 1:10 as

opposed to 1:100 in the case ofR. flavipes, would seem to necessitate a more active system for

the digestion of wood and similar materials.

This is precisely what the results seem to indicate among the workers, especially in the

case of exoglucanase and beta-glucosidase. While the overall distributions of carbohydrolase

activities are similar in these two species, there are a couple of differences; heightened enzyme

activities as mentioned above and the partial redistribution of endoglucanase from the foregut to

the midgut and hindgut in C. formosanus workers.

A reverse pattern was seen in the soldiers, with the C. formosanus soldiers having far less

enzyme activity in the case of endoglucanase, exoglucanase and xylanase. However, C.

formosanus soldier beta-glucosidase and beta-xylosidase were similar to the levels seen in R.

flavipes soldiers. The former three carbohydrolases are known to break down longer chains, thus

working in the earlier parts of cellulose and xylan degradation. The latter two break down

oligosaccharides, working in the final parts of cellulose and xylan degradation.

With its greatly enlarged frontal gland, the C. formosanus soldier has relatively little room

for its digestive system. Since the C. formosanus workers are so proficient at wood digestion,









there is little need for the earlier enzymes in the soldier digestive systems. Therefore, it makes

sense that these soldiers' limited digestive capacity would be almost entirely focused on the later

phases of wood digestion. This is supported by the less extreme case of the R. flavipes soldiers,

which also have relatively high levels of beta-glucosidase and beta-xylosidase compared with the

other three enzymes.

Formosan subterranean termites are not only more aggressive foragers than eastern

subterranean termites, but their digestive systems are apparently more capable of digesting wood,

particularly crystalline cellulose. This would suggest the possibility of natural cellulase

inhibitors or other obstacles to wood digestion in their native range. Some plants are known to

produce cellulase inhibitors in the form of proteins or polyphenols (Sineiro et al. 1997, York et

al. 2004). However, the increased digestive capacity seen in C. formosanus workers could also

simply be a result of the need to support a larger proportion of soldiers, as termite soldiers are

incapable of feeding themselves and Formosan soldiers have relatively little internal digestive

capacity.

Although there were clearly differences in overall activity levels, the patterns of

cellulolytic and xylanolytic enzyme activities were similar between the two species assayed, in

spite of the fact that these species were in two different genera from two different continents.

This suggests that findings regarding cellulose and xylan digestion in one species within the

Rhinotermitidae may be cautiously applied to the rest of the family. Our findings also suggest a

processive mechanism of amorphous cellulose degradation with a reliance on the termite

symbionts for the digestion of xylan and crystalline cellulose. This corroborates the need of

these termite species for their hindgut symbionts in spite of their production of endogenous









cellulolytic and xylanolytic enzymes. This pattern of digestion will be revisited in workers of

both species in subsequent chapters.









Table 4-1. A comparison of endoglucanase activities in Reticulitermesflavipes and Coptotermes
formosanus workers and soldiers
Caste Species Foregut Midgut Hindgut
worker R.flavipes 4.70 + 0.31a 1.39 + 0.08b 5.21 +0.28b
worker C.formosanus 2.84 + 0.22b 1.91 +0.19a 8.17 + 0.51a
soldier R. flavipes 0.36 + 0.07c 1.22 + 0.11b 2.81 +0.16c
soldier C. formosanus 0.00 + 0.01c 0.23 + 0.03c 0.79 + 0.07d
Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, a = 0.05,
SAS Institute 2001), n= 6 replicates.









Table 4-2. A comparison of exoglucanase activities in Reticulitermesflavipes and Coptotermes
formosanus workers and soldiers
Caste Species Foregut Midgut Hindgut
worker R.flavipes 0.0109 + 0.0001b 0.0121 + 0.0026c 0.3862 + 0.0067b
worker C.formosanus 0.0278 + 0.0002a 0.1182 + 0.0033a 0.6728 + 0.0140a
soldier R. flavipes 0.0013 + 0.0006d 0.0409 + 0.0008b 0.2184 + 0.0037c
soldier C. formosanus 0.0037 + 0.0002c 0.0377 + 0.0017b 0.1014 + 0.0019d
Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed
by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, a = 0.05, SAS Institute
2001), n= 4 replicates.









Table 4-3. A comparison of beta-glucosidase activities in Reticulitermesflavipes and
Coptotermesformosanus workers and soldiers
Caste Species Foregut Midgut Hindgut
worker R.flavipes 0.147 + 0.004b 0.176 + 0.004a 0.655 + 0.008b
worker C.formosanus 0.272 + 0.003a 0.891 + 0.033c 0.876 + 0.020a
soldier R. flavipes 0.037 + 0.001c 0.611 + 0.008b 0.348 + 0.001c
soldier C. formosanus 0.056 + 0.018c 0.560 + 0.016b 0.168 + 0.002d
Beta-glucosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, a = 0.05,
SAS Institute 2001), n = 4 replicates.









Table 4-4. A comparison of xylanase activities in Reticulitermesflavipes and Coptotermes
formosanus workers and soldiers
Caste Species Foregut Midgut Hindgut
worker R. flavipes -0.04 + 0.02a 0.02 + 0.02b 24.18 + 0.37a
worker C.formosanus -0.01+0.04a 0.48+0.11a 25.13+0.43a
soldier R. flavipes -0.03 + 0.03a 0.21 + 0.08b 21.51 + 0.27b
soldier C. formosanus -0.03 + 0.01a 0.01 + 0.01b 00.99 + 0.18c
Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by
the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, a = 0.05, SAS Institute
2001), n= 4 replicates.









Table 4-5. A comparison of beta-xylosidase activities in Reticulitermesflavipes and
Coptotermesformosanus workers and soldiers
Caste Species Foregut Midgut Hindgut
worker R.flavipes 0.0056 + 0.0006bc 0.0072 + 0.0003c 0.0399 + 0.0006b
worker C.formosanus 0.0075 + 0.0006a 0.0014 + 0.0004a 0.0645 + 0.0011a
soldier R. flavipes 0.0049 + 0.0004c 0.0011 + 0.0003b 0.0216 + 0.0003c
soldier C. formosanus 0.0067 + 0.0003ab 0.0015 + 0.0004a 0.0134 + 0.0002d
Beta-xylosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, a = 0.05,
SAS Institute 2001), n= 4 replicates.










Table 4-6. A comparison of soluble protein concentrations in Reticulitermesflavipes and
Coptotermesformosanus worker and soldier gut extracts


Species
R. flavipes
C. formosanus
R. flavipes
C. formosanus
R. flavipes
C. formosanus
R. flavipes
C. formosanus


Foregut
1.304 + 0.018
0.944 + 0.052
0.420 + 0.001
0.256 + 0.001
2.250 + 0.060
0.181 + 0.014
1.232 + 0.030
0.296 + 0.032


Midgut
1.126 + 0.046
1.714 + 0.032
0.676 + 0.014
0.354 + 0.016
4.192 + 0.128
3.952 + 0.088
2.234 + 0.052
0.556 + 0.016


Hindgut
6.954 + 0.208
11.312 + 0.060
3.808 + 0.156
0.622 + 0.078
15.726 + 0.852
21.522 + 0.228
9.832 + 0.500
2.312 + 0.052


Concentrations are in Ltg per termite gut region, n = 3 replicates.


Caste
Worker
Worker
Soldier
Soldier
Worker
Worker
Soldier
Soldier


























































Figure 4-1. Digestive systems of: A) R. flavipes worker, B) C. formosanus worker, C) R.
flavipes soldier, and D) C. formosanus soldier.



61



















































Figure 4-2. Heads and frontal glands of a R. flavipes soldier, and a C. formosanus soldier.









CHAPTER 5
CHANGES IN RETICULITERMES FLA VIPES GUT CELLULOLYTIC ACTIVITIES IN
RESPONSE TO DIET

Introduction

Subterranean termites, such as Reticulitermesflavipes (Kollar), subsist largely on a diet of

wood and similar material (Noirot and Noirot-Timothee 1969). Wood is not a substance that

most animals are capable of digesting to any significant degree. Termites are well known for the

ability to digest cellulose with the aid of microbial hindgut symbionts.

In wood, cellulose chains are typically arranged in parallel bundles known as microfibers

which are embedded in a matrix of lignin and hemicelluloses. The cellulose in the microfibers

may be broadly divided into two types: crystalline and amorphous. The crystalline form of

cellulose consists of tightly aligned parallel chains, held in a specific configuration by hydrogen

bonding. In the amorphous form of cellulose, the chains are more randomly arranged, and not so

closely bound together.

There are three major types of cellulase which work together to digest cellulose.

Exoglucanase (EC 3.2.1.91) cleaves the cellulose chain from the ends, typically producing

cellobiose, and is most active against crystalline cellulose. Endoglucanase (EC 3.2.1.4) cleaves

the cellulose chain randomly along its length and is most active against amorphous cellulose.

Beta-Glucosidase (3.2.1.21) cleaves cellobiose and other small cellulose fragments, hydrolyzing

them to glucose.

The gut ofR. flavipes and related termites consists of a foregut with a small crop and a

proventriculus, a fairly simple midgut, and a greatly expanded hindgut containing several species

of symbiotic protozoa, fungi, bacteria, and archaea (Yamin 1979, Lewis and Forschler 2004). A

pair of salivary glands is also present, emptying into the foregut anterior to the crop. To

effectively digest wood, a chemically demanding and nutritionally poor substrate, termites like









R. flavipes have evolved an array of enzymes. Some of the cellulases characterized from the

Rhinotermitidae have been shown to be endogenous, being produced naturally by the termite

(Watanabe et al. 1998, Watanabe et al. 1997). Others have been shown to be symbiotic in origin,

produced by flagellate symbionts within the hindgut (Nakashima et al. 2002a, Watanabe et al.

2002, Ohtoko et al. 2000). Zhou et al. (2007) demonstrated that exoglucanase activity is largely

localized to the hindgut of R. flavipes, strongly implicating the resident flagellates as the major

agents of crystalline cellulose digestion. Inoue et al. (1997) showed that the protozoan

composition of the Reticulitermes speratus (Kolbe) hindgut significantly changes when the

termites are fed on pure cellulose or pure xylan, as opposed to wood. It is probable that, with

changes in protozoan populations on differing diets, the levels of different cellulase activities

may also change.

Subterranean termites may encounter a number of potential food sources during foraging.

These may be broadly grouped into hardwoods, softwoods, and processed cellulosic materials

like paper. These three groups vary mainly in their hemicellulose content. In homes, hardwoods

may include furniture, flooring, and trim components. Structural timbers are nearly always

derived from softwoods.

Our objectives were to: 1) confirm the patterns of R. flavipes worker cellulolytic activity

seen in Chapter 4, 2) determine how the three major cellulase activities found in the gut of R.

flavipes workers change in response to three different simulated field diets.

Materials and Methods

Termite Collection

R. flavipes termites were field collected in termite traps consisting of a PVC bucket (20 cm

high by 20 cm diam.; Item # 811192-4, Ventura Packaging Inc., Monroeville, Ohio) with 11

holes drilled in the sides and base (3 cm diam.) placed vertically in the ground to a depth of









roughly 19 cm and covered with a PVC lid. Three rolls of single-faced corrugated cardboard (20

cm long by 10 cm diam.) were placed into the bucket side by side as a food source. Termites

were collected from the trap by removal of cardboard rolls, separated from the cardboard, and

either placed in feeding bioassays or immediately frozen and kept at -800C until dissection.

Collections were restricted to a single colony to eliminate colony as a source of variation in

enzyme activities.

Termite Diets and Feeding

Three diets were prepared for the termites to represent the probable food sources they

would encounter in the field. These simulated field diets were as follows: red oak (Quercus

spp.), pine (Pinus spp.), and filter paper. Wood diets were generated by drilling into craft wood

boards (0.635 x 5.08 x 60.96 cm) with a 2.54 cm spade drill bit. Sawdust was collected and

weighed. Filter paper consisted of a weighed number of crumpled cellulose filter paper disks

(42.5 mm diameter, Whatman, grade 4).

Each field diet (20 g) was added to a loosely capped 250 mL glass bottle (Pyrex) with 5

mL of deionized water. Field diets were not made from sterile materials. Because of this, the

bottles with field diets were autoclaved on a liquid cycle (30 min, 122C) to sterilize the diets

and the bottles were then allowed to cool. Termites were added (-300 workers and 3 soldiers per

bottle) after the bottles had cooled and kept in the dark at 210C for 6 wk, with deionized water (2

mL) added every 2 wk. Termites were collected, frozen and kept at -800C until dissection.

Termite Dissection and Enzyme Extraction

Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5,

and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the

endoglucanase assay, while sodium phosphate buffer was used for the exoglucanase and beta-

glucosidase assays.









Termites were removed from the freezer and kept on ice until dissection. Each termite's

gut was removed intact and separated into three regions: foregut (and salivary glands), midgut,

and hindgut. A single enzyme extract was prepared from a minimum of 35 dissected termites

from each feeding treatment for each assay type using an experimental design similar to previous

termite carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al.

2002b). For the endoglucanase assay, 50 termites from each feeding treatment were dissected in

sodium acetate buffer. For the exoglucanase and beta-glucosidase assays, 35 termites from each

feeding treatment were dissected in sodium phosphate buffer. The three gut regions were placed

into separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and

kept on ice. Final concentrations were 50 termite gut regions per mL in all cases.

Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of

each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and

manually homogenized on ice. The homogenates were centrifuged at 20,800 g at 40C for 15

min. The supernatants were collected, frozen, and kept at -80C until use in the enzyme assays.

Endoglucanase Assays

The endoglucanase assays were conducted using a method adapted from Han et al. (1995).

A 2% solution of carboxymethylcellulose (CMC; Sigma-Aldrich) was prepared in 0.1 M sodium

acetate buffer, pH 5.5.

Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of CMC solution. The solutions were allowed to react for 70 min at

23C. DNSA solution (100 [tL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each









microplate was read at 540 nm using a [tQuant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 10 min to

allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A

10 min reaction was used as a control to correct for any differences in initial sample reaction

rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated

using dilutions of glucose. Only the wells in the middle of the microplate were used for these

assays. Perimeter wells were filled with deionized water (200 p.L per well) to add temperature

stability and consistency during boiling. For all replicates, the control plates were used to adjust

for 540 nm absorbance in gut extracts and were replicated an equal number of times to the assay

plates, with one microplate well for each replicate.

Exoglucanase and Beta-glucosidase Assays

The exoglucanase and beta-glucosidase assay was conducted using a method adapted from

Han et al. (1995). Solutions of 4 mM p-nitrophenyl-P-D-cellobioside (pNPC) and 4 mM p-

nitrophenyl-P-D-glucopyranoside (pNPG) were prepared in 0.1 M sodium phosphate buffer, pH

6.5. Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of pNPC or pNPG solution. The reaction was allowed to proceed for

10 min before being placed in a [tQuant Universal Microplate Spectrophotometer (Bio-Tek

Instruments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 230C.

Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-

nitrophenol.

Data Analysis

The endoglucanase assays were set up as a one-factor split-plot design with four technical

replicates for each gut region, and one microplate per replicate. Four microplate wells were









assayed for each replicate of each gut region/diet combination. The exoglucanase and beta-

glucosidase assays were set up as one-factor designs with four technical replicates for each gut

region. Two homogenates were used for each gut/diet combination; one homogenate for

endoglucanase assays and another for exoglucanse and beta-glucosidase assays.

For the endoglucanase assays, the following formula was used to calculate specific

activities;

SA = Cs[(A-Ao)/t]NT

where: SA = specific activity (nmol reducing sugar per termite equivalent per min), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after

5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.

For the exoglucanase and beta-glucosidase assays, the following formula was used to

calculate specific activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA

mean velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard

(nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample.

The field diet data were analyzed using a mixed model analysis of variance. Fixed effects

were diet treatment and gut region. The Tukey-Kramer adjustment (c = 0.05) was used to

separate the mean activities on each diet within each gut region (SAS Institute 2001).

Results

Termite Gut Observations

During dissection, after the termites had been fed on the various diets, the color of the

termite guts reflected the color of the different diets. This was especially evident in the enlarged









hindguts (Fig. 1), which were typically filled with a mixture of partially digested food and

microbes. Termites fed on red oak had brownish-orange gut contents. Those fed on pine had

pale yellow gut contents. Those fed on paper had white gut contents. The termites collected

from the field and immediately frozen for dissection had relatively dark gut contents.

Endoglucanase Activities

Most of the endoglucanase activity was located in the foregut and the hindgut, with very

little in the midgut (Table 5-1). Foregut endoglucanase activity in the field-collected termites

was slightly lower than the foregut activity seen on paper, and greater than activity seen on the

other two diets (Table 5-1). Among the three field diets, foregut activity was highest on paper

and lowest on oak. Midgut endoglucanase activity in the field-collected termites was higher than

the midgut activity seen on all three diets. Among the three field diets, midgut activity was

highest on pine and lowest on paper. Hindgut endoglucanase activity in the field-collected

termites was higher than the hindgut activity seen on all three diets. Among the three field diets,

hindgut activity was highest on oak and lowest on paper.

Exoglucanase Activities

Foregut exoglucanase activity in the field collected termites was significantly higher than

the foregut activity seen all three diets (Table 5-2). Among the three diets, foregut activity was

highest on paper and lowest on oak. Midgut exoglucanase activity in the field collected termites

was intermediate between the midgut activities seen on oak and pine. Among the three diets,

midgut activity was highest on paper and lowest on oak. Hindgut exoglucanase activity in the

field collected termites was higher than the hindgut activity seen on all three diets. Among the

three diets, hindgut activity was highest on oak and lowest on paper. The activity seen on pine

was nearly as high as that seen on oak.









Beta-glucosidase Activities

Foregut beta-glucosidase activity in the field collected termites was intermediate between

the foregut activities seen on paper and pine (Table 5-3). Among the three diets, foregut activity

was highest on paper and lowest on oak. Midgut beta-glucosidase activity in the field collected

termites was intermediate between the midgut activities seen on paper and pine. Among the

three diets, midgut activity was highest on paper and lowest on oak. Hindgut beta-glucosidase

activity in the field collected termites was higher than the hindgut activity seen on all three diets.

Among the three diets, hindgut activity was highest on oak and lowest on paper.

Discussion

The overall gut morphology is consistent with that described for R. flavipes in previous

studies. The appearance of the termite guts upon dissection, particularly the hindguts, indicated

that the termites had fed on their respective diets. Based on the relatively dark gut contents of

the field termites, as well as the presence of dark oval objects in some cases, it is possible that

these termites were feeding on partially decayed materials.

Based on the observed locations of the activities, it is evident that both endoglucanases and

beta-glucosidases are produced by both the termite and its symbionts. Endogenous

endoglucanases are produced in the foregut, while symbiont endoglucanases are produced in the

hindgut. Beta-glucosidase activities have been seen throughout the termite gut, leading to the

conclusion that endogenous beta-glucosidases are produced in the foregut and midgut, while

symbiont beta-glucosidases are produced in the hindgut.

The overall cellulase activity pattern found in this study was similar to that described by

Inoue et al. (1997) for R. speratus, but the levels of activity are roughly 10 to 100-fold lower in

this study. These different results are due to a number of factors. First, rather than an

exoglucanase assay, Inoue et al. (1997) performed a total cellulase assay, so these data cannot be









directly compared. In addition, Inoue et al. used tetrazolium blue rather than DNSA to determine

the reducing sugars generated by endoglucanase. The advantage of tetrazolium blue is that it is

highly sensitive to reducing sugars. However, tetrazolium blue reacts continuously at room

temperature and the reacted reagent eventually precipitates out of solution. The DNSA reagent

requires boiling to react with reducing sugars. Therefore, it is possible to get more precise

reaction times, reducing the variance of the results. The enzymatic reactions in their study were

conducted at a higher incubation temperature (25C) than ours (23C).

In addition, we cannot directly compare the results because the two assays were carried out

on different species. The Inoue study used R. speratus collected from logs, while our study used

R. flavipes collected from cardboard traps. When these differences in methods are taken into

account, our findings are consistent with those of Inoue et al. (1997).

The overall endoglucanase activities did not differ significantly among the three diets, but

the source of the enzymes did. The activities of both endogenous endoglucanase, produced by

the termite in the foregut and midgut, and symbiotic endoglucanase, produced by microbes in the

hindgut, varied among diets. Endogenous activities were highest on paper and lowest on oak,

but the exact reverse trend was seen with the symbiont endoglucanase activities, which were

highest on the oak diet and lowest on paper. Based upon these data, it is probable that either the

termite is changing its endogenous enzyme output in response to fluctuations in its symbiont

enzymes, or else the symbiont population is fluctuating in response to the levels of endogenous

termite enzymes.

If the termite is responding to changes in its symbiont community composition, then some

qualities of the diet must be driving the symbiont population changes. It has previously been

demonstrated that symbionts are responsible for virtually all of the xylanolytic activity seen in R.









flavipes worker termites (Zhou et al. 2007, Smith and Koehler 2007). It is reasonable to suppose

that the xylan content of a given diet may affect the symbiont populations, and therefore affect

the activity levels of the enzymes produced by these symbionts. All symbiont enzyme activities

were significantly higher on diets with a higher xylan content. Hindgut activities in all cases

were the highest on the oak diet. Hardwoods typically contain more than twice as much xylan as

softwoods (Pettersen 1984). At the same time, hindgut activities were lowest on the paper diet, a

diet completely lacking in xylan.

Alternatively, the quality of the cellulose content in the diet may be affecting the termite's

ability to digest it without the aid of symbionts. While the termite produces its own

endoglucanases and beta-glucosidases, it relies on its symbionts for at least the majority of its

exoglucanase production. This means that the termite is most likely capable of degrading

amorphous cellulose, but relies heavily upon its symbionts to digest crystalline cellulose. The

proportion of crystalline cellulose may be anywhere from 50% to 70% of total cellulose content

in different wood species (Biermann 1996). The process of making paper partially denatures the

cellulose microfibers in wood, and may increase the ratio of amorphous cellulose to crystalline

cellulose. The increase in endogenous cellulolytic activities on the paper diet may therefore be

due to greater availability of substrates that the termite can digest without the aid of its

symbionts. With more of this digestion and absorption achieved before the food reaches the

hindgut, the resident symbiont population may be reduced by the more limited nutrient

availability.

Studies on Reticulitermes speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes

virginicus (Cook and Gold 2000) and Coptotermesformosanus (Mannesmann 1972, Waller and

La Fage 1987) have demonstrated significant changes in the hindgut protozoan communities in









response to different diets, including different wood species, pure cellulose and pure xylan. It is

probable that the hindgut enzyme changes in R. flavipes are due to changes in the protozoan

populations. Alternatively, there is the possibility that individual termite symbionts change their

levels of enzyme production in response to differing diets.

It is apparent that R. flavipes workers are quite capable of digesting cellulose, following the

same pattern seen in Chapter 4. Crystalline cellulose is mainly digested by hindgut symbionts

while amorphous cellulose and cellodextrins are digested by both the termites and its symbionts.

In addition, the balance of endogenous versus symbiont cellulolytic activities appears to change

in response to their diet, most likely by changes in the hindgut protozoan communities as well as

the termite enzyme expression. This flexibility allows them to efficiently utilize a variety of

wood species and wood-derived materials which have different qualities of cellulose. This

capacity for adaptation and partial balancing between the termite and its symbionts also makes

termite control by means of cellulase inhibition more difficult.









Table 5-1. Endoglucanase activities in the three gut regions of Reticulitermesflavipes workers in
response to different field diets
Diet Foregut Midgut Hindgut
Field 9.286 + 0.165a 0.471 + 0.107a 8.187 + 0.938a
Paper 9.649 + 0.246a 0.216 + 0.102a 5.789 + 0.610c
Pine 8.311 + 1.048ab 0.328 + 0.098a 6.765 + 0.166bc
Oak 7.438 + 0.664b 0.291 + 0.185a 7.663 + 0.842ab
Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n= 4 replicates.









Table 5-2. Exoglucanase activities in the three gut regions of Reticulitermesflavipes workers in
response to different field diets
Diet Foregut Midgut Hindgut
Field 0.0121 + 0.0004a 0.0062 + 0.001 Ibc 0.1654 + 0.0026a
Paper 0.0062 + 0.0006b 0.0083 + 0.0002a 0.1011 + 0.006d
Pine 0.0044 + 0.0004c 0.0078 + 0.0002ab 0.1486 + 0.0032c
Oak 0.0031 + 0.0008c 0.0054 + 0.0002c 0.1554 + 0.0032b
Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed
by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05, SAS
Institute 2001), n = 4 replicates.









Table 5-3. Beta-glucosidaase activities in the three gut regions ofReticulitermesflavipes
workers in response to different field diets
Diet Foregut Midgut Hindgut
Field 0.1196 + 0.0017b 0.2128 + 0.0016a 0.3229 + 0.0026a
Paper 0.1681 + 0.0016a 0.2161 + 0.0036a 0.2289 + 0.0067d
Pine 0.0991 + 0.0023c 0.1725 + 0.0056b 0.2674 + 0.0077c
Oak 0.0888 + 0.0015d 0.1445 + 0.0026c 0.3037 + 0.0042b
Beta-glucosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n = 4 replicates.









CHAPTER 6
CHANGES IN RETICULITERMES FLA VIPES GUT XYLANOLYTIC ACTIVITIES IN
RESPONSE TO DIET

Introduction

Subterranean termites, such as Reticulitermesflavipes (Kollar), subsist largely on a diet of

wood and similar material (Noirot and Noirot-Timothee 1969). Wood is not a substance that

most animals are capable of digesting to any significant degree. Termites are well known for the

ability to digest cellulose, but there are other polysaccharides in wood, such as xylan, which the

termites may utilize in order to get more energy out of this food source.

In wood, cellulose chains are typically arranged in parallel bundles known as microfibers,

which are embedded in a matrix composed mainly of hemicelluloses and lignin. Hemicelluloses

are polysaccharides like cellulose, but their chemical structures are far more variable. One of the

well-studied varieties of hemicellulose is xylan, which is mainly a polymer of the pentose sugar

xylose. Xylan is the predominant hemicellulose in hardwood. Softwood typically is much lower

in xylan content, along with significant quantities of other hemicelluloses (Pettersen 1984).

Subterranean termites may encounter a number of potential food sources during foraging.

These may be broadly grouped into hardwoods, softwoods, and man-made cellulosic materials

like paper. These three groups vary mainly in their xylan content, with hardwoods containing

the most xylan while man-made cellulosic materials contain virtually no hemicellulose. In

homes, hardwoods may include furniture, flooring, and trim components. Structural timbers are

nearly always derived from softwoods.

The gut ofR. flavipes and related termites consists of a foregut with a small crop and a

proventriculus, a fairly simple midgut and a greatly expanded hindgut containing several species

of symbiotic protozoa, fungi, bacteria, and archaea (Yamin 1979, Lewis and Forschler 2004). A

pair of salivary glands is also present, emptying into the foregut anterior to the crop. To









effectively digest wood, a chemically demanding and nutritionally poor substrate, termites like

R. flavipes have developed an array of enzymes. Some carbohydrolases characterized from the

Rhinotermitidae have been shown to be endogenous (Watanabe et al. 1998), while others are

produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al.

2000). Xylanolytic enzymes include xylanases (EC 3.2.1.8) which internally cleave the xylan

chain and beta-xylosidases (EC 3.2.1.37) which cleave xylan oligosaccharides into xylose. Inoue

et al. (1997) demonstrated that xylanase activity is largely localized to the hindgut of

Reticulitermes speratus (Kolbe), strongly implicating the resident protozoa as the major agents

of xylan digestion. In the same study, they showed that the protozoan composition of the R.

speratus hindgut significantly changes when the termites are fed on pure cellulose or pure xylan,

as opposed to wood. Through similar experiments in differential feeding, Azuma et al. (1993)

showed that, within the termite R. speratus, protozoan symbionts in the genera Pyrsonympha and

Dinenympha take part in xylan digestion. It is probable that, with the changes in protozoan

populations, the capacity for xylan digestion may also change to adapt to the xylan content of the

termite diet.

Our objectives were to determine 1) if R. flavipes can digest xylan, 2) the relative activities

of xylanase and beta-xylosidase in different regions of the gut of R. flavipes workers, 3) possible

correlations between these activities and dietary xylan content in five formulated cellulose/xylan

diets, and 4) how these activities change in response to three different field diets.

Materials and Methods

Termite Collection

R. flavipes termites were field collected in Gainesville, Florida in termite traps consisting

of a PVC bucket (20 cm high by 20 cm diam.; Item # 811192-4, Ventura Packaging Inc.,

Monroeville, Ohio) with 11 holes drilled in the sides and base (3 cm diam.) placed vertically in









the ground to a depth of roughly 19 cm and covered with a PVC lid. Three rolls of single-faced

corrugated cardboard (20 cm long by 10 cm diam.) were placed into the bucket side by side as a

food source. Termites were collected from the trap by removal of cardboard rolls, separated

from the cardboard, and either placed in feeding bioassays or immediately frozen and kept at -

80C until dissection. Termites were collected from one colony for field diet assays and another

colony for formulated diet assays. Collections were restricted to a single colony in each case to

eliminate colony as a source of variation in enzyme activities.

Termite Diets and Feeding

Five formulated diets consisting of microcrystalline cellulose and beechwood xylan (>90%

xylose residues) (Sigma-Aldrich, Atlanta, GA) were prepared for the termites: 0%, 5%, 10%,

20%, and 40% xylan. The remainder of the diet was composed of cellulose in all cases. The

cellulose and xylan were mixed together and water (1.5 mL/g) was added to make a paste. This

paste was thoroughly kneaded to achieve homogeneity.

For each formulated diet, a glass jar (125 mL, straight-sided, Fisherbrand, Fisher

Scientific, Pittsborough, PA) was prepared with 80 g of moist builders sand (10% moisture).

The bottom was cut out of a plastic cup (5.92 mL Souffle cup, Solo Cup Company, Urbana, IL),

leaving approximately 2 mm of cup wall intact. Formulated diet (1 g) was placed in the cup

bottom and this was placed on top of the sand in the jar. Termites were added (-200 workers

and 2 soldiers per jar) and kept in the dark at 21C for 2 wk. Termites were collected, frozen and

kept at -80C until dissection.

Three diets were prepared for the termites to represent the probable food sources they

would encounter in the field. These field diets were as follows: red oak (Quercus spp.), pine

(Pinus spp.), and filter paper. Wood diets were generated by drilling into craft wood boards

(0.635 x 5.08 x 60.96 cm, TOMS) with a 2.54 cm spade drill bit. Sawdust was collected and









weighed. Filter paper consisted of a weighed number of crumpled cellulose filter paper disks

(42.5 mm diameter, Whatman, grade 4).

Each field diet (20 g) was added to a loosely capped 250 mL glass bottle (Pyrex) with 5

mL of deionized water. Unlike the formulated diets, the field diets were not made from sterile

materials. Because of this, the bottles with field diets were autoclaved on a liquid cycle (30 min,

122C) to sterilize the diets and the bottles were then allowed to cool. Termites were added

(-300 workers and 3 soldiers per bottle) after the bottles had cooled and kept in the dark at 21C

for 6 wk, with deionized water (2 mL) added every 2 wk. Termites were collected, frozen and

kept at -80C until dissection.

Termite Dissection and Enzyme Extraction

Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5,

and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the xylanase

assay, while sodium phosphate buffer was used for the beta-xylosidase assays.

Termites were removed from the freezer and kept on ice until dissection. Each termite's

gut was removed intact and separated into three regions: foregut (and salivary glands), midgut,

and hindgut. A single enzyme extract was prepared from dissected termites from each feeding

treatment for each assay type using an experimental design similar to previous termite

carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For

the xylanase assay, 50 termites from each feeding treatment were dissected in sodium acetate

buffer. For the beta-xylosidase assays, 35 termites from each feeding treatment were dissected in

sodium phosphate buffer. The three gut regions were placed into separate 1.5 mL

microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ice. Final

concentrations were 50 termite gut regions per mL in all cases.









Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of

each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and

manually homogenized on ice. The homogenates were centrifuged at 20,800 g at 40C for 15

min. The supernatants were collected, frozen, and kept at -800C until use in the enzyme assays.

Xylanase Assays

The xylanase assays were conducted using a method adapted from Han et al. (1995). A

0.5% solution ofbeechwood xylan (>90% xylose residues; Sigma-Aldrich) was prepared in 0.1

M sodium acetate buffer, pH 5.5. The solution was boiled approximately 30 min, until xylan

particles were no longer visible. The solution was centrifuged at 1250 g for 5 min at 230C and

the supernatant was used as the xylan stock solution.

Assays were conducted in clear 96-well microplates. In each well, 10 pL of tissue extract

was combined with 90 pL of xylan solution. The solutions were allowed to react for 35 min at

23C. DNSA solution (100 [LL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each

microplate was read at 540 nm using a [[Quant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 5 min to allow

for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 5 min

reaction was used as a control to correct for any differences in initial sample reaction rates due to

slow mixture of enzyme and substrate solutions. Standards were generated using dilutions of

xylose. Only the wells in the middle of the microplate were used for these assays. Perimeter

wells were filled with deionized water (200 pL per well) to add temperature stability and

consistency during boiling. For all replicates, the control plates were used to adjust for 540 nm









absorbance in gut extracts and were replicated an equal number of times to the assay plates, with

one microplate well for each replicate.

Beta-Xylosidase Assays

Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-P-D-xylopyranoside (pNPX; Sigma-Aldrich) was prepared in

0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In

each well, 10 ptL of tissue extract was combined with 90 p.L of pNPX solution. The reaction was

allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 23C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

Data Analysis

The xylanase assays were set up as one-factor split-plot designs with seven technical

replicates per gut region for each formulated diet, four technical replicates per gut region for

each simulated field diet, and one microplate per replicate. Four microplate wells were assayed

for each replicate of each gut region/diet combination. The beta-xylosidase assays were set up as

one-factor designs with four technical replicates per gut region for each formulated diet and

simulated field diet. Two homogenates were used for each gut/diet combination; one

homogenate for xylanase assays and another for beta-xylosidase assays.

For the xylanase assays, the following formula was used to calculate specific activities;

SA = Cs[(A-Ao)/t]/NT

where: SA = specific activity (nmol reducing sugar per termite equivalent per min), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after









5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.

For the beta-xylosidase assays, the following formula was used to calculate specific

activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA

mean velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard

(nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample.

The field diet data were analyzed using a mixed model analysis of variance. Fixed effects

were diet treatment and gut region. The Tukey-Kramer adjustment (c = 0.05) was used to

separate the mean activities on each diet within each gut region (SAS Institute 2001).

Results

Termite Gut Observations

During dissection, after the termites had been fed on the various diets, the color of the

termite guts reflected the color of the different diets. This was especially evident in the enlarged

hindguts which were typically filled with a mixture of partially digested food and resident

microbes. Termites fed on formulated diets showed an increasing brown shade in their gut

contents on diets containing more xylan. Termites fed on red oak had brownish-orange gut

contents. Those fed on pine had pale yellow gut contents. Those fed on paper had white gut

contents. The termites collected from the field and immediately frozen for dissection had

relatively dark gut contents.

Xylanase Activities

Both foregut and midgut xylanase activities were insignificant in comparison with hindgut

xylanase activity on all of the formulated diets (Figure 6-1). Hindgut xylanase activity was









significantly greater in termites kept on 20% and 40% xylan diets than in termites kept on 0%,

5% and 10% xylan diets (Figure 6-1). Although there was higher activity in termites fed on 5%

xylan than 10% xylan, the difference was not significant.

Foregut xylanase activity was greater in the field-collected termites than activities in

termites fed on any of the three field diets (Table 6-1). Foregut activities on pine and paper diets

differed significantly, with activity on pine being the highest and activity on paper being the

lowest. Midgut xylanase activity in field-collected termites was between the activities on pine

and oak diets. Among the field diets, midgut activity was significantly highest on pine and

lowest on paper. Hindgut xylanase activity was >92% of total xylanase activity among the

different diets. The hindgut activity of field-collected termites was slightly higher than that on

paper, but not significantly so. The hindgut activity differed significantly among the three field

diets, being highest on oak and lowest on paper.

Beta-Xylosidase Activities

Overall beta-xylosidase activity showed a general increase from termites fed 0% xylan to

termites fed 40% xylan (Figure 6-2). There were no significant differences among the foregut

beta-xylosidase activities on the formulated diets. Midgut beta-xylosidase activities were

greatest in termites fed 10% xylan. Activities were intermediate and nearly equal on 5% and

20% xylan, and they were lowest and nearly equal on 0% and 40% xylan. Hindgut beta-

xylosidase activities showed a steady increase on the formulated diets from 5% xylan to 40%

xylan (Figure 6-2). Activities in termites fed 20% and 40% xylan were significantly greater than

those in termites fed 0% and 5% xylan. In addition, beta-xylosidase activities in termites fed

40% xylan were significantly greater than activities in termites fed 10% xylan.

There were no significant differences among the foregut beta-xylosidase activities in

termites fed field diets (Table 6-2). Midgut beta-xylosidase activity was lowest on the field-









collected termites, which did not differ significantly from the activity on pine. Among the diets,

midgut activity was highest on paper and lowest on pine. Similar to xylanase activity, beta-

xylosidase activity was predominantly located in the hindgut. However, this was not as

pronounced as with xylanase, as hindgut beta-xylosidase activity ranged from -50 to 75% of

total beta-xylosidase activity. Hindgut activity of the field-collected termites was intermediate

between the activities on oak and pine diets. Among the field diets, activity was highest on oak

and lowest on paper. Hindgut beta-xylosidase activities on all four of these treatments were

significantly different from one another.

Discussion

The overall gut morphology of R. flavipes workers was similar to that described for

Zootermopsis (Child 1946) and is consistent with that found in other lower termites (Noirot and

Noirot-Timothee 1969). The appearance of the termite guts upon dissection, particularly the

hindguts, indicated that the termites had fed on their respective diets. Based on the relatively

dark gut contents of the field-collected termites, it was apparent that these termites were feeding

on relatively dark materials, such as cardboard or pine bark mulch, associated with the termite

traps.

Among the termites in the colony fed on formulated diets, both xylanase and beta-

xylosidase total activities were significantly higher on diets with higher xylan content. The

hindgut activities in particular, which formed the majority of total activities, also followed this

pattern. A similar pattern was seen in termites from the colony fed on field diets. All

xylanolytic activities were highest on the oak diet. Hardwoods typically consist of roughly 20%

xylan, twice as much as the typical softwood xylan content of roughly 10% (Biermann 1996). At

the same time, hindgut activities were lowest on the paper diet, a diet completely lacking in

xylan. Just as termites fed on 20% xylan showed far greater xylanase activity than termites fed









on 10% xylan, so there was a similar jump in activity from pine fed termites to oak fed termites.

There was a much smaller gap between the hindgut xylanase activities of paper fed and pine fed

termites, as there was a much smaller gap between the activities of termites fed 0% and 10%

xylan. The hindgut beta-xylosidase patterns were also consistent between termites fed on

formulated diets and termites fed on field diets.

Our data showed that the majority of xylanase activity was in the R. flavipes hindgut. This

is consistent with previous findings where xylanase activities were almost exclusively located in

the hindgut of R. speratus and Coptotermes heimi (Wasmann), and associated with the symbionts

(Inoue et al. 1997, Mishra 1991). Therefore, it was evident that R. flavipes workers follow a

typical xylan digestion pattern for subterranean termites, where this hemicellulose is mainly

digested by hindgut symbionts.

The overall digestion pattern of xylanase and beta-xylosidase activity found in our study

using the field termites was similar to that found by Inoue et al. (1997), but the levels of activity

are roughly 10 to 100-fold lower in our study. These differences may be due to differences in

assay reagents or differences in termite species. Inoue et al. (1997) used tetrazolium blue rather

than DNSA to determine the reducing sugars generated by xylanase. We used the DNSA reagent

because it requires boiling to react with reducing sugars, in contrast with tetrazolium blue, which

reacts continuously at room temperature. This boiling requirement allowed more precise control

of reaction times, limiting the overestimation of activity. Our findings are consistent with those

of Inoue et al. (1997) when the differences in methods are taken into account. Inoue et al. (1997)

observed no beta-xylosidase activity in the foregut ofR. speratus, in contrast with our findings of

minor beta-xylosidase activity in the foregut of R. flavipes. We cannot directly compare these

results because the two assays were carried out on different species of termite; the Inoue et al.









(1997) study used R. speratus collected from logs, while our study used R. flavipes collected

from cardboard traps.

The low hindgut xylanolytic activities on the paper diet and the 0% xylan formulated diet

were consistent with the absence of xylan in the paper diet. However, significant xylanase and

beta-xylosidase activities were still present even in the absence of dietary xylan. This suggests

the presence of symbionts that constitutively produce xylanases and beta-xylosidases, but are

capable of subsisting solely on a cellulose diet. The relatively low hindgut xylanolytic activity in

the field-collected termites, similar to the activity on the paper diet, was most likely due either to

feeding on the cardboard in the termite traps, or feeding on some form of softwood such as pine

mulch.

Symbiont xylanase and beta-xylosidase activities change significantly in response to diet,

consistent with xylan content. It is apparent that the community of hindgut symbionts is able to

adapt to a wide range of dietary xylan content. Studies on R. speratus (Azuma et al. 1993, Inoue

et al. 1997), Reticulitermes virginicus (Banks) (Cook and Gold 2000) and Coptotermes

formosanus (Shiraki) (Mannesmann 1972, Waller and La Fage 1987) have demonstrated

significant changes in the hindgut protozoan communities in response to different diets. Many of

these studies focused on feeding termites different wood species. It is therefore probable that the

xylanolytic enzyme changes in R. flavipes are due to changes in hindgut symbiont populations.

Alternatively, it is possible that these enzyme changes are due to changes in xylanolytic enzyme

production within the hindgut symbionts.

R. flavipes workers are capable of digesting xylan, following the same pattern seen in

Chapter 4, where xylan is mainly digested in the hindgut. The xylanolytic activities of the two

termite colonies we investigated changed to accommodate dietary xylan content, most likely by









changes in the hindgut symbiont communities. These termites' capacity for xylan digestion

allows them to gain more energy from a wood diet, and their flexibility allows them to efficiently

utilize diets of varying xylan content.

Compared to cellulose, xylan is often overlooked as a starting compound for the

production of alternative fuels. However, xylan may comprise up to a quarter of wood,

depending on the species, and a significant proportion of other plant materials used for

alternative fuel production. These termites and their symbionts may provide candidate enzymes

for the degradation of xylan in this process.









Table 6-1. Xylanase activities in the three gut regions of Reticulitermesflavipes workers in
response to different field diets
Diet Foregut Midgut Hindgut
Field 0.833 + 0.048a 0.258 + 0.064a 12.672 + 0.249c
Paper 0.062 + 0.044b 0.030 + 0.078a 12.436 + 0.260c
Pine 0.216 + 0.051ab 0.324 + 0.088a 13.672 + 0.335b
Oak 0.017 + 0.045ab 0.196 + 0.052a 22.953 + 0.315a
Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by
the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05, SAS Institute
2001), n= 4 replicates.









Table 6-2. Beta-xylosidase activities in the three gut regions of Reticulitermesflavipes workers
in response to different field diets
Diet Foregut Midgut Hindgut
Field 2.50 + 0.22a 4.08 + 0.21b 21.08 + 0.20b
Paper 2.63 + 0.05a 9.25 + 0.09a 11.91 + 0.32d
Pine 1.82 + 0.23a 5.70 + 0.09b 16.91 + 0.32c
Oak 2.49 + 0.05a 7.04 + 0.32b 28.26 + 1.00a
Beta-xylosidase activities are in pmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n= 4 replicates.




















S- Foregut
o 5 Midgut
5 Hindgut
-5






-5 -

0 5 10 15 20 25 30 35 40

% Xylan in Diet



Figure 6-1. Xylanase activities in different gut regions of Reticulitermesflavipes in response to
dietary xylan content.


.3
E













0.04


0.03

I -

< I 0.02-

a 0

0.01 ,


Foregut
-Midgut
-Hindgut


0 5 10 15 20 25 30 35 40

% Xylan in Diet



Figure 6-2. Beta-Xylosidase activities in different gut regions of Reticulitermesflavipes in
response to dietary xylan content.


I









CHAPTER 7
CHANGES IN COPTOTERMES FORMOSANUS GUT CELLULOLYTIC ACTIVITIES IN
RESPONSE TO DIET

Introduction

Subterranean termites, such as Coptotermesformosanus (Shiraki), subsist largely on a diet

of wood and similar material. Wood is not a substance that most animals are capable of

digesting to any significant degree. Termites are well known for the ability to digest cellulose

with the aid of microbial hindgut symbionts.

In wood, cellulose chains are typically arranged in parallel bundles known as microfibers

which are embedded in a matrix of lignin and hemicelluloses. The cellulose in the microfibers

may be broadly divided into two types: crystalline and amorphous. The crystalline form of

cellulose consists of tightly aligned parallel chains, held in a specific configuration by hydrogen

bonding. In the amorphous form of cellulose, the chains are more randomly arranged, and not so

closely bound together.

There are three major types of cellulase which work together to digest cellulose.

Exoglucanase (EC 3.2.1.91) cleaves the cellulose chain from the ends, typically producing

cellobiose, and is most active against crystalline cellulose. Endoglucanase (EC 3.2.1.4) cleaves

the cellulose chain randomly along its length and is most active against amorphous cellulose.

Beta-Glucosidase (3.2.1.21) cleaves cellobiose and other small cellulose fragments, hydrolyzing

them to glucose.

The gut of C. formosanus and related termites consists of a foregut with a small crop and a

proventriculus, a fairly simple midgut, and a greatly expanded hindgut containing four major

species symbiotic protozoa, as well as several species of bacteria and archaea (Yamin 1979). A

pair of salivary glands is also present, emptying into the foregut anterior to the crop. To

effectively digest wood, a recalcitrant and nutritionally poor substrate, termites like C.









formosanus have developed an array of enzymes. Some of the cellulases characterized from the

Rhinotermitidae have been shown to be endogenous (Watanabe et al. 1998, Zhou et al. 2007),

while others are produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al.

2002, Ohtoko et al. 2000). Zhou et al. (2007) demonstrated that exoglucanase activity is largely

localized to the hindgut of C. formosanus, strongly implicating the resident flagellates as the

major agents of crystalline cellulose digestion. Inoue et al. (1997) showed that the protozoan

composition of the Reticulitermes speratus (Kolbe) hindgut significantly changes when the

termites are fed on pure cellulose or pure xylan, as opposed to wood. It is probable that, with

changes in protozoan populations on differing diets, the levels of different cellulase activities

may also change.

Subterranean termites may encounter a number of potential food sources during foraging.

These may be broadly grouped into hardwoods, softwoods, and processed cellulosic materials

like paper. These three groups vary mainly in their hemicellulose content. In homes, hardwoods

may include furniture, flooring, and trim components. Structural timbers are nearly always

derived from softwoods.

Our objectives were to determine: 1) the distribution of the three major cellulase activities

within the gut of C. formosanus workers and 2) how these activities change in response to three

different simulated field diets.

Materials and Methods

Termite Collection

Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in

Charleston, South Carolina. Termites were collected from the trap by removal of the wood

(southern yellow pine) in the bait stations and brought to the University of Florida. Termites









were then placed in feeding bioassays. Collections were restricted to a single colony to eliminate

colony as a source of variation in enzyme activities.

Termite Diets and Feeding

Three diets were prepared for the termites to represent the probable food sources they

would encounter in the field. These simulated field diets were as follows: red oak (Quercus

spp.), pine (Pinus spp.), and filter paper. Wood diets were generated by drilling into craft wood

boards (0.635 x 5.08 x 60.96 cm) with a 2.54 cm spade drill bit. Sawdust was collected and

weighed. Filter paper consisted of a weighed number of crumpled cellulose filter paper disks

(42.5 mm diameter, Whatman, grade 4).

For each simulated field diet, a mason jar (# mL, brand) was prepared with 40 g of moist

builders sand (10% moisture). Diet (10 g) was then added with 40 mL of deionized water and the

jar was loosely capped. The jars with simulated field diets were autoclaved on a liquid cycle (30

min, 122C) to sterilize the diets and the jars were then allowed to cool. Termites were added

(-270 workers and 30 soldiers per jar) after the jars had cooled and kept in the dark at 210C for 6

wk. Termites were collected, frozen and kept at -800C until dissection.

Termite Dissection and Enzyme Extraction

Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5,

and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the

endoglucanase assay, while sodium phosphate buffer was used for the exoglucanase and beta-

glucosidase assays.

Termites were removed from the freezer and kept on ice until dissection. Each termite's

gut was removed intact and separated into three regions: foregut (and salivary glands), midgut,

and hindgut. A single enzyme extract was prepared from dissected termites from each feeding

treatment for each assay type using an experimental design similar to the recent classic termite









carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For

the endoglucanase assay, 25 termites from each feeding treatment were dissected in sodium

acetate buffer. For the exoglucanase and beta-glucosidase assays, 25 termites from each feeding

treatment were dissected in sodium phosphate buffer. The three gut regions were placed into

separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept

on ice. Final concentrations were 50 termite gut regions per mL in all cases.

Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of

each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and

manually homogenized on ice. The homogenates were centrifuged at 20,800 g at 40C for 15

min. The supernatants were collected, frozen, and kept at -800C until use in the enzyme assays.

Endoglucanase Assays

The endoglucanase assays were conducted using a method adapted from Han et al. (1995).

A 2% solution of carboxymethylcellulose (CMC; Sigma-Aldrich) was prepared in 0.1 M sodium

acetate buffer, pH 5.5.

Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of xylan solution. The solutions were allowed to react for 70 min at

23C. DNSA solution (100 [tL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each

microplate was read at 540 nm using a [tQuant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 10 min to

allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A

10 min reaction was used as a control to correct for any differences in initial sample reaction









rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated

using dilutions of glucose. Only the wells in the middle of the microplate were used for these

assays. Perimeter wells were filled with deionized water (200 p.L per well) to add temperature

stability and consistency during boiling. For all replicates, the control plates were used to adjust

for 540 nm absorbance in gut extracts and were replicated an equal number of times to the assay

plates, with one microplate well for each replicate.

Exoglucanase and Beta-glucosidase Assays

The exoglucanase and beta-glucosidase assay was conducted using a method adapted from

Han et al. (1995). Solutions of 4 mM p-nitrophenyl-P-D-cellobioside (pNPC) and 4 mM p-

nitrophenyl-P-D-glucopyranoside (pNPG) were prepared in 0.1 M sodium phosphate buffer, pH

6.5. Assays were conducted in clear 96-well microplates. In each well, 10 ptL of tissue extract

was combined with 90 ptL of pNPC or pNPG solution. The reaction was allowed to proceed for

10 min before being placed in a [tQuant Universal Microplate Spectrophotometer (Bio-Tek

Instruments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 230C.

Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-

nitrophenol.

Data Analysis

The endoglucanase assays were set up as a one-factor design with four technical replicates

for each gut region. Four microplate wells were assayed for each replicate of each gut

region/diet combination. The exoglucanase and beta-glucosidase assays were set up as one-

factor designs with four technical replicates for each gut region. Two homogenates were used

for each gut/diet combination; one homogenate for endoglucanase assays and another for

exoglucanse and beta-glucosidase assays.









For the endoglucanase assays, the following formula was used to calculate specific

activities;

SA = Cs[(A-Ao)/t]NT

where: SA = specific activity (nmol reducing sugar per termite equivalent per min), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after

5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.

For the exoglucanase and beta-glucosidase assays, the following formula was used to

calculate specific activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA

mean velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard

(nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample.

The field diet data were analyzed using a mixed model analysis of variance. Fixed effects

were diet treatment and gut region. The Tukey-Kramer adjustment (c = 0.05) was used to

separate the mean activities on each diet within each gut region (SAS Institute 2001).

Results

Termite Gut Observations

During dissection, after the termites had been fed on the various diets, the color of the

termite guts reflected the color of the different diets. This was especially evident in the enlarged

hindguts, which were typically filled with a mixture of partially digested food and resident

microbes. Termites fed on red oak had brownish-orange gut contents. Those fed on pine had

pale yellow gut contents. Those fed on paper had white gut contents.









Endoglucanase Activities

Most of the endoglucanase activity was located in the foregut and the hindgut, with

relatively little in the midgut (Table 7-1). Among the three diets, there were no significant

differences in foregut activities. Midgut activities were significantly lower on the paper diet than

on the other two diets. Hindgut activities were significantly higher on the oak diet than on the

other two diets.

Exoglucanase Activities

Most of the exoglucanase activity was located in the hindgut, with very little in the foregut

or midgut (Table 6-2). Hindgut exoglucanase activity was significantly different among all three

diets, being highest on oak and lowest on pine.

Beta-glucosidase Activities

Most of the beta-glucosidase activity was located in the midgut and hindgut, with

relatively little in the foregut (Table 6-3). Foregut beta-glucosidase activity was significantly

different among all three diets, being highest on pine and lowest on paper. Midgut beta-

glucosidase activity was significantly different among all three diets, being highest on pine and

lowest on oak. Hindgut beta-glucosidase activity was significantly different among all three

diets, being highest on oak and lowest on paper.

Discussion

The overall gut morphology observed is consistent with that described for C. formosanus

in previous studies. The appearance of the termite guts upon dissection, particularly the

hindguts, indicated that the termites had fed on their respective diets.

Based on the observed locations of the activities, it is evident that both endoglucanases and

beta-glucosidases are produced by both the termite and its symbionts. Endogenous

endoglucanases are produced in the foregut, and such endoglucanases have been characterized in









a number of termite species from the genera Reticulitermes and Coptotermes (Watanabe et al.

1998, Nakashima et al. 2002b). The endoglucanase activities observed in the hindgut are most

likely derived from the hindgut flagellates, as a number of endoglucanases have been

characterized from these C. formosanus symbionts (Nakashima et al., 2002a; Watanabe et al.,

2002b). Beta-glucosidase activities have been seen throughout the termite gut, leading to the

conclusion that endogenous beta-glucosidases are produced in the foregut and midgut, while

symbiont beta-glucosidases are produced in the hindgut.

In contrast to the other cellulolytic activities, exoglucanase activity appears to be almost

entirely due to the symbionts. This is evident based on the almost exclusive distribution of

exoglucanase activity within the hindgut. Similar patterns have been observed in Coptotermes

lacteus (Hogan et al. 1988).

It is possible that endogenous cellulase expression is inducible by, or at least responsive to,

the quality of dietary cellulose. While the termite produces its own endoglucanases and beta-

glucosidases, it primarily relies on its symbionts for exoglucanase production. This means that

the termite is most likely capable of degrading amorphous cellulose, but relies upon its

symbionts to digest crystalline cellulose. The increase in endogenous cellulolytic activities on

the paper diet may therefore be due to greater availability of substrates, such as amorphous

cellulose, that the termite can digest without the aid of its symbionts.

Symbiont cellulase activities change significantly in response to diet, increasing

significantly in wood as opposed to paper, and in oak as opposed to pine. It is apparent that the

community of hindgut symbionts is able to adapt to a range of diets, with differences in cellulose

quality and hemicellulose content.









Studies on Reticulitermes speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes

virginicus (Cook and Gold 2000) and Coptotermesformosanus (Mannesmann 1972, Waller and

La Fage 1987) have demonstrated significant changes in the hindgut protozoan communities in

response to different diets, including different wood species, pure cellulose and pure xylan. It is

probable that the hindgut enzyme changes in C. formosanus are due to these changes in its

protozoan communities.

It is apparent that C. formosanus workers are quite capable of digesting cellulose,

following the same pattern seen in Chapter 4. Crystalline cellulose is mainly digested by hindgut

symbionts in while amorphous cellulose and cellodextrins are digested by both the termites and

its symbionts. Moreover, the balance of endogenous versus symbiont cellulolytic activities

changes in response to their diet, most likely by changes in the hindgut protozoan communities

as well as the termite enzyme expression. This flexibility allows these termites to efficiently

utilize a variety of wood species and wood-derived materials which have different qualities of

cellulose. This capacity for adaptation also makes termite control by means of cellulase

inhibition more difficult.









Table 7-1. Endoglucanase activities in the three gut regions of Coptotermesformosanus workers
in response to different field diets
Diet Foregut Midgut Hindgut
Paper 4.38 + 0.48a 2.91 + 0.21b 4.85 + 0.77b
Pine 4.52 + 0.52a 2.82 + 0.25b 7.94 + 0.60a
Oak 6.28 + 0.75a 4.06 + 0.36a 8.70 + 0.87a
Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n= 4 replicates.









Table 7-2. Exoglucanase activities in the three gut regions of Coptotermesformosanus workers
in response to different field diets
Diet Foregut Midgut Hindgut
Paper 0.022 + 0.003a 0.099 + 0.002a 0.448 + 0.027b
Pine 0.019 + 0.001a 0.099 + 0.002a 0.371 + 0.036c
Oak 0.018 + 0.002a 0.074 + 0.002b 0.589 + 0.047a
Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed
by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05, SAS
Institute 2001), n = 4 replicates.









Table 7-3. Beta-glucosidaase activities in the three gut regions of Coptotermesformosanus
workers in response to different field diets
Diet Foregut Midgut Hindgut
Paper 0.155 + 0.003c 0.852 + 0.005b 0.489 + 0.005c
Pine 0.221 + 0.003a 0.907 + 0.016a 0.623 + 0.012b
Oak 0.168 + 0.001b 0.770 + 0.010c 0.728 + 0.009a
Beta-glucosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n = 4 replicates.









CHAPTER 8
CHANGES IN COPTOTERMESFORMOSANUS GUT XYLANOLYTIC ACTIVITIES IN
RESPONSE TO DIET

Introduction

Subterranean termites, such as Coptotermesformosanus (Shiraki), subsist largely on a diet

of wood and similar material. Wood is not a substance that most animals are capable of

digesting to any significant degree. Termites are well known for the ability to digest cellulose,

but there are other polysaccharides in wood, such as xylan, which the termites may utilize in

order to get more energy out of this food source.

In wood, cellulose chains are typically arranged in parallel bundles known as microfibers,

which are embedded in a matrix composed mainly of hemicelluloses and lignin. Hemicelluloses

are polysaccharides like cellulose, but their chemical structures are far more variable. One of the

well-studied varieties of hemicellulose is xylan, which is mainly a polymer of the pentose sugar

xylose. Xylan is the predominant hemicellulose in hardwood. Softwood typically is much lower

in xylan content, along with significant quantities of other hemicelluloses (Pettersen 1984).

Subterranean termites may encounter a number of potential food sources during foraging.

These may be broadly grouped into hardwoods, softwoods, and man-made cellulosic materials

like paper. These three groups vary mainly in their xylan content, with hardwoods containing

the most xylan while man-made cellulosic materials contain virtually no hemicellulose. In

homes, hardwoods may include furniture, flooring, and trim components. Structural timbers are

nearly always derived from softwoods.

The gut of C. formosanus and related termites consists of a foregut with a small crop and a

proventriculus, a fairly simple midgut and a greatly expanded hindgut containing four main

species of symbiotic protozoa, as well as several species of bacteria and archaea (Yamin 1979).

A pair of salivary glands is also present, emptying into the foregut anterior to the crop. To









effectively digest wood, a recalcitrant and nutritionally poor substrate, termites like C.

formosanus have developed an array of enzymes. Some carbohydrolases characterized from the

Rhinotermitidae have been shown to be endogenous (Watanabe et al. 1998), while others are

produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al.

2000). Xylanolytic enzymes include xylanases (EC 3.2.1.8) which internally cleave the xylan

chain and beta-xylosidases (EC 3.2.1.37) which cleave xylan oligosaccharides into xylose. Inoue

et al. (1997) demonstrated that xylanase activity is largely localized to the hindgut of

Reticulitermes speratus (Kolbe), strongly implicating the resident protozoa as the major agents

of xylan digestion. In the same study, they showed that the protozoan composition of the R.

speratus hindgut significantly changes when the termites are fed on pure cellulose or pure xylan,

as opposed to wood. Through similar experiments in differential feeding, Azuma et al. (1993)

showed that, within the termite R. speratus, protozoan symbionts in the genera Pyrsonympha and

Dinenympha take part in xylan digestion. It is probable that, with the changes in protozoan

populations, the capacity for xylan digestion may also change to adapt to the xylan content of the

termite diet.

Our objectives were to determine 1) if C. formosanus can digest xylan, 2) the relative

activities of xylanase and beta-xylosidase in different regions of the gut of C. formosanus

workers, 3) possible correlations between these activities and dietary xylan content in five

formulated cellulose/xylan diets, and 4) how these activities change in response to three different

simulated field diets.

Materials and Methods

Termite Collection

Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in

Charleston, South Carolina. Termites were collected from the trap by removal of the wood









(southern yellow pine) in the bait stations and brought to the University of Florida. Termites

were then placed in feeding bioassays. Collections were restricted to a single colony for assays

with formulated diets and a single colony for assays with simulated field diets to eliminate

colony as a source of variation in enzyme activities.

Termite Diets and Feeding

Five formulated diets consisting of microcrystalline cellulose and beechwood xylan (>90%

xylose residues) (Sigma-Aldrich, Atlanta, GA) were prepared for the termites: 10%, 20%, and

40% xylan. The remainder of the diet was composed of cellulose in all cases. The cellulose and

xylan were mixed together and water (1.5 mL/g) was added to make a paste. This paste was

thoroughly kneaded to achieve homogeneity.

For each formulated diet, a glass jar (125 mL, straight-sided, Fisherbrand, Fisher

Scientific, Pittsborough, PA) was prepared with 80 g of moist builders sand (10% moisture).

The bottom was cut out of a plastic cup (5.92 mL Souffle cup, Solo Cup Company, Urbana, IL),

leaving approximately 2 mm of cup wall intact. Formulated diet (1 g) was placed in the cup

bottom and this was placed on top of the sand in the jar. Termites were added (-180 workers

and 20 soldiers perjar) and kept in the dark at 210C for 2 wk. Termites were collected, frozen

and kept at -800C until dissection.

Three diets were prepared for the termites to represent the probable food sources they

would encounter in the field. These field diets were as follows: red oak (Quercus spp.), pine

(Pinus spp.), and filter paper. Wood diets were generated by drilling into craft wood boards

(0.635 x 5.08 x 60.96 cm) with a 2.54 cm spade drill bit. Sawdust was collected and weighed.

Filter paper consisted of a weighed number of crumpled cellulose filter paper disks (42.5 mm

diameter, Whatman, grade 4).









For each field diet, a mason jar (# mL, brand) was prepared with 40 g of moist builders

sand (10% moisture). Field diet (10 g) was then added with 40 mL of deionized water and the jar

was loosely capped. Unlike the formulated diets, the field diets were not made from sterile

materials. Because of this, the jars with field diets were autoclaved on a liquid cycle (30 min,

122C) to sterilize the diets and the jars were then allowed to cool. Termites were added (-270

workers and 30 soldiers per jar) after the jars had cooled and kept in the dark at 21C for 6 wk.

Termites were collected, frozen and kept at -80C until dissection.

Termite Dissection and Enzyme Extraction

Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5,

and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the xylanase

assays, while sodium phosphate buffer was used for the beta-xylosidase assays.

Termites were removed from the freezer and kept on ice until dissection. Each termite's

gut was removed intact and separated into three regions: foregut (and salivary glands), midgut,

and hindgut. A single enzyme extract was prepared from dissected termites from each feeding

treatment for each assay type using an experimental design similar to the recent classic termite

carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For

the xylanase assays, 25 termites from each feeding treatment were dissected in sodium acetate

buffer. For the beta-xylosidase assays, 25 termites from each feeding treatment were dissected in

sodium phosphate buffer. The three gut regions were placed into separate 1.5 mL

microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ice. Final

concentrations were 50 termite gut regions per mL in all cases.

Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of

each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and









manually homogenized on ice. The homogenates were centrifuged at 20,800 g at 40C for 15

min. The supernatants were collected, frozen, and kept at -800C until use in the enzyme assays.

Xylanase Assays

The xylanase assays were conducted using a method adapted from Han et al. (1995). A

0.5% solution ofbeechwood xylan (>90% xylose residues; Sigma-Aldrich) was prepared in 0.1

M sodium acetate buffer, pH 5.5. The solution was boiled approximately 30 min, until xylan

particles were no longer visible. The solution was centrifuged at 1250 g for 5 min at 230C and

the supernatant was used as the xylan stock solution.

Assays were conducted in clear 96-well microplates. In each well, 10 pL of tissue extract

was combined with 90 pL of xylan solution. The solutions were allowed to react for 35 min at

230C. DNSA solution (100 [LL) consisting of 1% 3,5-dinitrosalicylic acid (DNSA), 0.4M sodium

hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

immediately placed in boiling water for 10 min and placed on ice for 15 min. After cooling, each

microplate was read at 540 nm using a [[Quant Universal Microplate Spectrophotometer (Bio-

Tek Instruments, Winooski, VT). Similar control plates were allowed to react for 5 min to allow

for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 5 min

reaction was used as a control to correct for any differences in initial sample reaction rates due to

slow mixture of enzyme and substrate solutions. Standards were generated using dilutions of

xylose. Only the wells in the middle of the microplate were used for these assays. Perimeter

wells were filled with deionized water (200 pL per well) to add temperature stability and

consistency during boiling. For all replicates, the control plates were used to adjust for 540 nm

absorbance in gut extracts and were replicated an equal number of times to the assay plates, with

one microplate well for each replicate.









Beta-Xylosidase Assays

Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A

solution of 4 mM p-nitrophenyl-P-D-xylopyranoside (pNPX; Sigma-Aldrich) was prepared in

0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96-well microplates. In

each well, 10 ptL of tissue extract was combined with 90 p.L of pNPX solution. The reaction was

allowed to proceed for 10 min before being placed in a [tQuant Universal Microplate

Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every

2 min for 30 min at 23C. Mean velocities (mOd/s) were recorded. Standards were generated

using dilutions of p-nitrophenol.

Data Analysis

The xylanase assays were set up as one-factor designs with four technical replicates per gut

region for each formulated diet and each simulated field diet. Four microplate wells were

assayed for each replicate of each gut region/diet combination. The beta-xylosidase assays were

set up as one-factor designs with four technical replicates per gut region for each formulated diet

and simulated field diet. Two homogenates were used for each gut/diet combination; one

homogenate for xylanase assays and another for beta-xylosidase assays.

For the xylanase assays, the following formula was used to calculate specific activities;

SA = Cs[(A-Ao)/t]/NT

where: SA = specific activity (nmol reducing sugar per termite equivalent per min), A =

absorbance (Od) after 35 min reaction, Ao = absorbance (Od) for the corresponding control after

5 min reaction, t = time (min), Cs = the coefficient derived from the standard (nmol reducing

sugar/mOd), and NT = the number of termite equivalents per sample.









For the beta-xylosidase assays, the following formula was used to calculate specific

activities;

SA = 60CsVA/NT

where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA

mean velocity of absorbance change (mOd/s), Cs = the coefficient derived from the standard

(nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample.

The field diet data were analyzed using a mixed model analysis of variance. Fixed effects

were diet treatment and gut region. The Tukey-Kramer adjustment (c = 0.05) was used to

separate the mean activities on each diet within each gut region (SAS Institute 2001).

Results

Termite Gut Observations

During dissection, after the termites had been fed on the various diets, the color of the

termite guts reflected the color of the different diets. This was especially evident in the enlarged

hindguts which were typically filled with a mixture of partially digested food and resident

microbes. Termites fed on formulated diets showed an increasing brown shade in their gut

contents on diets containing more xylan. Termites fed on red oak had brownish-orange gut

contents. Those fed on pine had pale yellow gut contents. Those fed on paper had white gut

contents. The termites collected from the field and immediately frozen for dissection had

relatively dark gut contents.

Xylanase Activities

Both foregut and midgut xylanase activities were insignificant in comparison with hindgut

xylanase activity on all of the formulated diets (Fig. 8-1). Hindgut xylanase activity was

significantly greater in termites kept on 40% xylan diets than in termites kept on 10% and 20%

xylan diets.









Both foregut and midgut xylanase activities were insignificant in comparison with hindgut

xylanase activity on all of the simulated field diets (Table 8-1). Hindgut xylanase activity

differed significantly among all of the three simulated field diets, being highest on oak and

lowest on paper.

Beta-Xylosidase Activities

Hindgut xylanase activity increased from termites fed 10% xylan to termites fed 40%

xylan, differing significantly on all three diets (Fig. 8-2). Midgut beta-xylosidase activities were

highest in termites fed 10% xylan, and were significantly lower in termites fed 40% xylan.

Activities were nearly equal on 10% and 20% xylan.

Hindgut beta-xylosidase activity differed significantly among all of the three simulated

field diets, being highest on oak and lowest on paper (Table 8-2). Midgut beta-xylosidase

activity was significantly greater on pine than on the other two simulated field diets.

Discussion

The overall gut morphology observed here for C. formosanus workers was similar to that

described for Zootermopsis (Child 1946) and is consistent with that found in other lower termites

(Noirot and Noirot-Timothee 1969). The appearance of the termite guts upon dissection,

particularly the hindguts, indicated that the termites had fed on their respective diets.

Among the termites in the colony fed on formulated diets, both xylanase and beta-

xylosidase hindgut activities were significantly higher on diets with higher xylan content. A

similar pattern was seen in termites fed on field diets. All hindgut xylanolytic activities were

highest on the oak diet. Hardwoods typically consist of roughly 20% xylan, twice as much as the

typical softwood xylan content of roughly 10% (Biermann 1996). At the same time, hindgut

activities were lowest on the paper diet, a diet completely lacking in xylan. Just as termites fed

on 40% xylan showed far greater xylanase activity than termites fed on 10% xylan, so there was









a similar jump in activity from pine fed termites to oak fed termites. The hindgut beta-

xylosidase patterns were also consistent between termites fed on formulated diets and termites

fed on field diets.

Our data showed that the majority of xylanase activity was in the C. formosanus hindguts.

This is consistent with previous findings where xylanase activities were almost exclusively

located in the hindgut of R. speratus and Coptotermes heimi (Wasmann), and associated with the

symbionts (Inoue et al. 1997, Mishra 1991). Therefore, it was evident that C. formosanus

workers follow an expected xylan digestion pattern for subterranean termites, in which xylan is

thought to be mainly digested by hindgut symbionts.

The low hindgut xylanolytic activities on the paper diet were consistent with the absence

of xylan. However, significant xylanase and beta-xylosidase activities were still present even in

the absence of dietary xylan. This suggests the presence of symbionts that constitutively produce

xylanases and beta-xylosidases, but are capable of subsisting solely on a cellulose diet.

Symbiont xylanase and beta-xylosidase activities were observed to change significantly in

response to diet, in a manner consistent with xylan content. These findings suggest that the

community of hindgut symbionts is able to adapt to a wide range of dietary xylan content.

Studies on R. speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes virginicus (Banks)

(Cook and Gold 2000) and Coptotermesformosanus (Shiraki) (Mannesmann 1972, Waller and

La Fage 1987) have demonstrated significant changes in the hindgut protozoan communities in

response to different diets. Many of these studies focused on feeding termites different wood

species. It is therefore probable that the xylanolytic enzyme changes in C. formosanus are due to

changes in hindgut symbiont populations. Alternatively, it is possible that these enzyme changes

are due to changes in xylanolytic enzyme production within the hindgut symbionts.









C. formosanus workers are capable of digesting xylan, following the same pattern seen in

Chapter 4, where xylan is mainly digested by in the hindgut. The xylanolytic activities of the

two termite colonies we investigated changed to accommodate dietary xylan content, most likely

by changes in the hindgut symbiont communities. This flexibility allows a termite colony to

efficiently utilize a variety of wood species and wood-derived materials.

Compared to cellulose, xylan is often overlooked as a starting compound for the

production of alternative fuels. However, xylan may comprise up to a quarter of wood,

depending on the species, and a significant proportion of other plant materials used for

alternative fuel production. These termites and their symbionts may provide candidate enzymes

for the degradation of xylan in this process.










Table 8-1. Xylanase activities in the three gut regions of Coptotermesformosanus workers in
response to different field diets
Diet Foregut Midgut Hindgut
Paper 0.14 + 0.06a 0.21 + 0.01a 12.75 + 0.15c
Pine 0.00 + 0.09a 0.20 + 0.03a 17.98 + 0.23b
Oak 0.07 + 0.04a 0.13 + 0.03a 26.95 + 0.45a
Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by
the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05, SAS Institute
2001), n= 4 replicates.









Table 8-2. Beta-xylosidase activities in the three gut regions of Coptotermesformosanus
workers in response to different field diets
Diet Foregut Midgut Hindgut
Paper 0.0020 + 0.0012a 0.0067 + 0.0004b 0.0252 + 0.001 1c
Pine 0.0027 + 0.0009a 0.0127 + 0.0003a 0.0408 + 0.0007b
Oak 0.0020 + 0.0008a 0.0073 + 0.0003b 0.0623 + 0.0008a
Beta-xylosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column
followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, a = 0.05,
SAS Institute 2001), n= 4 replicates.














20


S15
SForegut
S 10 --Midgut
i --Hindgut


0




-5
10 15 20 25 30 35 40

% Xylan in Diet


Figure 8-1. Xylanase activities in different gut regions of Coptotermesformosanus in response
to dietary xylan content.



















Foregut
- Midgut
- Hindgut


10 15 20 25 30 35 40

% Xylan in Diet


Figure 8-2. Beta-xylosidase activities in different gut regions of Coptotermesformosanus in
response to dietary xylan content.


0.05



0.04


0.03



0.02


0.01









CHAPTER 9
CONCLUSION

Termite gut carbohydrolase assays were optimized for buffer and pH, leading to the

selection of a 0.1 M pH 5.5 sodium acetate buffer for endoglucanase and xylanase assays and a

0.1M pH 6.5 sodium phosphate buffer for exoglucanase, beta-glucosidase, and beta-xylosidase

assays. This was in contrast with the almost universal use of 0.1M pH 5.5 sodium acetate buffer

in prior literature.

C. formosanus was found to have a more active array of gut carbohydrolases, particularly

exoglucanase and beta-glucosidase, than R. flavipes. This was consistent with increased

metabolic demands from more aggressive foraging and a larger soldier ratio within C.

formosanus colonies. Soldier carbohydrolase activities were generally lower than worker

carbohydrolase activities, consistent with a caste incapable of feeding itself.

Cellulolytic enzyme activity levels were found to significantly change on differing diets

for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to

change in a manner consistent with dietary xylan content, being increased on diets containing

more xylan. This has demonstrated dietary adaptability in both of these species.

The patterns of cellulolytic and xylanolytic enzyme activities and activity changes on

differing diets were similar between the two species assayed. General findings regarding

cellulose and xylan digestion in one species of Rhinotermitidae may be cautiously applied to the

rest of the family. Our findings suggest a processive endogenous mechanism of amorphous

cellulose degradation with a reliance on the termite symbionts for the digestion of xylan and

crystalline cellulose.

Crystalline cellulose and xylan may make up between 30% and 60% of wood dry weight,

while amorphous cellulose may make up between 15% and 25% of wood dry weight. Therefore,









the termite symbionts are vital to effective wood digestion. In addition, while the endogenous

termite enzymes may be of use in industrial digestion of amorphous cellulose, the symbiont

enzymes should prove far more valuable in the digestion of cellulose and xylan in general.

There is some redundancy with regards to amorphous cellulose digestion in the termites we

have investigated, as both the termites and their symbionts produce endoglucanases and beta-

glucosidases. This could complicate cellulase inhibition efforts. The enzymes responsible for

digestion of crystalline cellulose and xylan are largely produced by the hindgut symbionts, with

little contribution from the termite itself. Although this removes one level of redundancy, the

presence of multiple symbiont species producing differing enzymes may still complicate

cellulase and xylanase inhibition efforts. However, these very redundancies could make termites

a robust system to adapt for the industrial degradation of cellulosic waste.

Compared to cellulose, xylan is often overlooked as a starting compound for the

production of alternative fuels. However, xylan may comprise up to 25% of wood dry weight,

depending on the species, as well as a significant portion of other plant materials used for

alternative fuel production. The termites and termite symbionts investigated in this dissertation

may provide enzymes for the degradation of xylan in this process.

The balance of endogenous versus symbiont cellulolytic activities appears to change in

response to diet in both species, most likely by changes in the hindgut protozoan communities as

well as the termite enzyme expression. This capacity for adaptation and partial balancing

between the termite and its symbionts could make termite control by means of cellulase

inhibition more difficult, but may eventually yield a mechanism to increase efficiency in

industrial cellulose degradation.









The xylanolytic activities of the two termite species investigated changed to accommodate

dietary xylan content, most likely by changes in the hindgut symbiont communities. These

termites' capacity for xylan digestion allows them to gain more energy from a wood diet, and

their adaptability allows them to efficiently utilize diets of varying xylan content. This

adaptability could make termite xylanase inhibition difficult, but it may provide a means for

effective xylan degradation on an industrial scale using living termites or termite symbionts.

The enzymatic mechanisms of wood digestion in these subterranean termite pests are both

complex and effective. A greater understanding of these mechanisms may open new avenues in

safer termite control, and may certainly improve processes for recycling cellulosic waste and

development of alternative fuels.









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BIOGRAPHICAL SKETCH

Joseph Anthony Smith was born in Bremerton, Washington, October 14, 1977, the

youngest of six children. After graduating from Olympic High School in 1993, he entered the

baccalaureate program at Brigham Young University in Provo, Utah. He earned a Bachelor of

Science degree in zoology with an entomology emphasis in 1996.

After entering the doctoral program in the University of Washington Department of

Zoology in 2000, he transferred to the University of Florida Department of Entomology and

Nematology in 2002. He married Jessica Awad in August of 2006, and has been involved in a

variety of research, teaching and extension projects during the pursuit of his PhD in entomology.





PAGE 1

1 CELLULOLYTIC AND XYLANOLYTIC GUT ENZYME ACTIVITY PATTERNS IN MAJOR SUBTERRANEAN TERMITE PESTS By JOSEPH ANTHONY SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Joseph Anthony Smith

PAGE 3

3 To my family, friends and teachers, without w hom this degree would not have been possible

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank the fo llowing people and institutions for their support. I thank Procter and Gamble for the funding that allowed me to conduct this research. I thank my committee, Dr. Phil Koehler, Dr. Mike Scharf, Dr Lonnie Ingram, and Dr. Phil Brode, for their input and feedback on my research. I thank Br uce Ryser for supplying Formosan subterranean termites for my research. I thank my fellow student s, and Cynthia Tucker in particular, for their intelligent input and collaboration. I thank my family and friends for their moral support. Finally, I would like to especia lly thank Dr. Phil Koehler for hi s efforts as my advisor and committee chair.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............11 INTRODUCTION................................................................................................................... ......13 LITERATURE REVIEW..............................................................................................................15 Wood Molecular Structure.....................................................................................................15 Termite Taxonomy............................................................................................................... ..16 Termite Digestive System Anatomy.......................................................................................17 Symbiont Taxonomy and Anatomy........................................................................................18 Symbiont Assemblages in Termite Hindguts.........................................................................19 Termite Carbohydrolase Types and Locations.......................................................................20 Termite Carbohydrolase Structural and Functional Characteristics.......................................21 BUFFER AND PH OPTIMIZATION FOR TERMITE CARBOHYDROLASE ASSAYS.........28 Introduction................................................................................................................... ..........28 Materials and Methods.......................................................................................................... .29 Termite Collection, Dissection, and Enzyme Extraction................................................29 Buffer Optimization Assays............................................................................................29 Gut Region Assays Across PH........................................................................................31 Data Analysis.................................................................................................................. .32 Results........................................................................................................................ .............33 Buffer Optimization Assays............................................................................................33 Gut Region pH Optimization Assays..............................................................................34 Discussion..................................................................................................................... ..........34 Activity Magnitudes........................................................................................................34 Activity Changes with pH...............................................................................................35 Differences in Gut Regions.............................................................................................35 Conclusions.................................................................................................................... .36 CARBOHYDROLASE ACTI VITY PATTERNS IN RETICULITERMES FLAVIPES AND COPTOTERMES FORMOSANUS WORKERS AND SOLDIERS.......................................43 Introduction................................................................................................................... ..........43 Materials and Methods.......................................................................................................... .44 Termite Collection, Dissection, and Enzyme Extraction................................................44 Termite Photography.......................................................................................................45

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6 Soluble Protein Assays....................................................................................................45 Endoglucanase Assays.....................................................................................................45 Exoglucanase Assays.......................................................................................................46 Beta-Glucosidase Assays.................................................................................................46 Xylanase Assays..............................................................................................................47 Beta-Xylosidase Assays..................................................................................................48 Data Analysis.................................................................................................................. .48 Results........................................................................................................................ .............49 Termite Gut and Frontal Gland Comparisons.................................................................49 Worker Cellulolytic Activities........................................................................................49 Worker Xylanolytic Activities........................................................................................50 Soldier Cellulolytic Activities.........................................................................................50 Soldier Xylanolytic Activities.........................................................................................50 Gut Soluble Protein Levels..............................................................................................51 Discussion..................................................................................................................... ..........51 CHANGES IN RETICULITERMES FLAVIPES GUT CELLULOLYTIC ACTIVITIES IN RESPONSE TO DIET............................................................................................................63 Introduction................................................................................................................... ..........63 Materials and Methods.......................................................................................................... .64 Termite Collection...........................................................................................................64 Termite Diets and Feeding..............................................................................................65 Termite Dissection and Enzyme Extraction....................................................................65 Endoglucanase Assays.....................................................................................................66 Exoglucanase and Beta-glucosidase Assays...................................................................67 Data Analysis.................................................................................................................. .67 Results........................................................................................................................ .............68 Termite Gut Observations...............................................................................................68 Endoglucanase Activities................................................................................................69 Exoglucanase Activities..................................................................................................69 Beta-glucosidase Activities.............................................................................................70 Discussion..................................................................................................................... ..........70 CHANGES IN RETICULITERMES FLAVIPES GUT XYLANOLYTIC ACTIVITIES IN RESPONSE TO DIET............................................................................................................77 Introduction................................................................................................................... ..........77 Materials and Methods.......................................................................................................... .78 Termite Collection...........................................................................................................78 Termite Diets and Feeding..............................................................................................79 Termite Dissection and Enzyme Extraction....................................................................80 Xylanase Assays..............................................................................................................81 Beta-Xylosidase Assays..................................................................................................82 Data Analysis.................................................................................................................. .82 Results........................................................................................................................ .............83 Termite Gut Observations...............................................................................................83

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7 Xylanase Activities..........................................................................................................83 Beta-Xylosidase Activities..............................................................................................84 Discussion..................................................................................................................... ..........85 CHANGES IN COPTOTERMES FORMOSANUS GUT CELLULOLYTIC ACTIVITIES IN RESPONSE TO DIET............................................................................................................93 Introduction................................................................................................................... ..........93 Materials and Methods.......................................................................................................... .94 Termite Collection...........................................................................................................94 Termite Diets and Feeding..............................................................................................95 Termite Dissection and Enzyme Extraction....................................................................95 Endoglucanase Assays.....................................................................................................96 Exoglucanase and Beta-glucosidase Assays...................................................................97 Data Analysis.................................................................................................................. .97 Results........................................................................................................................ .............98 Termite Gut Observations...............................................................................................98 Endoglucanase Activities................................................................................................99 Exoglucanase Activities..................................................................................................99 Beta-glucosidase Activities.............................................................................................99 Discussion..................................................................................................................... ..........99 CHANGES IN COPTOTERMES FORMOSANUS GUT XYLANOLYTIC ACTIVITIES IN RESPONSE TO DIET..........................................................................................................105 Introduction................................................................................................................... ........105 Materials and Methods.........................................................................................................106 Termite Collection.........................................................................................................106 Termite Diets and Feeding............................................................................................107 Termite Dissection and Enzyme Extraction..................................................................108 Xylanase Assays............................................................................................................109 Beta-Xylosidase Assays................................................................................................110 Data Analysis.................................................................................................................110 Results........................................................................................................................ ...........111 Termite Gut Observations.............................................................................................111 Xylanase Activities........................................................................................................111 Beta-Xylosidase Activities............................................................................................112 Discussion..................................................................................................................... ........112 CONCLUSION..................................................................................................................... .......119 LIST OF REFERENCES.............................................................................................................122 BIOGRAPHICAL SKETCH.......................................................................................................126

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8 LIST OF TABLES Table page 4-1 A comparison of endoglucanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers.......................................................................................55 4-2 A comparison of exoglucanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers.......................................................................................56 4-3 A comparison of beta -glucosidase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers.................................................................57 4-4 A comparison of xylanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers.......................................................................................58 4-5 A comparison of beta -xylosidase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers.................................................................59 4-6 A comparison of soluble protein concentrations in Reticulitermes flavipes and Coptotermes formosanus worker and soldier gut extracts.................................................60 5-1 Endoglucanase activities in the three gut regions of Reticulitermes flavipes workers in response to different field diets......................................................................................74 5-2 Exoglucanase activities in the three gut regions of Reticulitermes flavipes workers in response to differe nt field diets..........................................................................................75 5-3 Beta-glucosidaase activities in the three gut regions of Reticulitermes flavipes workers in response to different field diets........................................................................76 6-1 Xylanase activities in the three gut regions of Reticulitermes flavipes workers in response to differe nt field diets..........................................................................................89 6-2 Beta-xylosidase activitie s in the three gut regions of Reticulitermes flavipes workers in response to different field diets......................................................................................90 7-1 Endoglucanase activities in the three gut regions of Coptotermes formosanus workers in response to different field diets......................................................................102 7-2 Exoglucanase activities in the three gut regions of Coptotermes formosanus workers in response to different field diets....................................................................................103 7-3 Beta-glucosidaase activities in the three gut regions of Coptotermes formosanus workers in response to different field diets......................................................................104 8-1 Xylanase activities in the three gut regions of Coptotermes formosanus workers in response to differe nt field diets........................................................................................115

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9 8-2 Beta-xylosidase activitie s in the three gut regions of Coptotermes formosanus workers in response to different field diets......................................................................116

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10 LIST OF FIGURES Figure page 2-1 Inverting cellulase mechanism...........................................................................................26 2-2 Retaining cellulase mechanism..........................................................................................27 3-1 Beta-glucosidase activities in Reticulitermes flavipes workers across pH and buffer.......38 3-2 Endoglucanase activities in different gut regions of Reticulitermes flavipes workers across pH...................................................................................................................... ......39 3-3 Beta-Glucosidase activities in differe nt gut regions of Reticulitermes flavipes workers across pH..............................................................................................................40 3-4 Hindgut exoglucanase activities in Reticulitermes flavipes workers across pH................41 3-5 Hindgut xylanase activities in Reticulitermes flavipes workers across pH........................42 4-1 Digestive systems of: A) R. flavipes worker, B) C. formosanus worker, C) R. flavipes soldier, and D) C. formosanus soldier...............................................................................61 4-2 Heads and frontal glands of a R. flavipes soldier, and a C. formosanus soldier................62 6-1 Xylanase activities in different gut regions of Reticulitermes flavipes in response to dietary xylan content..........................................................................................................91 6-2 Beta-Xylosidase activitie s in different gut regions of Reticulitermes flavipes in response to dietary xylan content.......................................................................................92 8-1 Xylanase activities in different gut regions of Coptotermes formosanus in response to dietary xylan content........................................................................................................117 8-2 Beta-xylosidase activitie s in different gut regions of Coptotermes formosanus in response to dietary xylan content.....................................................................................118

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CELLULOLYTIC AND XYLANOLYTIC GUT ENZYME ACTIVITY PATTERNS IN MAJOR SUBTERRANEAN TERMITE PESTS By Joseph Anthony Smith August 2007 Chair: Philip G. Koehler Major: Entomology and Nematology Cellulolytic and xylanolytic termite gut car bohydrolases were assayed for two major subterranean termite pest species; Coptotermes formosanus (Shiraki) and Reticulitermes flavipes (Kollar). Carbohydrolase assays were optimized for buffer and pH. This led to the selection of a 0.1 M pH 5.5 sodium acetate buffer for endoglucan ase and xylanase assays and a 0.1M pH 6.5 sodium phosphate buffer for exoglucanase, beta -glucosidase, and beta-xylosidase assays. Endoglucanase activity was found to be mainly localized in the foregut and hindgut of both species, reflecting both endogenous and symbiont enzyme origins. Beta-glucosidase activity was found throughout the gut in both species, but was mo st prevalent in the midgut or hindgut. Betaxylosidase activity was mostly re stricted to the hindgut and was lowest in the foregut in both species. Exoglucanase and xylanas e activities were almost entire ly confined to the hindgut in both species. C. formosanus workers were found to have a more active array of gut carbohydrolases, particularly exoglucanase a nd beta-glucosidase, than R. flavipes This was consistent with increased metabolic demands from more aggressive foraging and a larger soldier ratio within C. formosanus colonies. Soldier carbohydrol ase activities in bo th of these species were generally

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12 lower than worker carbohydrolase ac tivities, consistent with a caste incapable of feeding itself. C. formosanus soldier carbohydrolase activitie s were especially low, due to a reduced gut size to make room in the soldier abdomen for an enlarged frontal gland. Cellulolytic enzyme activity levels were found to significantly ch ange on differing diets for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to change in a manner consistent with dietary xylan content in both species, being increased on diets containing more xylan. This indicates some adaptability to dietary xylan content in both species. The presence of significant xylanolytic enzyme activities in termites kept on diets without xylan indicates constitutive production of xylanases by symbionts capable of subsisting wholly on cellulose. Alternatively, it is possible that the xylanolytic enzymes seen on xylan-free diets are bifunctional enzymes that al so have cellulolytic activities.

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13 CHAPTER 1 INTRODUCTION Two of the most economically significant pest termites in North America are the eastern subterranean termite, Reticulitermes flavipes (Kollar), and the Formosan subterranean termite, Coptotermes formosanus (Shiraki). One thing that distingu ishes termites from the majority of other insect pests is their ability to digest wood, and their conseque nt ability to cause significant structural damage to most types of buildings. This capacity for wood digestion is based upon a complex array of enzymes, mainly carbohydrolases, which allow termites to digest cellulose and hemicelluloses such as xylan. In all lower termites, including subterranean termites, some of these enzymes are endogenously produced, while others are produced by symbiotic flagellates found in the physiologically specialized, expanded hindgut. Perhaps more important than their status as st ructural pests is the enormous impact that termites have on terrestrial ecological processe s. Termites are among the major terrestrial recyclers of cellulose, on e of the most abundant substances in nature. Finding a way to disrupt or harness the enzymatic mechanisms that allow subterranean termites to digest cellulose and other polysaccharides may be a key to discove ring new approaches in termite control or industrial methods to generate alternative fuel s from cellulosic materials. As such, an understanding of the termite digestive system has significant applications. The first objective in this di ssertation is the optimization of the chosen carbohydrolase assays, which will be dealt with in Chapter 3. The next objectives concern the characterization of the activity levels of the various cellulolytic and xylanolytic gut enzymes, which will be dealt with in Chapter 4. The patte rns of activity for each carbohyd rolase along the gut must be determined, as well as the differences seen between the worker and soldier castes for R. flavipes and C. formosanus This will provide insight into the mechanisms of cellulose and xylan

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14 digestion for these two species of termites, a llowing a better understand ing of the interaction between the termites and their symbionts, as well as the interaction between the termite workers and soldiers. This understanding may facilitate the development of successful termite control strategies, or the discovery of novel enzyme s for industrial polysa ccharide degradation. Once these basic patterns of activ ity have been determined, the next main objective is the exploration of how these pattern s are affected by changes in termite diet, and whether the termites can adapt to their diets. Changes in patterns of R. flavipes worker cellulolytic and xylanolytic enzyme activities will be addres sed in Chapter 5 and Chapter 6, respectively. Changes in patterns of C. formosanus worker cellulolytic and xylanolytic enzyme activities will be addressed in Chapter 7 and Chapter 8, respectiv ely. In particular, the changes in xylanolytic enzyme activities across diets of varying xylan content will provide insight into the adaptive abilities of these termites with respect to diet In turn, this will pr ovide some idea of how termites may adapt to attempts to inhibit th eir digestive processes. Furthermore, any understanding of termite adaptation to diet would be useful if w hole, living termites were to be incorporated into industri al processes involving cellulo se and xylan degradation. The final aim of this dissertation is to facili tate an understanding of lower termite digestive processes, both in terms of enzyme activity pa tterns and adaptation to di fferent diets. This understanding may be used for development of cont rol strategies, or for the harnessing of these termites and their enzymes for the removal of ce llulosic waste and the ge neration of alternative fuels.

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15 CHAPTER 2 LITERATURE REVIEW Wood Molecular Structure The majority of wood consists of polysaccharides and lignin. The dominant polysaccharide is cellulose, which exists within the plant cell walls as microfibers roughly 5 nm in diameter (Astley et al. 1997). Each fiber cons ists of multiple chains of cellulose, which are largely aligned in tight, crystalline configuratio ns. These configurations make this molecule particularly difficult to digest. There are amorphous regions as we ll, where the configuration is disrupted. It is generally thought that these amorphous regions are cause d by interactions with other molecules in the cell walls. Each cell wa ll has several layers, cal led lamellae, roughly one microfiber thick. The microfibers in each layer are roughly parallel, but their orientation changes between layers. The surrounding matrix of the cell wall cont ains other polysacchar ides, predominantly hemicelluloses, and lignin, a non-repeating aroma tic polymer (Whistler and Chen 1991). Unlike cellulose, which is made up of beta-glucose un its, hemicelluloses contain a variety of sugar subunits. In addition, hemicellulases are much shor ter, only a few hundred units long, with short side chains (Timell 1964). These traits mean that they do not exist as mi crofibers, but instead form part of the matrix surrounding the cellulose mi crofibers. In some models, the ends of the hemicellulose chains are aligned with the cellulose microfibers while the bulk of the chains form cross-bridges between the microfibers (Whi stler and Chen 1991). The majority of hemicelluloses in hardwoods consist of modified polymers of xylose, commonly called xylans. Softwoods contain other hemicelluloses in addition to xylans, which occur in roughly comparable amounts. These are known as glucom annans and galactoglucom annans, and they are mainly polymers of mannose and glucose.

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16 Lignin is a highly complex, cross-linked polymer It is fundamentally different from the polysaccharides, as it is a polymer of various hydroxycinnamyl alcohols. In addition, the overall structure is far more amorphous and less predicta ble than that of polysaccharides. A limited degree of degradation of aromatic monomers ha s been demonstrated in the hindgut of lower termites (Brune et al. 1995). However, it has be en difficult to obtain evid ence of degradation of polymerized lignin in termites. Based on the high lignin content of fecal material and nest carton in multiple termite genera (Mishra and Sen-Sarma 1979), it may be assumed that lignin degradation is limited in most termites, and certainly in lower termites. As a result, the relevance of lignin to termite digestion is fairly limited and this paper will focus on the degradation of cellulose and hemicelluloses. Termite Taxonomy The order Isoptera is generally divided into six families. These are the Hodotermitidae, Termopsidae, Mastotermitidae, Kalotermitidae, Rhinotermitidae and Termitidae. Termitidae contains the so-called higher termites, and none of the termites in this family have symbiotic flagellates in their hindguts. The remaining five families of termites are commonly called lower termites, and they are a ll symbiotically associated with flagellates in their hindguts in addition to the more ubiquitous bacter ia and fungi found in termite guts. The Hodotermitidae and Termopsidae are most often found in the tropics and are often regarded as relatively primitive termites (Eggl eton 2001). The Mastotermitidae, generally considered to be the basal group, consists of the Australian species Mastotermes darwiniensis Kalotermitidae and Rhinotermitidae are the two termite families most commonly encountered in the temperate zones. The Ka lotermitidae include the dampwood termites and the drywood termites. They typically live in close as sociation with their food source, often nesting

PAGE 17

17 within the wood they consume (Noirot and Dar lington 2002). They are also considered to be more primitive in their behavior a nd morphology than the Rhinotermitidae. Rhinotermitidae are commonly called subterrane an termites. They are aptly named, as most species live within the soil and seek out dead wood and other sim ilar food sources. These termites are typically the worst st ructural pests in buildings (P otter 2004). A large number of studies on termite digestion and termite symbiosis have been carried out on genera from this family. The two genera of particular interest are Coptotermes and Reticulitermes Termite Digestive System Anatomy As with other insects, the termite gut ma y be divided into three major regions: the foregut, the midgut and the hindgut. The foregut is composed of the salivary glands, the crop and the proventriculus. The crop and proventriculus are lined with cuticle, which is shed at each molt along with the exoskeleton. Th e crop in termites is relatively small, but the proventriculus is fairly well-muscled and armed with scraping teeth and ridges on its inner surface (Noirot and Noirot-Timothee 1969). The second region is the midgut. This structur e is not lined with cu ticle, but instead has epithelial cells on the inner surf ace responsible for enzyme secretion and nutrient absorption. As with many insects, there is a peritrophic me mbrane present in the midgut, though it does not persist into the hindgut. The midgut of termites is fairly small, often without gastric caecae. This is mainly due to the degree of di gestion that takes place in the hindgut. At the junction between the midgut and the hi ndgut, there are severa l malpighian tubules which serve to excrete nitrogenous waste. It is thought by some that this waste may be recycled based on the presence of uricolytic bacteria (Potrikus and Breznak 1980).

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18 Symbiont Taxonomy and Anatomy The cellulolytic system of lower termites ha s been extensively studied since Clevelands early work on the hindgut symbionts (Cleveland 1924). The hindgut contains both protozoa and bacteria. These protozoa are basal flagellates. They lack mitochondria and so are anaerobic organisms, but some possess hydrogenosomes (C avalier-Smith 1993). Many have vacuoles for engulfing the wood fragments that enter the hi ndgut (Cleveland 1925). Cellulolytic bacteria have been documented in Zootermopsis angusticolis (Wenzel et al. 2002). However, the bulk of the evidence obtained thus far s uggests that the protozoa are th e major sources of cellulolytic enzymes. Removal of protozoa from Reticulitermes speratus has resulted in a significant loss of both cellulolytic and xylanolytic ac tivity (Inoue et al. 1997). The taxonomy of these flage llates is continually being revised as more powerful molecular techniques become available. As it is beyond the scope of this paper to provide a detailed phylogenetic analysis, only a basic overview of current groupings will be provided. The symbiotic flagellates may be broadly divi ded into three major classes. The first, class Anaeromonadea, consists of organisms ofte n referred to as Oxymonads. Most of these protozoa have some form of stru cture which allows them to attach to surfaces, most often the wall of the termite hindgut (Moriya et al. 2003). They tend to have a long, thin morphology and few flagellae. They are sometimes grouped w ithin the Kingdom Archezo a, and possess neither mitochondria nor hydrogenosomes (Cavalier-Smith 1993). The second and third major classes may be grouped within the phylum Parabasalia, a group which possesses hydrogenosomes, but not mitoc hondria. The first of these two classes is the class Trichomonadea. This is a very diverse group, containing families which include Monocercomonadidae, Trichomonadidae, De vescovinidae and Calonymphidae. The Monocercomonadidae and Trichomonadidae are gene rally small flagellates, and it is doubtful

PAGE 19

19 that they participate in cellulose digestion. The two families can be separated by the presence of a recumbent flagellum in the Trichomonadidae. The Devescovinidae and Calonymphidae appear to sort out as one group in recent phylogenetic studies (Delgado-Visc ogliosi et al. 2000, Ohkuma et al 2000). The flagellates in these two families tend to be larger, often with ma ny flagella, and in the case of Calonymphidae, many nuclei as well. In addition to these families, the class Trichomonadea also contains the orders Lophomonadida and the Spirotrichonymphida. In terms of overall structure, the Spirotrichonymphida are fairly similar to the flag ellates in the third clas s, and have been grouped with them in some earlier taxonomies. The third class is the clas s Trichonymphea. These flagellates are typically large and complex, with many flagella. They are wi dely distributed among the termites, with Trichonympha being one of the more well-known genera. Symbiont Assemblages in Termite Hindguts The species of flagellates vary with each species of termites. A particularly thorough documentation of these associations was unde rtaken by Yamin (1979). This study included several species of termites from the families Kalotermitidae and Rhinotermitidae. Upon examination, certain patter ns emerge. Most clearly evid ent is the presence of the families Devescovinidae and Calonymphidae in the Kalotermitidae, but not in either of the other termite families. Also noteworthy is the presence of Oxymonad flagellates in the family Pyrsonymphidae being restricted to the Rhi notermitidae. The order Spirotrichonymphida appears to be far more prevalent in the Rhinot ermitidae than in the Kalotermitidae. However, the genus Trichonympha is present in at least one species from all three of the termite families.

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20 In summary, each family of termites appears to have its own set of flagellate families associated with it. Some flagellate groups are more widespread among the termites. The overall pattern appears to be consistent with the coevolution of termite a nd flagellate lineages, with the possibility of occasiona l horizontal transfer. Termite Carbohydrolase Types and Locations Before comparing these enzymes between term ites and other creatures, it is necessary to define certain aspects of their st ructure and function. First of all, cellulases may be divided into three varieties, based on their mode of action (Breznak and Brune 1994). Any highly cellulolytic organism requires all three of these enzymes from one source or another. The first class of cellulase s consists of exoglucanases (EC 3.2.1.91). These enzymes are processive, binding to the end of a cellulose chain and moving along its length, breaking it down along the way. Most exoglucanases are cellobioh ydrolases, meaning that they reduce cellulose to cellobiose, a disaccharide of beta-glucose. These enzymes are most active against crystalline cellulose, where the cellulose ch ains are arranged in a tightly bound parallel configuration and held in place by hydrogen bonding. The second class of cellulases consists of the endoglucanases (EC 3.2.1.4). These enzymes bind anywhere along the cellulose chai n and break it up randomly, eventually reducing it to cellodextrins, oligosaccharides of beta-glucose. The endoglu canases are most active against amorphous cellulose, where the chains are not arranged in any partic ular configuration. The third class of cellulases is the beta-glucosidase s (EC 3.2.1.21). These enzymes break cellodextrins down into glucose. They are the final enzymes in the process of glucose liberation from cellulose. Aside from cellulose, the major polysaccharid es in wood are hemicelluloses. The most well known and thoroughly studied of these hemicelluloses is xylan. It is a polymer of xylose, a

PAGE 21

21 pentose sugar, with side molecules of 4-O-me thylglucuronic acid. The xylose molecules may have a varied degree of acetylati on. There is also the possibility of arabinose being incorporated into the polymer as an additional variety of si de group. Since xylan is far more complex than cellulose, there are several more enzymes required for its complete digestion. However, the major enzymes of concern are often simply known as xylanases (EC 3.2.1.8). They hydrolyze the bonds between xylose units in the polymer, much in the same fashion as endoglucanases hydrolyze cellulose. In addition, there are be ta-xylosidases (EC 3.2.1.37) which break down the resulting oligosaccharides. Functionally, the termite appears to have its enzymes spatially segregated. The amorphous cellulose is attacked first in the foregut and midgut. The hindgut symbionts then appear to digest the crystal line cellulose and the bulk of th e hemicelluloses (Hogan et al. 1988, Mishra 1991). The byproducts of this digestion, ma inly acetate, then diffuse out into the termite tissues. The xylanases of lower termites have not b een specifically characterized. However, the bulk of xylanase and beta-xylosidase activity has been shown to be located in the hindgut in multiple cases (Azuma et al. 1993, Inoue et al. 19 97). Mannanase activity appears to also be mainly located in the hindgut. Termite Carbohydrolase Structural and Functional Characteristics A vital structural aspect, in the case of cellula ses at least, is cleft vs. tunnel geometry. The catalytic site in exoglucanses is located within a tunnel in the enzyme. Th is allows the cellulose chain to travel through th e enzyme much as a thread may trav el through the eye of a needle. Of course, this particular needle has a guillotine incor porated into its structure. The cleft geometry, found in endoglucanases, has the catalytic site lo cated in a groove along the enzyme surface.

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22 This allows the enzyme to effectively clam p onto any available loca tion along the cellulose chain. The next structural aspect concerns reta ining enzymes and invert ing enzymes. Using cellulases as an example, an inverting cellula se inserts the hydroxyl group in the alpha position, opposite to the bond configuration in the cellulose chain. A retain ing cellulase inserts a hydroxyl group in the beta configuration, the same conf iguration as the bond in the cellulose chain. Inverting cellulases work in a single step (Fig 1). Th e catalytic nucleophile removes a hydrogen from a water molecule. The remaining hydroxide is bonded to the sugar molecule. At the same time, the proton donor donates its hyd rogen, allowing the oxygen bond between sugars to be broken, separating off as a hydroxyl group on the other sugar. The remaining hydrogen on the sugar is then brought back to the proton donor. Retaining cellulases work in two steps, commonly referred to as a double displacement mechanism (Davies et al. 1998). First, the catal ytic nucleophile forms a covalent bond to the substrate, while the proton donor allows the sepa ration of the leaving group (Fig 2). In the second step, a water molecule interacts with the bonded substrate, donating a hydroxyl group to the substrate and effectively breaking the bond between it and the nucle ophilic residue. The remaining proton is accepted by the proton donor, restoring the enzyme to its original configuration. Another structural consider ation is the nature of the proton donor and the catalytic nucleophile. Glycosyl hydrolases function by placing the polysaccharide between two acidic amino acid residues. If these residues are fairly fa r apart, there is room for a water molecule to be incorporated into the confi guration. This results in a direct trade of bonds, leading to an inverting mechanism. If the residues are closer together, there is no room for a water molecule

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23 in an occupied site, and so the sugar becomes temporarily bonded to th e nucleophilic residue. After this step, there is room for a water molecule to enter the site, and it is attached in the beta configuration. This leads to a retaining mechanism. There are only two amino acids with acidic side chains. These are aspartic acid and glutamic acid. Each of these has a carboxyl group at the end of a short carbon chain. The glutamic acid chain is one carbon long er than that of the aspartic ac id, and so leaves less space. As a result, nearly every carbohydrolase with a glut amic acid in each of the two key positions is a retaining enzyme, while nearly every one with aspartic acid in each position is an inverting enzyme. In enzymes with one of each residue, th e glutamic acid is nearly always the proton donor. A final consideration, particularly applicab le to the exoglucanases, is the positioning of aromatic residues. Aromatic residues at ce rtain locations on the en zyme surface allow the cellulose chain to slide more easil y through the catalytic site. This is often noticeable as a row of tryptophan residues locate d within the tunnel of exoglucanases (Parsiegla et al. 2000). While there are other aspects of structure, such as di sulfide bonds, alpha helices and beta sheets, these are generally more significant to enzyme grouping and relatedness. So far, all of the endogenous cellulases of lower termites have been in glycosyl hydrolase family 9 (Watanabe et al. 1998, Zhou et al. 2007). The enzymes in th is family have aspartic acid as a catalytic nucleophile and glutamic acid as a proton donor. They are inverting cellulases with structures consisting mainly of alpha helices. The catal ytic site follows the cleft configuration, allowing the endoglucanases to bind anywhere along the cellulose chai n. In addition, there appears to be a high density of aromatic re sidues on the enzyme surface near the catalytic residues.

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24 The second family found in lower termites, family 7, contains both exoglucanases and endoglucanases. Their structures ar e dominated by beta sheets and ar e rich in disulf ide linkages. They have glutamic acid at both positions of th e catalytic site, and are retaining cellulases. Unlike the other cellulases, the exoglucanases in th is family have their catalytic sites located in tunnels. In addition, they have several tryptophan residues located along the length of the tunnel. The exoglucanases generally have four tryptopha n residues in a row, while the endoglucanases typically have two or thr ee tryptophan residues along the length of the cleft. So far, these enzymes have been found mainly in the Coptotermes symbionts Pseudotrichonympha grassii and Holomastigotoides mirabile The majority of sequences recovered have indicated an endoglucanase activity (Watanabe et al. 2002). However, there have been some promising indications of exoglu canases as well (Nakashima et al. 2002). Glycosyl hydrolase family 45 consists of endoglucanases, which have aspartic acid in both positions in the catalyt ic site. As may be expected, these are inverting cellulases. Like the family 7 enzymes, their structures are dominated by beta sheets and are rich in disulfide linkages. Like the termite cellulases, they have an open cl eft structure. However, they differ significantly from these enzymes in that they have very few alpha helices, and instead consist mainly of beta sheets. In addition, they have se veral disulfide linkages, while family 9 cellulases tend to have only one or two. Enzymes in family 45 have been found in the protozoan symbionts of Reticulitermes speratus (Ohtoko et al., 2000). Two of the clones were localized to Trichonympha agilis and Teranympha mirabilis Both of these are large parabasalian flagellates. Glycosyl hydrolase family 5 contains of exoglucanases, endoglucanases, xylanases, mannanases, and beta-mannosidases. These enzyme s have glutamic acid in both positions in the catalytic site and follow a retaining mechanism. These enzymes consist of a combination of

PAGE 25

25 alpha helices and beta sheets. These enzymes have been characterized from Coptotermes formosanus symbionts (Inoue et al. 2005).

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26 Figure 2-1. Inverting cellulase mechanism.

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27 Figure 2-2. Retaining cellulase mechanism.

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28 CHAPTER 3 BUFFER AND PH OPTIMIZATION FOR TERMITE CARBOHYDROLASE ASSAYS Introduction The gut of the typical subterranean termite consists of a foregut with a small crop and a proventriculus, a fairly simple midgut and a gr eatly expanded hindgut containing several species of symbiotic protozoa, fungi, bacteria, and ar chaea (Yamin 1979, Lewis and Forschler 2004). A pair of salivary glands is al so present, emptying into the foregut anterior to the crop. To effectively digest wood, a r ecalcitrant and nutritionally poor substrate, termites like R. flavipes have evolved an array of enzymes. So me carbohydrolases characterized from the Rhinotermitidae have been shown to be endogeno us (Watanabe et al. 1998), while others are produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al. 2000). In most prior studies, the gut carbohydrolases of subterranean termites have been assayed using 0.1 M sodium acetate buffer, pH 5.5, as a sta ndard buffer. Substrates have either been based upon the generation of reducing sugars from specific polymers or the hydrolysis of smaller molecules consisting of a monosaccharide or o ligosaccharide complexed with p-nitrophenol, a molecule that is yellow when isolated and clear when complexed with a sugar. Prior to using these substrates for carbohydrolase activity evalua tions, it was deemed necessary to evaluate a range of possible pH values and buffers to dete rmine whether the standard sodium acetate buffer would produce optimal results. Our objectives were to determine 1) the optim al buffer types for p-nitrophenol generating substrate-based assays, 2) the optimal buffer ty pes for reducing sugar-dependent assays, 3) the optimal pH values for p-nitrophenol generating substrate-based assays for each major gut region, and 4) the optimal pH values for reducing s ugar-dependent assays for each major gut region.

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29 Materials and Methods Termite Collection, Dissection, and Enzyme Extraction Reticulitermes flavipes (Kollar) termites were field collected in termite traps consisting of a polyvinyl chloride bucket (20 cm high by 20 cm diam.; Item # 811192-4, Ventura Packaging Inc., Monroeville, Ohio) with 11 holes drilled in the sides and base (3 cm diam.) placed vertically in the ground to a dept h of roughly 19 cm and covered with a PVC lid. Three rolls of single-faced corrugated cardboard (10 cm diam. by 20 cm length) were placed into the tube side by side as a food source. Termites were collect ed from the trap by removal of cardboard rolls, separated from the cardboard, immedi ately frozen, and kept at -80oC until dissection. Termites were removed from the freezer and kept on ice until use. Termites were either homogenized intact or dissected before hom ogenization. In the case of dissections, each termites gut was removed intact and then sepa rated into three regions: foregut (and salivary glands), midgut and hindgut. The three gut regions were placed into separate 1.5-mL microcentrifuge tubes (Eppendorf, Westbury, NY) c ontaining the appropriate buffer, and kept on ice. Enzymes were extracted using a method adapte d from Inoue et al. (1997). Whole termites or gut contents of each microcentrifuge tube were placed in a 2-mL Tenbroeck glass tissue grinder and manually homogenized on ice. Th e homogenates were then centrifuged at 14,000 rpm at 4oC for 15 min. The supernatants were collected, frozen, and kept at -80oC until use in the enzyme assays. Buffer Optimization Assays Five buffers were prepared at 0.1M in all case s. The buffers and their pH values were as follows: sodium acetate (4.0, 4.5, 5.0, 5.5), sodium phosphate (6.0, 6.5, 7.0, 7.5), MES (methylethyl sulfide; 5.5, 6.0, 6.5), bis-tris (2-[bis(2-hydroxyethyl)i mino]-2-(hydroxymethyl)-

PAGE 30

30 1,3-propanediol; 6.0, 6.5, 7.0), and PIPES (piper azine-N,N-bis(2-ethan esulfonic acid); 6.5, 7.0, 7.5). Two substrate solutions were prepared for each of the buffer/pH combinations. The first was 2% carboxymethylcellulose (CMC; SigmaAldrich) and the sec ond was 4 mM pNPG. Whole termite extracts were prepared at a concentration of 50 termites/mL. Endoglucanase assays were conducted using a method adapted from Han et al. (1995). Assays were conducted in clear, flat-botto med 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of CMC solution. Only the wells more than 2 wells away from the edge of the microplate were used for these assa ys. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boiling. Identical plates were prepared as controls. The enzyme and substrate solutions were allowed to react for 10 min at 23oC. DNSA solution (100 L), consisting of 1% 3,5-di nitrosalicylic acid (DNS A), 0.4M sodium hydroxide and 30% sodium potassium tartrate, was then adde d to each enzyme/substrate well of the control plates. After an additional 60 min, the DNSA so lution was added in the same manner to the sample plates. In both cases, immediately af ter addition of DNSA solution, the microplate was placed in boiling water for 10 min to induce an oxidation/reduction reaction with the DNSA, resulting in a color change. A ll plates were placed on ice imme diately after boiling to cool for 15 min. Each microplate was read at 540 nm immediatel y after cooling using a Quant Universal Microplate Spectrophotometer (Bio -Tek Instruments, Winooski, VT ). Standards were generated using dilutions of glucose in sodium acetate buffer (0.1M, pH 5.5) combined with equal volumes of DNSA solution. Standards were boile d, cooled, and read as described above. Beta-glucosidase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-glucopyranoside (pNPG; Sigma-Aldrich) was prepared in

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31 0.1 M sodium phosphate buffer, pH 6.5. Assays we re conducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPG solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. Gut Region Assays Across PH Assays were conducted using a method adapted from Han et al. (1995). For endoglucanase assays, both sodium acetate and s odium phosphate CMC solutions were prepared as described above for the buffer optimization assa ys. Sodium acetate solutions were prepared at pH values of 4, 4.5, 5, and 5.5. Sodium phosphate so lutions were prepared at pH values of 6, 6.5, 7, and 7.5. For xylanase assays, solutions consisted of 0.5% beechwood xylan (>90% xylose residues; Sigma-Aldrich) prepared in the same sodium acetate and sodium phosphate buffers. The solutions were boiled approximately 30 min, until xylan particles were no longer visible. The solutions were then centrifuged at 2,500 rpm for 5 min at 23oC and the supernatants were used as the final xylan substrate solutions. Termite tissues were extracted into the eight pH buffers described above. Final tissue extracts consisted of foregut, midgut, and hindgut at a concentration of 50 termite equivalents/mL in all cases. The assays were conducted as described above for endoglucanase buffer optimization assays. In each well, 10 L of tissue extract was combined with 90 l of CMC or xylan solution. CMC solutions were allowed to react for 70 min at 23oC. Xylan solutions were allowed to react for 35 min at 23oC. Control plates were prepared in th e same manner, but were allowed to react 10 min for endoglucanase assays or 5 min before xylanase assays before boiling. Standards were

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32 generated using dilutions of glucose for endoglucanase assays and dilutions of xylose for xylanase assays. In both cases the standards we re prepared in sodium acetate buffer (0.1M, pH 5.5), combined with equal volumes of DNSA so lution, boiled, cooled, and read at 540 nm. Exoglucanase, beta-glucosidase, and beta-x ylosidase assays were conducted using a method adapted from Han et al. (1995) Solutions of 4 mM p-nitrophenyl-D-cellobioside (pNPC; Sigma-Aldrich) 4 mM p-nitrophenyl-D-glucopyranoside (pNPG; Sigma-Aldrich), and 4 mM p-nitrophenyl-D-xylopyranoioside (pNPX; Sigma-Aldr ich) were prepared as described above for the buffer optimization assays. Sodium acetate solutions were pr epared at pH values of 4, 4.5, 5, and 5.5. Sodium phosphate solutions we re prepared at pH values of 6, 6.5, 7, and 7.5. Assays were conducted in clear 96well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPC solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectr ophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorde d. Standards were generated us ing dilutions of p-nitrophenol. Data Analysis The buffer optimization assays were set up as one-factor designs with a single homogenate for each buffer/pH combination and three technical replicates. The pH assays across gut region were set up as one-factor designs with a singl e homogenate for each pH and eight technical replicates for each gut region. For the endoglucanase and xylanase assays, th e following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT

PAGE 33

33 where: SA = specific activity (nmol reducing sugar/termite equivalent/minute), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample. For the beta-glucosidase and exoglucanase assays, the following formula was used to calculate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol/termite equivalent/min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol pnitrophenol/mOd), and NT = the number of termite equivalents per sample. For the time/concentration analyses, the mean specific activities were calculated for each combination of time and concentration. The va riance in mean specific activity was then calculated among all times for each concentration and among all concentrations for each time. For the remaining assays, means and standard erro rs were calculated for each enzyme specific activity at each combination of pH and buffe r, pH and gut region, or concentration and temperature. Results Buffer Optimization Assays Beta-glucosidase activities incr eased with pH from 5.5 to 7 (F igure 3-1). Activities were negligible below pH 5.5. MES buffer caused a gr eater activity level than phosphate or PIPES buffers at pH 6.5. Bis-Tris cau sed significantly lower activities across its f unctional pH range. Activities on PIPES buffer were slightly lo wer than those for phosphate buffer across its functional pH range. With the ex ception of the bis-tris buffer, the greatest changes in betaglucosidase activity over pH we re seen between pH 5.5 and 7.

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34 Gut Region pH Optimization Assays Foregut endoglucanase activity was greatest at a pH value of 6.5, with a smaller activity peak at 5.5 (Figure 3-2). Midgut and hindgut endoglucanas e activities were grea test at a pH of 5.5. Hindgut activity was significantly lower at pH values above 5.5 and below 5. Changes in foregut endoglucanase ac tivities were high throughout the pH range tested, while the greatest change in hindgut endoglucanase act ivities was the decline in activ ity from a pH of 5.5 to 6. Foregut and midgut beta-glucosidase activit ies increased with pH up to pH 6.5, then declined at higher pH values (Figure 3-3). Be ta-glucosidase activities in the hindgut extract showed a steady increase with pH up to pH 7. All activities were neg ligible below pH 5.5. Changes in foregut and midgut beta-glucosidase activities were greatest between pH 6 and 6.5. Changes in hindgut beta-glucosidase activ ities were greatest between pH 5.5 and 7. Hindgut exoglucanase activities were consiste ntly greater with increasing pH value, following a sigmoid curve (Figure 3-4). The grea test differences in activity were between pH 5.5 and 7. Activity was negligible below a pH of 6. Hindgut xylanase activity was great est at pH 4.5 (Figure 3-5). However, there was little difference in hindgut xylanase activ ities from pH 4 to 5.5. The greatest change in hindgut xylanase activity was seen from a pH of 5.5 to 7.5. Discussion Activity Magnitudes Three qualities of enzymatic activity are of pa rticular interest when selecting buffers for termite carbohydrolase assays. The first is the magnitude of activ ity yielded by a given buffer. Experiments with a goal of quantifying enzymatic activity may underestimate actual activities if the buffer does not maximize in vitro enzyme activity levels. Lower levels of actual activity may

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35 be missed altogether with some buffers. For comparative studies, lower overall activities may make differences in activity leve ls more difficult to distinguish. For pNPG-based beta-glucosidase assays, th e highest activities ranged from 6.5 to 7.5 on MES, PIPES, and sodium phosphate buffers. So dium acetate and bis-tris buffers yielded far lower activities. In the past, investigators ha ve used pH 5.5 sodium acetate buffer for pNPGbased termite beta-glucosidase assays (Inoue et al. 1997). This may have caused betaglucosidase activity to be undere stimated within the termite. For symbiont exoglucanase from the hindgut, activity was greatest on pH 7.5 sodium phosphate buffer. The activity of symbiont xyl anase from the hindgut was greatest on pH 4.5 sodium acetate buffer, and nearly as high on all sodium acetate buffers tested. Activity Changes with pH The second quality of interest is the rate of change in activity with changing pH. If the selected buffer is in a region of high change, smaller changes in buffer pH will cause greater changes in activity levels. This can increase va riation in experimental results and, in extreme cases, cause difficulties with expe rimental repeatability. Theref ore, the optimal buffer should maximize activity within a regi on of minimal change in ac tivity with change in pH. Exoglucanase activity declined sharply be low pH 6.5 sodium phosphate buffer and was nearly nonexistent on sodium acetate buffer. Ho wever, xylanase activity declined sharply with increasing pH on sodium phosphate buffer. Differences in Gut Regions Different activity maxima were seen for diffe rent gut regions in beta-glucosidase and endoglucanase assays. In addition, pH ranges wi th the highest changes in activity differed between gut regions for beta-gluco sidase and endoglucanase assays.

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36 In the case of beta-glucosidases, the differe nces were mainly seen between the endogenous enzymes from the foregut and midgut, and the symb iont enzymes from the hindgut. In contrast, the differences in the endoglucanase assays we re mainly seen between the foregut and the hindgut, with midgut activity levels far lower than those seen in the other gut regions. Because of these differences, the optimal buffers for carbohydrolase assays may not be the same for endogenous (termite) and symbiont produced enzymes. Conclusions Based upon our experimental data, the optimal pH buffer for predicting overall xylanase or endoglucanase activities is pH 5.5 sodium acetate bu ffer, which is the standard buffer used in past termite carbohydrolase assays (Hogan et al. 1988, Inoue et al. 1997). Although endoglucanase activity shifts considerably from pH 5.5 to pH 6, the prox imity of pH 5.5 sodium acetate buffer to this shift presents no concern. In this case, the observed shift is not only between two pH values, but also occurs in a shift between sodium acetate and sodium phosphate buffers. Previous experiments have generally used pH 5.5 sodium acetate buffer as a universal buffer for termite carbohydrolase assays. Base d on our p-nitrophenol-gen erating assays, the optimal pH buffer for predicting overall beta-glu cosidase activity using pNPG is pH 6.5 sodium phosphate buffer. Both beta-glucosidase and exoglucanase activit ies on pH 6.5 sodium phosphate buffer are roughly 10-fold higher than on pH 5.5 sodium acetate buffer. The optimal pH buffer for predicting exoglu canase activity or hindgut beta-g lucosidase activity is pH 7.5 sodium phosphate buffer. Hindgut exoglucanase activity is roughly 15-fold higher and betaglucosidase activity roughly 20-fo ld higher on pH 7.5 buffer than on pH 5.5 buffer. We believe that prior pNPG or pNPC-based assays using pH 5.5 sodium acetate buffer may have underestimated actual beta-gluco sidase or exoglucanase activity levels within the termites.

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37 Based upon these findings, we intend to conduct pNPC, pNPG, and pNPX-based assays using 0.1 M sodium phosphate buffer, pH 6.5, in th e following chapters. We intend to conduct DNSA-based assays using 0.1 M sodium acetate buffer, pH 5.5.

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38 0 0.4 0.8 1.2 1.6 2 2.4 3.544.555.566.577.58pHSpecific Activity (nmol/min/termite) Acetate Phosphate MES Bis-Tris PIPES Figure 3-1. Beta-glucosidase activities in Reticulitermes flavipes workers across pH and buffer.

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39 0 1 2 3 4 5 6 7 44.555.566.577.5pHSpecific Activity (nmol/min/termite) Foregut Midgut Hindgut Figure 3-2. Endoglucanase activitie s in different gut regions of Reticulitermes flavipes workers across pH.

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40 0 0.3 0.6 0.9 44.555.566.577.5pHSpecific Activity (nmol/min/termite) Foregut Midgut Hindgut Figure 3-3. Beta-Glucosidase ac tivities in different gut region s of Reticulitermes flavipes workers across pH.

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41 0 0.1 0.2 0.3 0.4 0.5 44.555.566.577.5pHSpecific Activity (nmol/min/termite) Figure 3-4. Hindgut exog lucanase activities in Reticulitermes flavipes workers across pH.

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42 0 5 10 15 20 25 30 35 44.555.566.577.5pHSpecific Activity (nmol/min/termite) Figure 3-5. Hindgut xyl anase activities in Reticulitermes flavipes workers across pH.

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43 CHAPTER 4 CARBOHYDROLASE AC TIVITY PATTERNS IN RETICULITERMES FLAVIPES AND COPTOTERMES FORMOSANUS WORKERS AND SOLDIERS Introduction Reticulitermes flavipes (Kollar) and Coptotermes formosanus (Shiraki) are two of the most economically significant pest termites in North Am erica. Both are subterranean termites, but R. flavipes is a native termite with relatively sma ll colonies and a low soldier ratio while C. formosanus is an invasive termite with relatively larg e colonies and a higher soldier ratio. Such differences in colony structure may be acco mpanied by differences in gut carbohydrolase profiles. Termites and their symbionts are known to pr oduce three major types of cellulase which work together to digest cellulose. Exoglucan ase (EC 3.2.1.91) cleaves the cellulose chain from the ends, typically producing cell obiose, and is most active ag ainst crystalline cellulose. Endoglucanase (EC 3.2.1.4) cleaves the cellulose chain randomly along its length and is most active against amorphous cellulose. Beta-Gluco sidase (3.2.1.21) cleave s cellobiose and other small cellulose fragments, hydrolyzing them to gl ucose. Xylanolytic enzymes include xylanases (EC 3.2.1.8) which internally cleave the xylan chain and beta-xylosid ases (EC 3.2.1.37) which cleave xylan oligosaccharides into xylose. The objectives of this study were 1) determin e the patterns of cellulolytic and xylanolytic enzymes in different termite gut regions, 2) de termine the relative cellu lolytic and xylanolytic activities of R. flavipes and C. formosanus workers, 3) determine the relative cellulolytic and xylanolytic activities of R. flavipes and C. formosanus soldiers, and 4) determine the relative cellulolytic and xylanolytic activit ies of the soldiers of each speci es compared to the workers.

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44 Materials and Methods Termite Collection, Dissection, and Enzyme Extraction Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in Charleston, South Carolina. Termites were coll ected from the trap by removal of the wood (southern yellow pine) in the bait stations and brought to the Un iversity of Florida. Termites were then placed in feeding bioassays. Term ites were collected, frozen and kept at -80oC until dissection. Collections were rest ricted to a single colony for each species to eliminate colony as a source of variation in enzyme activities. Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5, and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the endoglucanase and xylanase assays, while s odium phosphate buffer was used for the exoglucanase, beta-glucosidase and beta-xylosidase assays. Termites were removed from the freezer and ke pt on ice until dissecti on. Each termites gut was removed intact and separated into thr ee regions: foregut (and sa livary glands), midgut, and hindgut. A single enzyme extract was prepared from dissected termites from each feeding treatment for each assay type usi ng an experimental design similar to the recent previous termite carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For the endoglucanase and xylanase assays, 25 termites of each caste and species were dissected in sodium acetate buffer. For the exoglucanase, beta-glucosidase and beta-xylosidase assay, 25 termites of each caste and species were dissected in sodium phosphate buffer. The three gut regions were placed into separate 1.5 mL mi crocentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ic e. Final concentrations were 50 termite gut regions per mL in all cases.

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45 Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and manually homogenized on ice. The homoge nates were centrifuged at 20,800 g at 4oC for 15 min. The supernatants were colle cted, frozen, and kept at -80oC until use in the enzyme assays. Termite Photography Guts and frontal glands from both termite species were dissected into 0.1 M sodium phosphate buffer, pH 6.5, using the methods descri bed above. Termites were then photographed on an auto-montage system (Syncroscopy, Frederick, MD). Soluble Protein Assays Assays for soluble protein content were conduc ted using the Bradford reagent. Enzyme extracts (10 L) were combined with 250 L of Bradford reagent (Bio-Rad, Hercules, CA) in a 96-well microplate. Absorbances were then read at 595 nm using a Quant Universal Microplate Spectrophotometer (Bio -Tek Instruments, Winooski, VT ). Standards were generated using BSA standards (Bio-Rad, Hercules, CA) comb ined with the Bradford reagent in the same proportion as the enzyme extracts. Endoglucanase Assays The endoglucanase assays were conducted usi ng a method adapted from Han et al. (1995). A 2% solution of carboxymethylcellulose (CMC; Si gma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of CMC solution. The solutions were allowed to react for 70 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was

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46 immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar contro l plates were allowed to react for 10 min to allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 10 min reaction was used as a control to correct for any differences in initial sample reaction rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated using dilutions of glucose. Only the wells in the middle of the microplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boiling. Exoglucanase Assays Exoglucanase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-cellobioside (pNPC; Sigma-Al drich) was prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays were co nducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPC solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. Beta-Glucosidase Assays Beta-glucosidase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-glucopyranoside (pNPG; Sigma-Aldrich) was prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays we re conducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPG solution. The reaction was

PAGE 47

47 allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. Xylanase Assays The xylanase assays were conducted using a method adapted from Han et al. (1995). A 0.5% solution of beechwood xylan (>90% xylose re sidues; Sigma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. The soluti on was boiled approximately 30 min, until xylan particles were no longer visible. The solu tion was centrifuged at 1250 g for 5 min at 23oC and the supernatant was used as the xylan stock solution. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of xylan solution. The solutions were allowed to react for 35 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar control pl ates were allowed to react for 5 min to allow for passive mixing of solutions before boiling with DNSA soluti on (Zhou et al. 2007). A 5 min reaction was used as a control to correct for any di fferences in initial sample reaction rates due to incomplete mixture of enzyme and substrate solu tions. Standards were generated using dilutions of xylose. Only the wells in the middle of the microplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boiling.

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48 Beta-Xylosidase Assays Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-xylopyranoside (pNPX; SigmaAldrich) was prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays we re conducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPX solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. The experiment with formulated diets was set up as a two-factor design with four replicates. The two factors were gut region a nd diet treatments. On e microplate well was assayed for each replicate of each gut region/diet combination. Thre e gut sections and five diet treatments were assayed for each replicate, yielding a total of 15 wells per replicate. Data Analysis Experiments were set up as one-factor de signs with two homogenates for each caste/species combination; one homogenate for endoglucanase and xylanase assays and another for exoglucanse, beta-glucosidase, and beta-xylos idase assays. The endoglucanase assays had 6 technical replicates for each gut region while the other assays had 4 technical replicates for each gut region. The protein assays had 3 technical replicates for each gut region, species and caste. For the endoglucanase and xylanase assays, th e following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT

PAGE 49

49 where: SA = specific activity (nmol reducing su gar per termite equivalent per min), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample. For the exoglucanase, beta-glucosidase, and beta-xylosidase assays, the following formula was used to calc ulate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample. The data were analyzed using analysis of variance. Student -Neuman-Keuls test, = 0.05, was used to separate the mean activities within each gut region (SAS Institute 2001). Results Termite Gut and Frontal Gland Comparisons Soldiers in both species had smaller, less developed hindguts than workers (Figure 4-1). Compared to R. flavipes workers, C. formosanus workers had a smaller crop, a larger rectum, and a small internal sclerotization just behind the malpighian tubules. The frontal gland in C. formosanus soldiers was far larger and appare ntly more developed than that in R. flavipes soldiers (Figure 4-2). Formosan soldier term ites had a prolonged pharynx, to accommodate the enlarged frontal gland. Worker Cellulolytic Activities Endoglucanase activities were mainly confined to the foregut and hindgut (Table 4-1). Activities were significantly greater in the R. flavipes worker foregut than in the C. formosanus worker foregut, but they were significantly greater in the C. formosanus midgut and hindgut.

PAGE 50

50 Exoglucanase activities were almost exclusive to the hindgut, with C. formosanus worker activities being significan tly greater in all gut regions (Table 4-2). Beta-glucosidase activities were distributed across the gut, be ing most prominent in the midgut in C. formosanus workers and most prominent in the hindgut in R. flavipes workers (Table 4-3). Differences in Betaglucosidase activities were significant in all three gut regions among all species-caste combinations. Worker Xylanolytic Activities Xylanase activities were almo st exclusive to the hindgut, wi th no significant differences between worker hindgut xylanase ac tivities (Table 4-4). Beta-xylosidase activ ities were mainly restricted to the hindgut, and were significantly higher in C. formosanus workers in all gut regions (Table 4-5). Soldier Cellulolytic Activities Soldier cellulolytic activities were lower than those seen in workers in most cases. Endoglucanase activities were mainly confined to the hindgut, with midgut and hindgut activities significantly greater in R. flavipes soldiers (Table 4-1). Exoglu canase activities were mainly confined to the hindgut, and hindgut activ ities were signifi cantly greater in R. flavipes soldiers (Table 4-2). Beta-glucosidase ac tivities were distributed across the gut, being most prominent in the midgut and significantly greater in R. flavipes soldier hindguts (Table 4-3). Soldier Xylanolytic Activities Soldier xylanolytic activities were lower than those seen in workers in most cases. Xylanase activities were almost exclusive to the hindgut, and were significantly higher in the R. flavipes soldier hindgut (Table 4-4). Beta-xylosidase activities were mainly restricted to the hindgut, and were significantly higher in the R. flavipes soldier hindgut, wh ile midgut activities were significantly higher in the C. formosanus soldier hindgut (Table 4-5).

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51 Gut Soluble Protein Levels The hindgut extracts had far more soluble protein than the fore gut or midgut extracts in all cases except for the C. formosanus soldier extract in pH 5.5 buffer (Table 4-6). This difference was smaller, but the hindgut still contained more protein than the foregut or midgut, even for the pH 5.5 C. formosanus soldier extract. Soldier protein levels were universally lower than worker protein levels for each gut region. C. formosanus worker extracts had high er protein levels than R. flavipes worker extracts in most cases. C. formosanus soldier extracts had lower protein levels than R. flavipes soldier extracts in all cases. Th e extracts in pH 5.5 buffer had lower protein levels than the ex tracts in pH 6.5 buffer. Discussion The patterns of cellulolytic enzymes within the R. flavipes workers appeared to indicate a processive degradation of amorphous cellulose along the gut. Endoglucan ase activity was seen to be higher in the foregut than in the midgut, whereas beta-glu cosidase activity appeared to increase progressively through the gut. The majority of exoglucanase and xylanase activities were located in the hindgut of workers soldiers of both species assayed. Simila r exoglucanase patterns have been observed in Coptotermes lacteus (Hogan et al. 1988). This indicates a large dependence of the termite on its hindgut symbionts for the digestion of crysta lline cellulose and xyl an. Our findings are consistent with prior characterizatio ns of symbiont exoglucanases in C. formosanus and R. flavipes (Watanabe et al. 2002, Nakashima et al. 2002a, Zhou et al. 2007). Th is localization of exoglucanase and xylanase activities to the hindgut is significant, as up to 70% of wood cellulose may be crystalline in nature and xylan may make up as much as 25% of wood dry weight (Biermann 1996). This could explain past obs ervations of reliance on hindgut symbionts (Cleveland 1924) in spite of the charact erization of endogenous cellulases in Reticulitermes and

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52 Coptotermes termites (Watanabe et al. 1998, Nakashim a et al. 2002b, Zhou et al. 2007). It should be noted that, thus far, no endogenous ce llulases in characterized lower termites have been exoglucanases. Formosan subterranean termites are widely re garded as more aggressive foragers than eastern subterranean termites, with far larger col onies. Their soldiers have a larger frontal gland than the soldiers of the easter n subterranean termite, producing a significant amount of chemical exudate in defense of the colony (need to take ph otos of the frontal glands). This increased metabolic demand, combined with the larger rati o of soldiers to workers, roughly 1:10 as opposed to 1:100 in the case of R. flavipes would seem to necessitate a more active system for the digestion of wood and similar materials. This is precisely what the results seem to indicate among the workers, especially in the case of exoglucanase and beta-g lucosidase. While the overal l distributions of carbohydrolase activities are similar in these tw o species, there are a couple of differences; heightened enzyme activities as mentioned above and the partial redi stribution of endoglucanas e from the foregut to the midgut and hindgut in C. formosanus workers. A reverse pattern was seen in the soldiers, with the C. formosanus soldiers having far less enzyme activity in the case of endoglucanas e, exoglucanase and xylanase. However, C. formosanus soldier beta-glucosidase and beta-xylosidase were similar to the levels seen in R. flavipes soldiers. The former three carbohydrolases are known to break down longer chains, thus working in the earlier parts of cellulose and xylan degradation. The latter two break down oligosaccharides, working in the final pa rts of cellulose a nd xylan degradation. With its greatly enlarged frontal gland, the C. formosanus soldier has relatively little room for its digestive system. Since the C. formosanus workers are so profic ient at wood digestion,

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53 there is little need for the earlier enzymes in th e soldier digestive system s. Therefore, it makes sense that these soldiers limited digestive capacit y would be almost entirely focused on the later phases of wood digestion. This is su pported by the less extreme case of the R. flavipes soldiers, which also have relatively high levels of beta-g lucosidase and beta-xylosidase compared with the other three enzymes. Formosan subterranean termites are not only more aggressive foragers than eastern subterranean termites, but their digestive systems are apparently more capable of digesting wood, particularly crystalline cellulose. This woul d suggest the possibility of natural cellulase inhibitors or other obstacles to wood digestion in their native ra nge. Some plants are known to produce cellulase inhibitors in th e form of proteins or polyphenol s (Sineiro et al. 1997, York et al. 2004). However, the increase d digestive capacity seen in C. formosanus workers could also simply be a result of the need to support a larger proportion of so ldiers, as termite soldiers are incapable of feeding themselves and Formosan sold iers have relatively little internal digestive capacity. Although there were clearly differences in overall activity levels, the patterns of cellulolytic and xylanolytic enzyme activities we re similar between the two species assayed, in spite of the fact that these speci es were in two different genera from two different continents. This suggests that findings rega rding cellulose and xylan diges tion in one species within the Rhinotermitidae may be cautiously applied to the re st of the family. Our findings also suggest a processive mechanism of amorphous cellulose degradation with a reliance on the termite symbionts for the digestion of xylan and crystall ine cellulose. This corroborates the need of these termite species for their hindgut symbiont s in spite of their production of endogenous

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54 cellulolytic and xylanolytic enzymes. This patter n of digestion will be revisited in workers of both species in subsequent chapters.

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55 Table 4-1. A comparison of endoglucanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers Caste Species Foregut Midgut Hindgut worker R. flavipes 4.70 + 0.31a 1.39 + 0.08b 5.21 + 0.28b worker C. formosanus 2.84 + 0.22b 1.91 + 0.19a 8.17 + 0.51a soldier R. flavipes 0.36 + 0.07c 1.22 + 0.11b 2.81 + 0.16c soldier C. formosanus 0.00 + 0.01c 0.23 + 0.03c 0.79 + 0.07d Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, = 0.05, SAS Institute 2001), n = 6 replicates.

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56 Table 4-2. A comparison of exoglucanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers Caste Species Foregut Midgut Hindgut worker R. flavipes 0.0109 + 0.0001b 0.0121 + 0.0026c 0.3862 + 0.0067b worker C. formosanus 0.0278 + 0.0002a 0.1182 + 0.0033a 0.6728 + 0.0140a soldier R. flavipes 0.0013 + 0.0006d 0.0409 + 0.0008b 0.2184 + 0.0037c soldier C. formosanus 0.0037 + 0.0002c 0.0377 + 0.0017b 0.1014 + 0.0019d Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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57 Table 4-3. A comparison of be ta-glucosidase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers Caste Species Foregut Midgut Hindgut worker R. flavipes 0.147 + 0.004b 0.176 + 0.004a 0.655 + 0.008b worker C. formosanus 0.272 + 0.003a 0.891 + 0.033c 0.876 + 0.020a soldier R. flavipes 0.037 + 0.001c 0.611 + 0.008b 0.348 + 0.001c soldier C. formosanus 0.056 + 0.018c 0.560 + 0.016b 0.168 + 0.002d Beta-glucosidase activities are in nmol p-nitrophenol pe r termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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58 Table 4-4. A comparison of xylanase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers Caste Species Foregut Midgut Hindgut worker R. flavipes -0.04 + 0.02a 0.02 + 0.02b 24.18 + 0.37a worker C. formosanus -0.01 + 0.04a 0.48 + 0.11a 25.13 + 0.43a soldier R. flavipes -0.03 + 0.03a 0.21 + 0.08b 21.51 + 0.27b soldier C. formosanus -0.03 + 0.01a 0.01 + 0.01b 00.99 + 0.18c Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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59 Table 4-5. A comparison of be ta-xylosidase activities in Reticulitermes flavipes and Coptotermes formosanus workers and soldiers Caste Species Foregut Midgut Hindgut worker R. flavipes 0.0056 + 0.0006bc 0.0072 + 0.0003c 0.0399 + 0.0006b worker C. formosanus 0.0075 + 0.0006a 0.0014 + 0.0004a 0.0645 + 0.0011a soldier R. flavipes 0.0049 + 0.0004c 0.0011 + 0.0003b 0.0216 + 0.0003c soldier C. formosanus 0.0067 + 0.0003ab 0.0015 + 0.0004a 0.0134 + 0.0002d Beta-xylosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Student-Neuman-Keuls Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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60 Table 4-6. A comparison of sol uble protein concentrations in Reticulitermes flavipes and Coptotermes formosanus worker and soldier gut extracts pH Caste Species Foregut Midgut Hindgut 5.5 Worker R. flavipes 1.304 + 0.018 1.126 + 0.046 0 6.954 + 0.208 5.5 Worker C. formosanus 0.944 + 0.052 1.714 + 0.032 11.312 + 0.060 5.5 Soldier R. flavipes 0.420 + 0.001 0.676 + 0.014 0 3.808 + 0.156 5.5 Soldier C. formosanus 0.256 + 0.001 0.354 + 0.016 0 0.622 + 0.078 6.5 Worker R. flavipes 2.250 + 0.060 4.192 + 0.128 15.726 + 0.852 6.5 Worker C. formosanus 0.181 + 0.014 3.952 + 0.088 21.522 + 0.228 6.5 Soldier R. flavipes 1.232 + 0.030 2.234 + 0.052 0 9.832 + 0.500 6.5 Soldier C. formosanus 0.296 + 0.032 0.556 + 0.016 0 2.312 + 0.052 Concentrations are in g per termite gut region, n = 3 replicates.

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61 Figure 4-1. Digestive systems of: A) R. flavipes worker, B) C. formosanus worker, C) R. flavipes soldier, and D) C. formosanus soldier.

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62 Figure 4-2. Heads and frontal glands of a R. flavipes soldier, and a C. formosanus soldier.

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63 CHAPTER 5 CHANGES IN RETICULITERMES FLAVIPES GUT CELLULOLYTIC ACTIVITIES IN RESPONSE TO DIET Introduction Subterranean termites, such as Reticulitermes flavipes (Kollar), subsist la rgely on a diet of wood and similar material (Noi rot and Noirot-Timothee 1969). Wood is not a substance that most animals are capable of digesting to any sign ificant degree. Termites are well known for the ability to digest cellulose with th e aid of microbial hindgut symbionts. In wood, cellulose chains are typically arrang ed in parallel bundles known as microfibers which are embedded in a matrix of lignin and hemi celluloses. The cellulose in the microfibers may be broadly divided into two types: crystalline and amorphous. The crystalline form of cellulose consists of tightly a ligned parallel chains, held in a specific configuration by hydrogen bonding. In the amorphous form of cellulose, the chains are more randomly arranged, and not so closely bound together. There are three major types of cellulase whic h work together to digest cellulose. Exoglucanase (EC 3.2.1.91) cleaves the cellulose chain from the ends, typically producing cellobiose, and is most active against crysta lline cellulose. Endoglucanase (EC 3.2.1.4) cleaves the cellulose chain randomly al ong its length and is most activ e against amorphous cellulose. Beta-Glucosidase (3.2.1.21) cleaves cellobiose a nd other small cellulose fragments, hydrolyzing them to glucose. The gut of R. flavipes and related termites consists of a foregut with a small crop and a proventriculus, a fairly simple midgut, and a gr eatly expanded hindgut containing several species of symbiotic protozoa, fungi, bacteria, and ar chaea (Yamin 1979, Lewis and Forschler 2004). A pair of salivary glands is al so present, emptying into the foregut anterior to the crop. To effectively digest wood, a chemically demanding and nutritionally poor substrate, termites like

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64 R. flavipes have evolved an array of enzymes. Some of the cellulases characterized from the Rhinotermitidae have been shown to be endoge nous, being produced naturally by the termite (Watanabe et al. 1998, Watanabe et al. 1997). Others have been show n to be symbiotic in origin, produced by flagellate symbionts within the hi ndgut (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al. 2000). Zhou et al. (2007) demons trated that exoglucanase activity is largely localized to the hindgut of R. flavipes strongly implicating the resident flagellates as the major agents of crystalline cellulose digestion. I noue et al. (1997) showed that the protozoan composition of the Reticulitermes speratus (Kolbe) hindgut significantly changes when the termites are fed on pure cellulose or pure xylan, as opposed to wood. It is probable that, with changes in protozoan populations on differing diet s, the levels of different cellulase activities may also change. Subterranean termites may encounter a numbe r of potential food sources during foraging. These may be broadly grouped into hardwoods, softwoods, and processed cellulosic materials like paper. These three groups vary mainly in th eir hemicellulose content. In homes, hardwoods may include furniture, flooring, a nd trim components. Structural timbers are nearly always derived from softwoods. Our objectives were to: 1) confirm the patterns of R. flavipes worker cellulolytic activity seen in Chapter 4, 2) determine how the three major cellulase activities found in the gut of R. flavipes workers change in response to thr ee different simulated field diets. Materials and Methods Termite Collection R. flavipes termites were field collected in termite traps consisting of a PVC bucket (20 cm high by 20 cm diam.; Item # 811192-4, Ventura P ackaging Inc., Monroeville, Ohio) with 11 holes drilled in the sides and base (3 cm diam.) placed vertically in the ground to a depth of

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65 roughly 19 cm and covered with a PVC lid. Three rolls of single-faced corrugated cardboard (20 cm long by 10 cm diam.) were place d into the bucket side by side as a food source. Termites were collected from the trap by removal of cardb oard rolls, separated from the cardboard, and either placed in feeding bioassays or immediately frozen and kept at -80oC until dissection. Collections were restricted to a single colony to eliminate colony as a so urce of variation in enzyme activities. Termite Diets and Feeding Three diets were prepared for the termites to represent the probable food sources they would encounter in the field. These simulated field diets were as follows: red oak (Quercus spp.), pine (Pinus spp.), and filte r paper. Wood diets were gene rated by drilling into craft wood boards (0.635 x 5.08 x 60.96 cm) with a 2.54 cm spad e drill bit. Sawdus t was collected and weighed. Filter paper consisted of a weighed num ber of crumpled cellulose filter paper disks (42.5 mm diameter, Whatman, grade 4). Each field diet (20 g) was added to a l oosely capped 250 mL glass bottle (Pyrex) with 5 mL of deionized water. Field di ets were not made from sterile ma terials. Because of this, the bottles with field diets were auto claved on a liquid cycle (30 min, 122oC) to sterilize the diets and the bottles were then allowe d to cool. Termites were added (~300 workers and 3 soldiers per bottle) after the bottles had cooled and kept in the dark at 21oC for 6 wk, with deionized water (2 mL) added every 2 wk. Termites were collected, frozen and kept at -80oC until dissection. Termite Dissection and Enzyme Extraction Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5, and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the endoglucanase assay, while sodium phosphate buffe r was used for the exoglucanase and betaglucosidase assays.

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66 Termites were removed from the freezer and ke pt on ice until dissecti on. Each termites gut was removed intact and separated into thr ee regions: foregut (and sa livary glands), midgut, and hindgut. A single enzyme extract was prepar ed from a minimum of 35 dissected termites from each feeding treatment for each assay type us ing an experimental design similar to previous termite carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For the endoglucanase assay, 50 termites from each feeding treatment were dissected in sodium acetate buffer. For the exoglucanase and beta-glucosidase assays, 35 termites from each feeding treatment were dissected in sodium phospha te buffer. The three gut regions were placed into separate 1.5 mL microcentr ifuge tubes (Eppendorf) containi ng the appropriate buffer, and kept on ice. Final concentrations were 50 termite gut regions per mL in all cases. Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and manually homogenized on ice. The homoge nates were centrifuged at 20,800 g at 4oC for 15 min. The supernatants were colle cted, frozen, and kept at -80oC until use in the enzyme assays. Endoglucanase Assays The endoglucanase assays were conducted usi ng a method adapted from Han et al. (1995). A 2% solution of carboxymethylcellulose (CMC; Si gma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of CMC solution. The solutions were allowed to react for 70 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each

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67 microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar contro l plates were allowed to react for 10 min to allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 10 min reaction was used as a control to correct for any differences in initial sample reaction rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated using dilutions of glucose. Only the wells in the middle of the microplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boili ng. For all replicates, the contro l plates were used to adjust for 540 nm absorbance in gut extracts and were re plicated an equal number of times to the assay plates, with one microplate well for each replicate. Exoglucanase and Beta-glucosidase Assays The exoglucanase and beta-glucosidase assa y was conducted using a method adapted from Han et al. (1995). Solutions of 4 mM p-nitrophenyl-D-cellobioside (p NPC) and 4 mM pnitrophenyl-D-glucopyranoside (pNPG) were prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPC or pNPG solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was r ead at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Sta ndards were generated using dilutions of pnitrophenol. Data Analysis The endoglucanase assays were set up as a one-f actor split-plot design with four technical replicates for each gut region, and one microplate per replicate. Four microplate wells were

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68 assayed for each replicate of each gut region/di et combination. The exoglucanase and betaglucosidase assays were set up as one-factor designs with four t echnical replicates for each gut region. Two homogenates were used for each gut/diet combination; one homogenate for endoglucanase assays and another for exogl ucanse and beta-glucosidase assays. For the endoglucanase assays, the following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT where: SA = specific activity (nmol reducing su gar per termite equivalent per min), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample. For the exoglucanase and beta-glucosidase assays, the following formula was used to calculate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample. The field diet data were analyzed using a mixe d model analysis of variance. Fixed effects were diet treatment and gut regi on. The Tukey-Kramer adjustment = 0.05) was used to separate the mean activities on each diet within each gut region (SAS Institute 2001). Results Termite Gut Observations During dissection, after the termites had been fed on the various diets, the color of the termite guts reflected the color of the different diets. This was es pecially evident in the enlarged

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69 hindguts (Fig. 1), which were typically filled w ith a mixture of partially digested food and microbes. Termites fed on red oak had brownish -orange gut contents. Those fed on pine had pale yellow gut contents. Those fed on paper ha d white gut contents. The termites collected from the field and immediately frozen for di ssection had relatively dark gut contents. Endoglucanase Activities Most of the endoglucanase activity was located in the foregut and th e hindgut, with very little in the midgut (Table 5-1). Foregut endoglucanase activity in the field-collected termites was slightly lower than the foregut activity seen on paper, and greater th an activity seen on the other two diets (Table 5-1). Among the three field diets, fore gut activity was highest on paper and lowest on oak. Midgut endogl ucanase activity in the field-co llected termites was higher than the midgut activity seen on all th ree diets. Among the three fi eld diets, midgut activity was highest on pine and lowest on paper. Hindgut endoglucanase ac tivity in the field-collected termites was higher than the hindgut activity seen on all th ree diets. Among th e three field diets, hindgut activity was highest on oak and lowest on paper. Exoglucanase Activities Foregut exoglucanase activity in the field coll ected termites was significantly higher than the foregut activity seen all thr ee diets (Table 5-2). Among the three diets, foregut activity was highest on paper and lowest on oa k. Midgut exoglucanas e activity in the fiel d collected termites was intermediate between the midgut activities seen on oak and pine. Among the three diets, midgut activity was highest on pa per and lowest on oak. Hindgut exoglucanase activity in the field collected termites was higher than the hind gut activity seen on all three diets. Among the three diets, hindgut activity was highest on oak a nd lowest on paper. The activity seen on pine was nearly as high as that seen on oak.

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70 Beta-glucosidase Activities Foregut beta-glucosidase activity in the field collected termites was intermediate between the foregut activities seen on paper and pine (Tab le 5-3). Among the three diets, foregut activity was highest on paper and lowest on oak. Midgut beta-glucosidase ac tivity in the field collected termites was intermediate between the midgut activities seen on pape r and pine. Among the three diets, midgut activity was highest on paper and lowest on oak. Hindgut beta-glucosidase activity in the field collected termites was higher th an the hindgut activity seen on all three diets. Among the three diets, hindgut activity wa s highest on oak and lowest on paper. Discussion The overall gut morphology is consis tent with that described for R. flavipes in previous studies. The appearance of the termite guts upon dissection, particularly the hindguts, indicated that the termites had fed on their respective diets. Based on the relatively dark gut contents of the field termites, as well as the presence of dark oval objects in some cases, it is possible that these termites were feeding on partially decayed materials. Based on the observed locations of the activities, it is evident that both endoglucanases and beta-glucosidases are produced by both the termite and its symbionts. Endogenous endoglucanases are produced in the foregut, while symbiont endoglucanases are produced in the hindgut. Beta-glucosidase activi ties have been seen throughout the termite gut, leading to the conclusion that endogenous beta-glucosidases are produced in the foregut and midgut, while symbiont beta-glucosidases are produced in the hindgut. The overall cellulase activity pa ttern found in this study was similar to that described by Inoue et al. (1997) for R. sperat us, but the levels of activity ar e roughly 10 to 100-fold lower in this study. These different results are due to a number of factors. Fi rst, rather than an exoglucanase assay, Inoue et al. (1997) performed a total cellulase assay, so these data cannot be

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71 directly compared. In addition, I noue et al. used tetrazolium blue rather than DNSA to determine the reducing sugars generated by en doglucanase. The advantage of tetrazolium blue is that it is highly sensitive to reducing sugars. However, tetrazolium blue reacts continuously at room temperature and the reacted reagent eventually precipitates out of solution. The DNSA reagent requires boiling to react with reducing sugars. Th erefore, it is possible to get more precise reaction times, reducing the varian ce of the results. The enzymatic reactions in their study were conducted at a higher incubation temperature (25oC) than ours (23oC). In addition, we cannot directly compare the resu lts because the two assays were carried out on different species. The Inoue study used R. sp eratus collected from lo gs, while our study used R. flavipes collected from cardboard traps. When th ese differences in methods are taken into account, our findings are cons istent with those of Inoue et al. (1997). The overall endoglucanase activities did not di ffer significantly among the three diets, but the source of the enzymes did. The activitie s of both endogenous endoglucanase, produced by the termite in the foregut and midgut, and symb iotic endoglucanase, produced by microbes in the hindgut, varied among diets. Endogenous activitie s were highest on paper and lowest on oak, but the exact reverse trend was seen with the symbiont endoglucanase activities, which were highest on the oak diet and lowest on paper. Based upon these data it is probable that either the termite is changing its endogenous enzyme output in response to fluctuatio ns in its symbiont enzymes, or else the symbiont population is fluc tuating in response to th e levels of endogenous termite enzymes. If the termite is responding to changes in its symbiont community composition, then some qualities of the diet must be driving the symbi ont population changes. It has previously been demonstrated that symbionts are responsible for vi rtually all of the xylanolytic activity seen in R.

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72 flavipes worker termites (Zhou et al. 2007, Smith and Koehler 2007). It is reasonable to suppose that the xylan content of a give n diet may affect the symbiont populations, and therefore affect the activity levels of the enzymes produced by thes e symbionts. All symbiont enzyme activities were significantly higher on diets with a higher xy lan content. Hindgut activities in all cases were the highest on the oak diet. Hardwoods typica lly contain more than twice as much xylan as softwoods (Pettersen 1984). At the same time, hindgut activities were lowe st on the paper diet, a diet completely lacking in xylan. Alternatively, the quality of the cellulose conten t in the diet may be affecting the termites ability to digest it without the aid of sy mbionts. While the termite produces its own endoglucanases and beta-glucosidases, it relies on its symbionts for at least the majority of its exoglucanase production. This means that the termite is most likely capable of degrading amorphous cellulose, but relies heavily upon its symb ionts to digest crystalline cellulose. The proportion of crystalline cellulose may be anywhere from 50% to 70% of total cellulose content in different wood species (Biermann 1996). The pr ocess of making paper partially denatures the cellulose microfibers in wood, a nd may increase the ratio of amor phous cellulose to crystalline cellulose. The increase in endoge nous cellulolytic activities on the paper diet may therefore be due to greater availability of substrates that the termite can digest without the aid of its symbionts. With more of this digestion and absorption achieved before the food reaches the hindgut, the resident symbiont population may be reduced by the more limited nutrient availability. Studies on Reticulitermes speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes virginicus (Cook and Gold 2000) and Coptotermes formosanus (Mannesmann 1972, Waller and La Fage 1987) have demonstrated significant ch anges in the hindgut protozoan communities in

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73 response to different diets, incl uding different wood species, pure ce llulose and pure xylan. It is probable that the hindgut enzyme changes in R. flavipes are due to changes in the protozoan populations. Alternatively, there is the possibility that individual termite symbionts change their levels of enzyme production in response to differing diets. It is apparent that R. flavipes workers are quite capable of digesting cellulose, following the same pattern seen in Chapter 4. Crystalline ce llulose is mainly digested by hindgut symbionts while amorphous cellulose and cello dextrins are digested by both th e termites and its symbionts. In addition, the balance of endogenous versus symb iont cellulolytic activi ties appears to change in response to their diet, most likely by change s in the hindgut protozoan communities as well as the termite enzyme expression. This flexibility a llows them to efficiently utilize a variety of wood species and wood-derived materials which ha ve different qualities of cellulose. This capacity for adaptation and partia l balancing between the termite and its symbionts also makes termite control by means of cellulase inhibition more difficult.

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74 Table 5-1. Endoglucanase activitie s in the three gut regions of Reticulitermes flavipes workers in response to different field diets Diet Foregut Midgut Hindgut Field 9.286 + 0.165a 0.471 + 0.107a 8.187 + 0.938a Paper 9.649 + 0.246a 0.216 + 0.102a 5.789 + 0.610c Pine 8.311 + 1.048ab 0.328 + 0.098a 6.765 + 0.166bc Oak 7.438 + 0.664b 0.291 + 0.185a 7.663 + 0.842ab Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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75 Table 5-2. Exoglucanase activitie s in the three gut regions of Reticulitermes flavipes workers in response to different field diets Diet Foregut Midgut Hindgut Field 0.0121 + 0.0004a 0.0062 + 0.0011bc 0.1654 + 0.0026a Paper 0.0062 + 0.0006b 0.0083 + 0.0002a 0.1011 + 0.006d Pine 0.0044 + 0.0004c 0.0078 + 0.0002ab 0.1486 + 0.0032c Oak 0.0031 + 0.0008c 0.0054 + 0.0002c 0.1554 + 0.0032b Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly differe nt, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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76 Table 5-3. Beta-glucosidaase activities in the three gut regions of Reticulitermes flavipes workers in response to different field diets Diet Foregut Midgut Hindgut Field 0.1196 + 0.0017b 0.2128 + 0.0016a 0.3229 + 0.0026a Paper 0.1681 + 0.0016a 0.2161 + 0.0036a 0.2289 + 0.0067d Pine 0.0991 + 0.0023c 0.1725 + 0.0056b 0.2674 + 0.0077c Oak 0.0888 + 0.0015d 0.1445 + 0.0026c 0.3037 + 0.0042b Beta-glucosidase activities are in nmol p-nitrophenol pe r termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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77 CHAPTER 6 CHANGES IN RETICULITERMES FLAVIPES GUT XYLANOLYTIC ACTIVITIES IN RESPONSE TO DIET Introduction Subterranean termites, such as Reticulitermes flavipes (Kollar), subsist la rgely on a diet of wood and similar material (Noi rot and Noirot-Timothee 1969). Wood is not a substance that most animals are capable of digesting to any sign ificant degree. Termites are well known for the ability to digest cellulose, but there are other polysaccharides in wood, such as xylan, which the termites may utilize in order to get mo re energy out of this food source. In wood, cellulose chains are ty pically arranged in parallel bundles known as microfibers, which are embedded in a matrix composed mainly of hemicelluloses and lignin. Hemicelluloses are polysaccharides like cellulose, but their chemical structures are far more variable. One of the well-studied varieties of hemicellulose is xylan, which is mainly a polymer of the pentose sugar xylose. Xylan is the predominant hemicellulose in hardwood. Softwood t ypically is much lower in xylan content, along with significant quantit ies of other hemicelluloses (Pettersen 1984). Subterranean termites may encounter a numbe r of potential food sources during foraging. These may be broadly grouped into hardwoods, softwoods, and man-made cellulosic materials like paper. These three groups vary mainly in their xylan content, wi th hardwoods containing the most xylan while man-made cellulosic mate rials contain virtually no hemicellulose. In homes, hardwoods may include furniture, flooring, and trim components. Structural timbers are nearly always derived from softwoods. The gut of R. flavipes and related termites consists of a foregut with a small crop and a proventriculus, a fairly simple midgut and a gr eatly expanded hindgut containing several species of symbiotic protozoa, fungi, bacteria, and ar chaea (Yamin 1979, Lewis and Forschler 2004). A pair of salivary glands is al so present, emptying into the foregut anterior to the crop. To

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78 effectively digest wood, a chemically demanding and nutritionally poor substrate, termites like R. flavipes have developed an array of enzymes. Some carbohydrolases characterized from the Rhinotermitidae have been shown to be endogeno us (Watanabe et al. 1998), while others are produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al. 2000). Xylanolytic enzymes include xylanases (EC 3.2.1.8) which internally cleave the xylan chain and beta-xylosidases (EC 3.2.1.37) which cleave xylan oligosaccharides into xylose. Inoue et al. (1997) demonstrated that xylanase activity is largel y localized to the hindgut of Reticulitermes speratus (Kolbe), strongly implicating the resi dent protozoa as the major agents of xylan digestion. In the same study, they s howed that the protozoan composition of the R. speratus hindgut significantly changes when the termites are fed on pure cellulose or pure xylan, as opposed to wood. Through similar experiments in differential feeding, Azuma et al. (1993) showed that, within the termite R. speratus protozoan symbionts in the genera Pyrsonympha and Dinenympha take part in xylan digest ion. It is probable that, w ith the changes in protozoan populations, the capacity for xylan digestion may also change to adapt to the xylan content of the termite diet. Our objectives were to determine 1) if R. flavipes can digest xylan, 2) the relative activities of xylanase and beta-xylosidase in different regions of the gut of R. flavipes workers, 3) possible correlations between these activitie s and dietary xylan content in fi ve formulated cellulose/xylan diets, and 4) how these activiti es change in response to three different field diets. Materials and Methods Termite Collection R. flavipes termites were field collected in Gainesville, Florida in termite traps consisting of a PVC bucket (20 cm high by 20 cm diam .; Item # 811192-4, Ventura Packaging Inc., Monroeville, Ohio) with 11 holes dril led in the sides and base (3 cm diam.) placed vertically in

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79 the ground to a depth of roughly 19 cm and covered with a PVC lid. Three rolls of single-faced corrugated cardboard (20 cm long by 10 cm diam.) were placed into the buc ket side by side as a food source. Termites were collected from th e trap by removal of cardboard rolls, separated from the cardboard, and either placed in feeding bioassays or immediately frozen and kept at 80oC until dissection. Termites were collected from one colony for field diet assays and another colony for formulated diet assays. Collections we re restricted to a sing le colony in each case to eliminate colony as a source of variation in en zyme activities. Termite Diets and Feeding Five formulated diets consisting of microcrystalline cellulose and beechwood xylan (>90% xylose residues) (Sigma-Aldrich, Atlanta, GA) were prepared for the termites: 0%, 5%, 10%, 20%, and 40% xylan. The remainder of the diet wa s composed of cellulose in all cases. The cellulose and xylan were mixed together and wate r (1.5 mL/g) was added to make a paste. This paste was thoroughly kneaded to achieve homogeneity. For each formulated diet, a glass jar ( 125 mL, straight-sided, Fisherbrand, Fisher Scientific, Pittsborough, PA) was prepared with 80 g of moist builders sand (10% moisture). The bottom was cut out of a plastic cup (5.92 mL Souffl cup, Solo Cup Company, Urbana, IL), leaving approximately 2 mm of cup wall intact. Formulated diet (1 g) was placed in the cup bottom and this was placed on top of the sand in the jar. Termites were added (~200 workers and 2 soldiers per jar) an d kept in the dark at 21oC for 2 wk. Termites were collected, frozen and kept at -80oC until dissection. Three diets were prepared for the termites to represent the probable food sources they would encounter in the field. Th ese field diets were as follows: red oak (Quercus spp.), pine (Pinus spp.), and filter paper. Wood diets we re generated by drilling into craft wood boards (0.635 x 5.08 x 60.96 cm, TOMS) with a 2.54 cm spad e drill bit. Sawdust was collected and

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80 weighed. Filter paper consisted of a weighed num ber of crumpled cellulose filter paper disks (42.5 mm diameter, Whatman, grade 4). Each field diet (20 g) was added to a l oosely capped 250 mL glass bottle (Pyrex) with 5 mL of deionized water. Unlike the formulated di ets, the field diets were not made from sterile materials. Because of this, the bottles with fiel d diets were autoclaved on a liquid cycle (30 min, 122oC) to sterilize the diets and the bottles were then allowed to cool. Termites were added (~300 workers and 3 soldiers per bot tle) after the bottles had cooled and kept in the dark at 21oC for 6 wk, with deionized water (2 mL) added ev ery 2 wk. Termites were collected, frozen and kept at -80oC until dissection. Termite Dissection and Enzyme Extraction Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5, and 0.1 M sodium phosphate buffer, pH 6.5. Sodi um acetate buffer was used for the xylanase assay, while sodium phosphate buffer was used for the beta-xylosidase assays. Termites were removed from the freezer and ke pt on ice until dissecti on. Each termites gut was removed intact and separated into thr ee regions: foregut (and sa livary glands), midgut, and hindgut. A single enzyme extract was prepared from dissected termites from each feeding treatment for each assay type using an experi mental design similar to previous termite carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For the xylanase assay, 50 termites from each feeding treatment were dissected in sodium acetate buffer. For the beta-xylosidase assays, 35 termite s from each feeding treatment were dissected in sodium phosphate buffer. The three gut regions were placed into separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ice. Final concentrations were 50 termite gut regions per mL in all cases.

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81 Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and manually homogenized on ice. The homoge nates were centrifuged at 20,800 g at 4oC for 15 min. The supernatants were colle cted, frozen, and kept at -80oC until use in the enzyme assays. Xylanase Assays The xylanase assays were conducted using a method adapted from Han et al. (1995). A 0.5% solution of beechwood xylan (>90% xylose re sidues; Sigma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. The soluti on was boiled approximately 30 min, until xylan particles were no longer visible. The solu tion was centrifuged at 1250 g for 5 min at 23oC and the supernatant was used as the xylan stock solution. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of xylan solution. The solutions were allowed to react for 35 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar control pl ates were allowed to react for 5 min to allow for passive mixing of solutions before boiling with DNSA soluti on (Zhou et al. 2007). A 5 min reaction was used as a control to correct for any di fferences in initial sample reaction rates due to slow mixture of enzyme and substrate solutions. Standards were generated using dilutions of xylose. Only the wells in the middle of the mi croplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boiling. For all replicates, the control plates we re used to adjust for 540 nm

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82 absorbance in gut extracts and were replicated an equal number of times to the assay plates, with one microplate well for each replicate. Beta-Xylosidase Assays Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-xylopyranoside (pNPX; SigmaAldrich) was prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays we re conducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPX solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. Data Analysis The xylanase assays were set up as one-factor split-plot designs with seven technical replicates per gut region for each formulated diet four technical replicates per gut region for each simulated field diet, and one microplate per replicate. Four microplate wells were assayed for each replicate of each gut region/diet combinat ion. The beta-xylosidase assays were set up as one-factor designs with four technical replicates per gut region for each formulated diet and simulated field diet. Two homogenates were used for each gut/diet combination; one homogenate for xylanase assays and another for beta-xylosidase assays. For the xylanase assays, the following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT where: SA = specific activity (nmol reducing su gar per termite equivalent per min), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after

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83 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample. For the beta-xylosidase assa ys, the following formula was used to calculate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample. The field diet data were analyzed using a mixe d model analysis of variance. Fixed effects were diet treatment and gut regi on. The Tukey-Kramer adjustment = 0.05) was used to separate the mean activities on each diet within each gut region (SAS Institute 2001). Results Termite Gut Observations During dissection, after the termites had been fed on the various diets, the color of the termite guts reflected the color of the different diets. This was es pecially evident in the enlarged hindguts which were typically fill ed with a mixture of partially digested food and resident microbes. Termites fed on formulated diets sh owed an increasing brown shade in their gut contents on diets containing mo re xylan. Termites fed on red oak had brownish-orange gut contents. Those fed on pine had pale yellow gut contents. Those fed on paper had white gut contents. The termites collected from the fi eld and immediately frozen for dissection had relatively dark gut contents. Xylanase Activities Both foregut and midgut xylanase activities were insignificant in comparison with hindgut xylanase activity on all of the formulated diets (Figure 6-1) Hindgut xylanase activity was

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84 significantly greater in termites kept on 20% and 40% xylan diets than in termites kept on 0%, 5% and 10% xylan diets (Figure 6-1). Although there was higher activity in termites fed on 5% xylan than 10% xylan, the diffe rence was not significant. Foregut xylanase activity was greater in the field-collected termites than activities in termites fed on any of the three field diets (Table 6-1). Foregut activities on pine and paper diets differed significantly, with activity on pine be ing the highest and activ ity on paper being the lowest. Midgut xylanase activity in field-collected termites was between the activities on pine and oak diets. Among the field diets, midgut activity was significantly highest on pine and lowest on paper. Hindgut xylanase activity wa s >92% of total xylan ase activity among the different diets. The hindgut activ ity of field-collected termites was slightly higher than that on paper, but not significantly so. The hindgut acti vity differed significantly among the three field diets, being highest on oa k and lowest on paper. Beta-Xylosidase Activities Overall beta-xylosidase activity showed a general increase from termites fed 0% xylan to termites fed 40% xylan (Figure 6-2). There we re no significant differe nces among the foregut beta-xylosidase activities on the formulated di ets. Midgut beta-xylosidase activities were greatest in termites fed 10% xylan. Activities we re intermediate and nearly equal on 5% and 20% xylan, and they were lowest and nearly equal on 0% and 40% xylan. Hindgut betaxylosidase activities showed a steady increase on the formulated diets from 5% xylan to 40% xylan (Figure 6-2). Activities in termites fed 20 % and 40% xylan were si gnificantly greater than those in termites fed 0% and 5% xylan. In ad dition, beta-xylosidase ac tivities in termites fed 40% xylan were significantly greater than activities in termites fed 10% xylan. There were no significant di fferences among the foregut be ta-xylosidase activities in termites fed field diets (Table 6-2). Midgut beta-xylosidase ac tivity was lowest on the field-

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85 collected termites, which did not differ significan tly from the activity on pine. Among the diets, midgut activity was highest on pape r and lowest on pine. Sim ilar to xylanase activity, betaxylosidase activity was predominantly located in the hindgut. However, this was not as pronounced as with xylanase, as hindgut beta-xyl osidase activity ranged from ~50 to 75% of total beta-xylosidase activity. Hindgut activity of the field-collected termites was intermediate between the activities on oak and pi ne diets. Among the field di ets, activity was highest on oak and lowest on paper. Hindgut beta-xylosidase ac tivities on all four of these treatments were significantly different from one another. Discussion The overall gut morphology of R. flavipes workers was similar to that described for Zootermopsis (Child 1946) and is consis tent with that found in othe r lower termites (Noirot and Noirot-Timothee 1969). The appearance of the termite guts upon dissect ion, particularly the hindguts, indicated that the termite s had fed on their respective diets. Based on the relatively dark gut contents of the field-collected termites, it was apparent that these termites were feeding on relatively dark materials, such as cardboard or pine bark mulch, associated with the termite traps. Among the termites in the colony fed on form ulated diets, both xylanase and betaxylosidase total activities were significantly hi gher on diets with highe r xylan content. The hindgut activities in particular, wh ich formed the majority of total activities, also followed this pattern. A similar pattern was seen in term ites from the colony fed on field diets. All xylanolytic activities were highest on the oak diet. Hardwoods typically consist of roughly 20% xylan, twice as much as the typical softwood xyl an content of roughly 10% (Biermann 1996). At the same time, hindgut activities were lowest on the paper diet, a diet completely lacking in xylan. Just as termites fed on 20% xylan showed far greater xylanase activity than termites fed

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86 on 10% xylan, so there was a similar jump in activity from pine fed termites to oak fed termites. There was a much smaller gap be tween the hindgut xylanas e activities of paper fed and pine fed termites, as there was a much smaller gap betw een the activities of termites fed 0% and 10% xylan. The hindgut beta-xylosid ase patterns were also consis tent between termites fed on formulated diets and term ites fed on field diets. Our data showed that the majority of xylanase activity was in the R. flavipes hindgut. This is consistent with previous fi ndings where xylanase activities were almost exclusively located in the hindgut of R. speratus and Coptotermes heimi (Wasmann), and associated with the symbionts (Inoue et al. 1997, Mishra 1991). Th erefore, it was evident that R. flavipes workers follow a typical xylan digestion pattern fo r subterranean termites, where this hemicellulose is mainly digested by hindgut symbionts. The overall digestion pattern of xylanase and beta-xylosidase activity found in our study using the field termites was similar to that found by Inoue et al. (1997), but the levels of activity are roughly 10 to 100-fold lower in our study. Th ese differences may be due to differences in assay reagents or differences in termite species. Inoue et al. (1997) used tetrazolium blue rather than DNSA to determine the reducing sugars ge nerated by xylanase. We used the DNSA reagent because it requires boiling to reac t with reducing sugars, in contra st with tetrazolium blue, which reacts continuously at room temperature. This boiling requirement allowed more precise control of reaction times, limiting the overestimation of activity. Our findings are consistent with those of Inoue et al. (1997) when the differences in me thods are taken into acco unt. Inoue et al. (1997) observed no beta-xylosidase ac tivity in the foregut of R. speratus in contrast with our findings of minor beta-xylosidase activity in the foregut of R. flavipes We cannot directly compare these results because the two assays we re carried out on different speci es of termite; the Inoue et al.

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87 (1997) study used R. speratus collected from logs, while our study used R. flavipes collected from cardboard traps. The low hindgut xylanolytic activities on the pa per diet and the 0% xyl an formulated diet were consistent with the absence of xylan in the paper diet. Ho wever, significant xylanase and beta-xylosidase activities were st ill present even in the absence of dietary xylan. This suggests the presence of symbionts that constitutively produce xylanases and beta-xylosidases, but are capable of subsisting solely on a cellulose diet. The relatively low hindgut xylanolytic activity in the field-collected termites, similar to the activity on the paper diet, was mo st likely due either to feeding on the cardboard in the termite traps, or feeding on some form of softwood such as pine mulch. Symbiont xylanase and beta-xylos idase activities change signif icantly in response to diet, consistent with xylan content. It is apparent that the community of hindgut symbionts is able to adapt to a wide range of diet ary xylan content. Studies on R. speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes virginicus (Banks) (Cook and Gold 2000) and Coptotermes formosanus (Shiraki) (Mannesmann 1972, Waller and La Fage 1987) have demonstrated significant changes in the hindgut protozoan commun ities in response to different diets. Many of these studies focused on feeding termites different wood species. It is ther efore probable that the xylanolytic enzyme changes in R. flavipes are due to changes in hindgut symbiont populations. Alternatively, it is possible that these enzyme ch anges are due to changes in xylanolytic enzyme production within the hindgut symbionts. R. flavipes workers are capable of digesting xyla n, following the same pattern seen in Chapter 4, where xylan is mainly digested in th e hindgut. The xylanolytic activities of the two termite colonies we investigated changed to accommodate dietary xylan content, most likely by

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88 changes in the hindgut symbiont communities. These termites capacity for xylan digestion allows them to gain more energy from a wood diet, and their flexibility allows them to efficiently utilize diets of varying xylan content. Compared to cellulose, xylan is often overlooked as a starting compound for the production of alternative fuels. However, xylan may comprise up to a quarter of wood, depending on the species, and a significant pr oportion of other plan t materials used for alternative fuel production. Thes e termites and their symbionts may provide candidate enzymes for the degradation of xylan in this process.

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89 Table 6-1. Xylanase activities in the three gut regions of Reticulitermes flavipes workers in response to different field diets Diet Foregut Midgut Hindgut Field 0.833 + 0.048a 0.258 + 0.064a 12.672 + 0.249c Paper 0.062 + 0.044b 0.030 + 0.078a 12.436 + 0.260c Pine 0.216 + 0.051ab 0.324 + 0.088a 13.672 + 0.335b Oak 0.017 + 0.045ab 0.196 + 0.052a 22.953 + 0.315a Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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90 Table 6-2. Beta-xylosidase activit ies in the three gut regions of Reticulitermes flavipes workers in response to different field diets Diet Foregut Midgut Hindgut Field 2.50 + 0.22a 4.08 + 0.21b 21.08 + 0.20b Paper 2.63 + 0.05a 9.25 + 0.09a 11.91 + 0.32d Pine 1.82 + 0.23a 5.70 + 0.09b 16.91 + 0.32c Oak 2.49 + 0.05a 7.04 + 0.32b 28.26 + 1.00a Beta-xylosidase activities are in pmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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91 -5 0 5 10 15 20 0510152025303540% Xylan in DietSpecific Activity (nmol/termite/min) Foregut Midgut Hindgut Figure 6-1. Xylanase activities in different gut regions of Reticulitermes flavipes in response to dietary xylan content.

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92 0 0.01 0.02 0.03 0.04 0510152025303540% Xylan in DietSpecific Activity (nmol/termite/min) Foregut Midgut Hindgut Figure 6-2. Beta-Xylosidase activit ies in different gut regions of Reticulitermes flavipes in response to dietary xylan content.

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93 CHAPTER 7 CHANGES IN COPTOTERMES FORMOSANUS GUT CELLULOLYTIC ACTIVITIES IN RESPONSE TO DIET Introduction Subterranean termites, such as Coptotermes formosanus (Shiraki), subsist largely on a diet of wood and similar material. Wood is not a substance that most animals are capable of digesting to any significant degr ee. Termites are well known for th e ability to digest cellulose with the aid of microbi al hindgut symbionts. In wood, cellulose chains are typically arrang ed in parallel bundles known as microfibers which are embedded in a matrix of lignin and hemi celluloses. The cellulose in the microfibers may be broadly divided into two types: crystalline and amorphous. The crystalline form of cellulose consists of tightly a ligned parallel chains, held in a specific configuration by hydrogen bonding. In the amorphous form of cellulose, the chains are more randomly arranged, and not so closely bound together. There are three major types of cellulase whic h work together to digest cellulose. Exoglucanase (EC 3.2.1.91) cleaves the cellulose chain from the ends, typically producing cellobiose, and is most active against crysta lline cellulose. Endoglucanase (EC 3.2.1.4) cleaves the cellulose chain randomly al ong its length and is most activ e against amorphous cellulose. Beta-Glucosidase (3.2.1.21) cleaves cellobiose a nd other small cellulose fragments, hydrolyzing them to glucose. The gut of C. formosanus and related termites consists of a foregut with a small crop and a proventriculus, a fairly simple midgut, and a greatly expanded hindgut containing four major species symbiotic protozoa, as well as several species of bacteria and archaea (Yamin 1979). A pair of salivary glands is al so present, emptying into the foregut anterior to the crop. To effectively digest wood, a r ecalcitrant and nutritionally poor substrate, termites like C.

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94 formosanus have developed an array of enzymes. So me of the cellulases ch aracterized from the Rhinotermitidae have been shown to be endoge nous (Watanabe et al. 1998, Zhou et al. 2007), while others are produce d by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al. 2000). Zhou et al. (2007) demons trated that exoglucanase activity is largely localized to the hindgut of C. formosanus strongly implicating the resident flagellates as the major agents of crystalline cellulose digestion. Inoue et al. (1997) show ed that the protozoan composition of the Reticulitermes speratus (Kolbe) hindgut significantly changes when the termites are fed on pure cellulose or pure xylan, as opposed to wood. It is probable that, with changes in protozoan populations on differing diet s, the levels of different cellulase activities may also change. Subterranean termites may encounter a numbe r of potential food sources during foraging. These may be broadly grouped into hardwoods, softwoods, and processed cellulosic materials like paper. These three groups vary mainly in th eir hemicellulose content. In homes, hardwoods may include furniture, flooring, a nd trim components. Structural timbers are nearly always derived from softwoods. Our objectives were to determine: 1) the dist ribution of the three major cellulase activities within the gut of C. formosanus workers and 2) how these activities change in response to three different simulated field diets. Materials and Methods Termite Collection Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in Charleston, South Carolina. Termites were coll ected from the trap by removal of the wood (southern yellow pine) in the bait stations and brought to the Un iversity of Florida. Termites

PAGE 95

95 were then placed in feeding bioassays. Collections were restricted to a single colony to eliminate colony as a source of varia tion in enzyme activities. Termite Diets and Feeding Three diets were prepared for the termites to represent the probable food sources they would encounter in the field. These simulated field diets were as follows: red oak (Quercus spp.), pine (Pinus spp.), and filte r paper. Wood diets were gene rated by drilling into craft wood boards (0.635 x 5.08 x 60.96 cm) with a 2.54 cm spad e drill bit. Sawdus t was collected and weighed. Filter paper consisted of a weighed num ber of crumpled cellulose filter paper disks (42.5 mm diameter, Whatman, grade 4). For each simulated field diet, a mason jar (# mL, brand) was prepared with 40 g of moist builders sand (10% moisture). Diet (10 g) was th en added with 40 mL of deionized water and the jar was loosely capped. The jars with simulated field diets were autoclaved on a liquid cycle (30 min, 122oC) to sterilize the diets and the jars were then allowed to cool. Termites were added (~270 workers and 30 soldiers per jar) after the jars had cooled and kept in the dark at 21oC for 6 wk. Termites were collected, frozen and kept at -80oC until dissection. Termite Dissection and Enzyme Extraction Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5, and 0.1 M sodium phosphate buffer, pH 6.5. Sodium acetate buffer was used for the endoglucanase assay, while sodium phosphate buffe r was used for the exoglucanase and betaglucosidase assays. Termites were removed from the freezer and ke pt on ice until dissecti on. Each termites gut was removed intact and separated into thr ee regions: foregut (and sa livary glands), midgut, and hindgut. A single enzyme extract was prepared from dissected termites from each feeding treatment for each assay type usi ng an experimental design simila r to the recent classic termite

PAGE 96

96 carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For the endoglucanase assay, 25 termites from each f eeding treatment were dissected in sodium acetate buffer. For the exoglucanase and beta-glu cosidase assays, 25 termites from each feeding treatment were dissected in sodium phosphate buffe r. The three gut regions were placed into separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ice. Final concentrations were 50 te rmite gut regions per mL in all cases. Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and manually homogenized on ice. The homoge nates were centrifuged at 20,800 g at 4oC for 15 min. The supernatants were colle cted, frozen, and kept at -80oC until use in the enzyme assays. Endoglucanase Assays The endoglucanase assays were conducted usi ng a method adapted from Han et al. (1995). A 2% solution of carboxymethylcellulose (CMC; Si gma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of xylan solution. The solutions were allowed to react for 70 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar contro l plates were allowed to react for 10 min to allow for passive mixing of solutions before boiling with DNSA solution (Zhou et al. 2007). A 10 min reaction was used as a control to correct for any differences in initial sample reaction

PAGE 97

97 rates due to incomplete mixture of enzyme and substrate solutions. Standards were generated using dilutions of glucose. Only the wells in the middle of the microplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boili ng. For all replicates, the contro l plates were used to adjust for 540 nm absorbance in gut extracts and were re plicated an equal number of times to the assay plates, with one microplate well for each replicate. Exoglucanase and Beta-glucosidase Assays The exoglucanase and beta-glucosidase assa y was conducted using a method adapted from Han et al. (1995). Solutions of 4 mM p-nitrophenyl-D-cellobioside (p NPC) and 4 mM pnitrophenyl-D-glucopyranoside (pNPG) were prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays were conducted in clear 96well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPC or pNPG solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT). Absorbance was r ead at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Sta ndards were generated using dilutions of pnitrophenol. Data Analysis The endoglucanase assays were set up as a one-f actor design with four technical replicates for each gut region. Four microplate wells we re assayed for each replicate of each gut region/diet combination. The e xoglucanase and beta-glucosidase assays were set up as onefactor designs with four tec hnical replicates for each gut re gion. Two homogenates were used for each gut/diet combination; one homogenate for endoglucanase assays and another for exoglucanse and beta-glucosidase assays.

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98 For the endoglucanase assays, the following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT where: SA = specific activity (nmol reducing su gar per termite equivalent per min), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample. For the exoglucanase and beta-glucosidase assays, the following formula was used to calculate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample. The field diet data were analyzed using a mixe d model analysis of variance. Fixed effects were diet treatment and gut regi on. The Tukey-Kramer adjustment = 0.05) was used to separate the mean activities on each diet within each gut region (SAS Institute 2001). Results Termite Gut Observations During dissection, after the termites had been fed on the various diets, the color of the termite guts reflected the color of the different diets. This was es pecially evident in the enlarged hindguts, which were typically f illed with a mixture of partia lly digested food and resident microbes. Termites fed on red oak had brownish -orange gut contents. Those fed on pine had pale yellow gut contents. Those fe d on paper had white gut contents.

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99 Endoglucanase Activities Most of the endoglucanase activity was loca ted in the foregut and the hindgut, with relatively little in the midgut (T able 7-1). Among the three di ets, there were no significant differences in foregut activities. Midgut activitie s were significantly lower on the paper diet than on the other two diets. Hindgut activities were significantly hi gher on the oak diet than on the other two diets. Exoglucanase Activities Most of the exoglucanase activity was located in the hindgut, with very little in the foregut or midgut (Table 6-2). Hindgut exoglucanase activ ity was significantly different among all three diets, being highest on oak and lowest on pine. Beta-glucosidase Activities Most of the beta-glucosidase activity wa s located in the midgut and hindgut, with relatively little in the foregut (Table 6-3). Fo regut beta-glucosidase activity was significantly different among all three diets, being highest on pine and lo west on paper. Midgut betaglucosidase activity was significan tly different among all three diet s, being highest on pine and lowest on oak. Hindgut beta-glucosidase activit y was significantly different among all three diets, being highest on oa k and lowest on paper. Discussion The overall gut morphology observed is c onsistent with that described for C. formosanus in previous studies. The a ppearance of the termite guts upon dissection, particularly the hindguts, indicated that the termites had fed on their respective diets. Based on the observed locations of the activities, it is evident that both endoglucanases and beta-glucosidases are produced by both the termite and its symbionts. Endogenous endoglucanases are produced in the foregut, and su ch endoglucanases have been characterized in

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100 a number of termite species from the genera Reticulitermes and Coptotermes (Watanabe et al. 1998, Nakashima et al. 2002b). The endoglucanase activities observed in the hindgut are most likely derived from the hindgut flagellates, as a number of endoglucanases have been characterized from these C. formosanus symbionts (Nakashima et al., 2002a; Watanabe et al., 2002b). Beta-glucosidase activities have been seen throughout the termite gut, leading to the conclusion that endogenous beta-glucosidases are produced in the foregut and midgut, while symbiont beta-glucosidases are produced in the hindgut. In contrast to the other cellulolytic activities, exoglucanase activity appears to be almost entirely due to the symbionts. This is evid ent based on the almost ex clusive distribution of exoglucanase activity within the hindgut. Similar patterns have been observed in Coptotermes lacteus (Hogan et al. 1988). It is possible that endogenous cel lulase expression is inducible by, or at least responsive to, the quality of dietary cellulose. While the termite produces its own endoglucanases and betaglucosidases, it primarily relies on its symbionts for exoglucanase production. This means that the termite is most likely capable of degr ading amorphous cellulose, but relies upon its symbionts to digest crystalline cellulose. Th e increase in endogenous ce llulolytic activities on the paper diet may therefore be due to greater availability of substrates, such as amorphous cellulose, that the termit e can digest without the aid of its symbionts. Symbiont cellulase activitie s change significantly in re sponse to diet, increasing significantly in wood as opposed to pa per, and in oak as opposed to pi ne. It is apparent that the community of hindgut symbionts is ab le to adapt to a range of diets, with differences in cellulose quality and hemicellulose content.

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101 Studies on Reticulitermes speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes virginicus (Cook and Gold 2000) and Coptotermes formosanus (Mannesmann 1972, Waller and La Fage 1987) have demonstrated significant ch anges in the hindgut protozoan communities in response to different diets, incl uding different wood species, pure ce llulose and pure xylan. It is probable that the hindgut enzyme changes in C. formosanus are due to these changes in its protozoan communities. It is apparent that C. formosanus workers are quite capable of digesting cellulose, following the same pattern seen in Chapter 4. Cr ystalline cellulose is mainly digested by hindgut symbionts in while amorphous cellulose and cellode xtrins are digested by both the termites and its symbionts. Moreover, the balance of endoge nous versus symbiont cellulolytic activities changes in response to their diet, most likel y by changes in the hindgut protozoan communities as well as the termite enzyme expression. This flexibility allows these termites to efficiently utilize a variety of wood species and wood-derived materials whic h have different qualities of cellulose. This capacity for adaptation also makes termite control by means of cellulase inhibition more difficult.

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102 Table 7-1. Endoglucanase activitie s in the three gut regions of Coptotermes formosanus workers in response to different field diets Diet Foregut Midgut Hindgut Paper 4.38 + 0.48a 2.91 + 0.21b 4.85 + 0.77b Pine 4.52 + 0.52a 2.82 + 0.25b 7.94 + 0.60a Oak 6.28 + 0.75a 4.06 + 0.36a 8.70 + 0.87a Endoglucanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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103 Table 7-2. Exoglucanase activitie s in the three gut regions of Coptotermes formosanus workers in response to different field diets Diet Foregut Midgut Hindgut Paper 0.022 + 0.003a 0.099 + 0.002a 0.448 + 0.027b Pine 0.019 + 0.001a 0.099 + 0.002a 0.371 + 0.036c Oak 0.018 + 0.002a 0.074 + 0.002b 0.589 + 0.047a Exoglucanase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly differe nt, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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104 Table 7-3. Beta-glucosidaase activities in the three gut regions of Coptotermes formosanus workers in response to different field diets Diet Foregut Midgut Hindgut Paper 0.155 + 0.003c 0.852 + 0.005b 0.489 + 0.005c Pine 0.221 + 0.003a 0.907 + 0.016a 0.623 + 0.012b Oak 0.168 + 0.001b 0.770 + 0.010c 0.728 + 0.009a Beta-glucosidase activities are in nmol p-nitrophenol pe r termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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105 CHAPTER 8 CHANGES IN COPTOTERMES FORMOSANUS GUT XYLANOLYTIC ACTIVITIES IN RESPONSE TO DIET Introduction Subterranean termites, such as Coptotermes formosanus (Shiraki), subsist largely on a diet of wood and similar material. Wood is not a substance that most animals are capable of digesting to any significant degr ee. Termites are well known for th e ability to digest cellulose, but there are other polysaccharides in wood, such as xylan, which the termites may utilize in order to get more energy out of this food source. In wood, cellulose chains are ty pically arranged in parallel bundles known as microfibers, which are embedded in a matrix composed mainly of hemicelluloses and lignin. Hemicelluloses are polysaccharides like cellulose, but their chemical structures are far more variable. One of the well-studied varieties of hemicellulose is xylan, which is mainly a polymer of the pentose sugar xylose. Xylan is the predominant hemicellulose in hardwood. Softwood t ypically is much lower in xylan content, along with significant quantit ies of other hemicelluloses (Pettersen 1984). Subterranean termites may encounter a numbe r of potential food sources during foraging. These may be broadly grouped into hardwoods, softwoods, and man-made cellulosic materials like paper. These three groups vary mainly in their xylan content, wi th hardwoods containing the most xylan while man-made cellulosic mate rials contain virtually no hemicellulose. In homes, hardwoods may include furniture, flooring, and trim components. Structural timbers are nearly always derived from softwoods. The gut of C. formosanus and related termites consists of a foregut with a small crop and a proventriculus, a fairly simple midgut and a greatly expanded hindgut containing four main species of symbiotic protozoa, as well as severa l species of bacteria and archaea (Yamin 1979). A pair of salivary glands is also present, empt ying into the foregut ante rior to the crop. To

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106 effectively digest wood, a r ecalcitrant and nutritionally poor substrate, termites like C. formosanus have developed an array of enzymes. Some carbohydrolases characterized from the Rhinotermitidae have been shown to be endogeno us (Watanabe et al. 1998), while others are produced by microbial symbionts (Nakashima et al. 2002a, Watanabe et al. 2002, Ohtoko et al. 2000). Xylanolytic enzymes include xylanases (EC 3.2.1.8) which internally cleave the xylan chain and beta-xylosidases (EC 3.2.1.37) which cleave xylan oligosaccharides into xylose. Inoue et al. (1997) demonstrated that xylanase activity is largel y localized to the hindgut of Reticulitermes speratus (Kolbe), strongly implicating the resi dent protozoa as the major agents of xylan digestion. In the same study, they s howed that the protozoan composition of the R. speratus hindgut significantly changes when the termites are fed on pure cellulose or pure xylan, as opposed to wood. Through similar experiments in differential feeding, Azuma et al. (1993) showed that, within the termite R. speratus protozoan symbionts in the genera Pyrsonympha and Dinenympha take part in xylan digest ion. It is probable that, w ith the changes in protozoan populations, the capacity for xylan digestion may also change to adapt to the xylan content of the termite diet. Our objectives were to determine 1) if C. formosanus can digest xylan, 2) the relative activities of xylanase and be ta-xylosidase in different regions of the gut of C. formosanus workers, 3) possible correlations between thes e activities and dietary xylan content in five formulated cellulose/xylan diets, and 4) how these activities change in response to three different simulated field diets. Materials and Methods Termite Collection Termites were field collected in First Line Smartdisc monitors (FMC Corporation) in Charleston, South Carolina. Termites were coll ected from the trap by removal of the wood

PAGE 107

107 (southern yellow pine) in the bait stations and brought to the Un iversity of Florida. Termites were then placed in feeding bioassays. Collections were restricted to a single colony for assays with formulated diets and a si ngle colony for assays with simu lated field diets to eliminate colony as a source of varia tion in enzyme activities. Termite Diets and Feeding Five formulated diets consisting of microcrystalline cellulose and beechwood xylan (>90% xylose residues) (Sigma-Aldrich, Atlanta, GA) were prepared for the termites: 10%, 20%, and 40% xylan. The remainder of the diet was composed of cellulose in all ca ses. The cellulose and xylan were mixed together and water (1.5 mL/g) was added to make a paste. This paste was thoroughly kneaded to achieve homogeneity. For each formulated diet, a glass jar ( 125 mL, straight-sided, Fisherbrand, Fisher Scientific, Pittsborough, PA) was prepared with 80 g of moist builders sand (10% moisture). The bottom was cut out of a plastic cup (5.92 mL Souffl cup, Solo Cup Company, Urbana, IL), leaving approximately 2 mm of cup wall intact. Formulated diet (1 g) was placed in the cup bottom and this was placed on top of the sand in the jar. Termites were added (~180 workers and 20 soldiers per jar) and kept in the dark at 21oC for 2 wk. Termites were collected, frozen and kept at -80oC until dissection. Three diets were prepared for the termites to represent the probable food sources they would encounter in the field. Th ese field diets were as follows: red oak (Quercus spp.), pine (Pinus spp.), and filter paper. Wood diets we re generated by drilling into craft wood boards (0.635 x 5.08 x 60.96 cm) with a 2.54 cm spade drill bit. Sawdust was collected and weighed. Filter paper consisted of a wei ghed number of crumpled cellulose filter paper disks (42.5 mm diameter, Whatman, grade 4).

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108 For each field diet, a mason jar (# mL, bra nd) was prepared with 40 g of moist builders sand (10% moisture). Field diet (10 g) was then added with 40 mL of deionized water and the jar was loosely capped. Unlike the formulated diets, the field diets were not made from sterile materials. Because of this, the jars with fiel d diets were autoclaved on a liquid cycle (30 min, 122oC) to sterilize the diets and th e jars were then allowed to cool. Termites were added (~270 workers and 30 soldiers per jar) after the jars had cooled and kept in the dark at 21oC for 6 wk. Termites were collected, frozen and kept at -80oC until dissection. Termite Dissection and Enzyme Extraction Two buffers were prepared for use in the dissections: 0.1 M sodium acetate buffer, pH 5.5, and 0.1 M sodium phosphate buffer, pH 6.5. Sodi um acetate buffer was used for the xylanase assays, while sodium phosphate buffer wa s used for the beta-xylosidase assays. Termites were removed from the freezer and ke pt on ice until dissecti on. Each termites gut was removed intact and separated into thr ee regions: foregut (and sa livary glands), midgut, and hindgut. A single enzyme extract was prepared from dissected termites from each feeding treatment for each assay type usi ng an experimental design simila r to the recent classic termite carbohydrolase experiments (Hogan et al. 1988, Inoue et al. 1997, Nakashima et al. 2002b). For the xylanase assays, 25 termites from each feeding treatment were dissected in sodium acetate buffer. For the beta-xylosidase assays, 25 termite s from each feeding treatment were dissected in sodium phosphate buffer. The three gut regions were placed into separate 1.5 mL microcentrifuge tubes (Eppendorf) containing the appropriate buffer, and kept on ice. Final concentrations were 50 termite gut regions per mL in all cases. Enzymes were extracted using a method adapted from Inoue et al. (1997). The contents of each microcentrifuge tube were placed in a 2 mL Tenbroeck glass tissue grinder (Pyrex) and

PAGE 109

109 manually homogenized on ice. The homoge nates were centrifuged at 20,800 g at 4oC for 15 min. The supernatants were colle cted, frozen, and kept at -80oC until use in the enzyme assays. Xylanase Assays The xylanase assays were conducted using a method adapted from Han et al. (1995). A 0.5% solution of beechwood xylan (>90% xylose re sidues; Sigma-Aldrich) was prepared in 0.1 M sodium acetate buffer, pH 5.5. The soluti on was boiled approximately 30 min, until xylan particles were no longer visible. The solu tion was centrifuged at 1250 g for 5 min at 23oC and the supernatant was used as the xylan stock solution. Assays were conducted in clear 96-we ll microplates. In each well, 10 L of tissue extract was combined with 90 L of xylan solution. The solutions were allowed to react for 35 min at 23oC. DNSA solution (100 L) consisting of 1% 3,5-dinitrosal icylic acid (DNSA), 0.4M sodium hydroxide and 30% sodium potassium tartrate was added to each well. The microplate was immediately placed in boiling water for 10 min a nd placed on ice for 15 min. After cooling, each microplate was read at 540 nm using a Quant Universal Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). Similar control pl ates were allowed to react for 5 min to allow for passive mixing of solutions before boiling with DNSA soluti on (Zhou et al. 2007). A 5 min reaction was used as a control to correct for any di fferences in initial sample reaction rates due to slow mixture of enzyme and substrate solutions. Standards were generated using dilutions of xylose. Only the wells in the middle of the mi croplate were used for these assays. Perimeter wells were filled with deionized water (200 L per well) to add temperature stability and consistency during boiling. For all replicates, the control plates we re used to adjust for 540 nm absorbance in gut extracts and were replicated an equal number of times to the assay plates, with one microplate well for each replicate.

PAGE 110

110 Beta-Xylosidase Assays Beta-xylosidase assays were conducted using a method adapted from Han et al. (1995). A solution of 4 mM p-nitrophenyl-D-xylopyranoside (pNPX; SigmaAldrich) was prepared in 0.1 M sodium phosphate buffer, pH 6.5. Assays we re conducted in clear 96-well microplates. In each well, 10 L of tissue extract was combined with 90 L of pNPX solution. The reaction was allowed to proceed for 10 min before being placed in a Quant Universal Microplate Spectrophotometer (Bio-Tek Instru ments, Winooski, VT). Absorbance was read at 420 nm every 2 min for 30 min at 23oC. Mean velocities (mOd/s) were recorded. Standards were generated using dilutions of p-nitrophenol. Data Analysis The xylanase assays were set up as one-factor de signs with four technical replicates per gut region for each formulated diet and each simu lated field diet. Four microplate wells were assayed for each replicate of each gut region/diet combination. The beta-xylosidase assays were set up as one-factor designs with four technical replicates per gut region for each formulated diet and simulated field diet. Tw o homogenates were used for each gut/diet combination; one homogenate for xylanase assays and another for beta-xylosidase assays. For the xylanase assays, the following formula was used to calculate specific activities; SA = CS[(A-A0)/t]/NT where: SA = specific activity (nmol reducing su gar per termite equivalent per min), A = absorbance (Od) after 35 min reaction, A0 = absorbance (Od) for th e corresponding control after 5 min reaction, t = time (min), CS = the coefficient derived from the standard (nmol reducing sugar/mOd), and NT = the number of termite equivalents per sample.

PAGE 111

111 For the beta-xylosidase assa ys, the following formula was used to calculate specific activities; SA = 60CSVA/NT where: SA = specific activity (nmol p-nitrophenol per termite equivalent per min), VA = mean velocity of absorbance change (mOd/s), CS = the coefficient derived from the standard (nmol p-nitrophenol/mOd), and NT = the number of termite equivalents per sample. The field diet data were analyzed using a mixe d model analysis of variance. Fixed effects were diet treatment and gut regi on. The Tukey-Kramer adjustment = 0.05) was used to separate the mean activities on each diet within each gut region (SAS Institute 2001). Results Termite Gut Observations During dissection, after the termites had been fed on the various diets, the color of the termite guts reflected the color of the different diets. This was es pecially evident in the enlarged hindguts which were typically fill ed with a mixture of partially digested food and resident microbes. Termites fed on formulated diets sh owed an increasing brown shade in their gut contents on diets containing mo re xylan. Termites fed on red oak had brownish-orange gut contents. Those fed on pine had pale yellow gut contents. Those fed on paper had white gut contents. The termites collected from the fi eld and immediately frozen for dissection had relatively dark gut contents. Xylanase Activities Both foregut and midgut xylanase activities were insignificant in comparison with hindgut xylanase activity on all of the formulated di ets (Fig. 8-1). Hindgut xylanase activity was significantly greater in termites kept on 40% xylan diets than in termites kept on 10% and 20% xylan diets.

PAGE 112

112 Both foregut and midgut xylanase activities were insignificant in comparison with hindgut xylanase activity on all of the simulated field diets (Table 8-1). Hi ndgut xylanase activity differed significantly among all of the three simulated field diets, being highest on oak and lowest on paper. Beta-Xylosidase Activities Hindgut xylanase activity increased from te rmites fed 10% xylan to termites fed 40% xylan, differing significantly on all three diets (Fig. 8-2) Midgut beta-xylosidase activities were highest in termites fed 10% xylan, and were si gnificantly lower in termites fed 40% xylan. Activities were nearly equal on 10% and 20% xylan. Hindgut beta-xylosidase activity differed si gnificantly among all of the three simulated field diets, being highe st on oak and lowest on paper (Table 8-2). Midgut beta-xylosidase activity was significantly greater on pine than on the other two simulated field diets. Discussion The overall gut morphology observed here for C. formosanus workers was similar to that described for Zootermopsis (Child 1946) and is consistent wi th that found in other lower termites (Noirot and Noirot-Timothee 1969). The app earance of the termite guts upon dissection, particularly the hindguts, indi cated that the termites had fe d on their respective diets. Among the termites in the colony fed on form ulated diets, both xylanase and betaxylosidase hindgut activities were significantly higher on diets with higher xylan content. A similar pattern was seen in termites fed on field diets. All hindgut xylan olytic activities were highest on the oak diet. Hardwoods typically consist of roughly 20% xylan, twice as much as the typical softwood xylan content of roughly 10% (Biermann 1996). At the same time, hindgut activities were lowest on the pape r diet, a diet completely lacking in xylan. Just as termites fed on 40% xylan showed far greater xylanase activity than termites fed on 10% xylan, so there was

PAGE 113

113 a similar jump in activity from pine fed te rmites to oak fed termites. The hindgut betaxylosidase patterns were also c onsistent between termites fed on formulated diets and termites fed on field diets. Our data showed that the majority of xylanase activity was in the C. formosanus hindguts. This is consistent with previous findings wher e xylanase activities were almost exclusively located in the hindgut of R. speratus and Coptotermes heimi (Wasmann), and associated with the symbionts (Inoue et al. 1997, Mishra 1991) Therefore, it was evident that C. formosanus workers follow an expected xylan digestion patter n for subterranean termites, in which xylan is thought to be mainly digested by hindgut symbionts. The low hindgut xylanolytic activities on the pa per diet were consistent with the absence of xylan. However, significant xy lanase and beta-xylosidase activities were still present even in the absence of dietary xylan. Th is suggests the presen ce of symbionts that constitutively produce xylanases and beta-xylosidases, but are capable of subsisting solely on a cellulose diet. Symbiont xylanase and beta-xylos idase activities were observed to change significantly in response to diet, in a manner consistent with xy lan content. These fi ndings suggest that the community of hindgut symbionts is able to adapt to a wide range of dietary xylan content. Studies on R. speratus (Azuma et al. 1993, Inoue et al. 1997), Reticulitermes virginicus (Banks) (Cook and Gold 2000) and Coptotermes formosanus (Shiraki) (Mannesmann 1972, Waller and La Fage 1987) have demonstrated significant ch anges in the hindgut protozoan communities in response to different diets. Many of these studies focused on feeding termites different wood species. It is therefore probable that the xylanolytic enzyme changes in C. formosanus are due to changes in hindgut symbiont populations. Alternativ ely, it is possible that these enzyme changes are due to changes in xylanolytic enzyme production within the hindgut symbionts.

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114 C. formosanus workers are capable of digesting xyla n, following the same pattern seen in Chapter 4, where xylan is mainly digested by in the hindgut. The xylanol ytic activities of the two termite colonies we investigated changed to accommodate dietary xylan content, most likely by changes in the hindgut symbiont communities. This flexibility allows a termite colony to efficiently utilize a variety of wood species and wood-derived materials. Compared to cellulose, xylan is often overlooked as a starting compound for the production of alternative fuels. However, xylan may comprise up to a quarter of wood, depending on the species, and a significant pr oportion of other plan t materials used for alternative fuel production. Thes e termites and their symbionts may provide candidate enzymes for the degradation of xylan in this process.

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115 Table 8-1. Xylanase activities in the three gut regions of Coptotermes formosanus workers in response to different field diets Diet Foregut Midgut Hindgut Paper 0.14 + 0.06a 0.21 + 0.01a 12.75 + 0.15c Pine 0.00 + 0.09a 0.20 + 0.03a 17.98 + 0.23b Oak 0.07 + 0.04a 0.13 + 0.03a 26.95 + 0.45a Xylanase activities are in nmol reducing sugar per termite equivalent per min. Means within a column followed by the same letter are not significantly different (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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116 Table 8-2. Beta-xylosidase activit ies in the three gut regions of Coptotermes formosanus workers in response to different field diets Diet Foregut Midgut Hindgut Paper 0.0020 + 0.0012a 0.0067 + 0.0004b 0.0252 + 0.0011c Pine 0.0027 + 0.0009a 0.0127 + 0.0003a 0.0408 + 0.0007b Oak 0.0020 + 0.0008a 0.0073 + 0.0003b 0.0623 + 0.0008a Beta-xylosidase activities are in nmol p-nitrophenol per termite equivalent per min. Means within a column followed by the same letter are not significantly different, (Tukey-Kramer Adjusted Means Separation, = 0.05, SAS Institute 2001), n = 4 replicates.

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117 -5 0 5 10 15 20 25 10152025303540% Xylan in DietSpecific Activity (nmol/termite/min) Foregut Midgut Hindgut Figure 8-1. Xylanase activities in different gut regions of Coptotermes formosanus in response to dietary xylan content.

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118 0 0.01 0.02 0.03 0.04 0.05 10152025303540% Xylan in DietSpecific Activity (nmol/termite/min) Foregut Midgut Hindgut Figure 8-2. Beta-xylosidase activit ies in different gut regions of Coptotermes formosanus in response to dietary xylan content.

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119 CHAPTER 9 CONCLUSION Termite gut carbohydrolase assays were optim ized for buffer and pH, leading to the selection of a 0.1 M pH 5.5 sodium acetate buffe r for endoglucanase and xylanase assays and a 0.1M pH 6.5 sodium phosphate buffer for exoglucan ase, beta-glucosidase and beta-xylosidase assays. This was in contrast with the almost universal use of 0.1M pH 5.5 sodium acetate buffer in prior literature. C. formosanus was found to have a more active arra y of gut carbohydrolas es, particularly exoglucanase and beta-glucosidase, than R. flavipes This was consistent with increased metabolic demands from more aggressive fo raging and a larger soldier ratio within C. formosanus colonies. Soldier carbohydrolase activi ties were generally lower than worker carbohydrolase activities, consistent with a caste incapable of feeding itself. Cellulolytic enzyme activity levels were found to significantly ch ange on differing diets for both C. formosanus and R. flavipes workers. Xylanolytic enzyme activities were found to change in a manner consistent with dietary xyl an content, being incr eased on diets containing more xylan. This has demonstrated dietar y adaptability in both of these species. The patterns of cellulolytic and xylanolytic enzyme activities and activity changes on differing diets were similar between the two sp ecies assayed. General findings regarding cellulose and xylan digestion in one species of Rhinotermitidae may be cautiously applied to the rest of the family. Our findings suggest a processive endogenous mechanism of amorphous cellulose degradation with a reliance on the term ite symbionts for the digestion of xylan and crystalline cellulose. Crystalline cellulose and xylan may make up between 30% and 60% of wood dry weight, while amorphous cellulose may make up between 15% and 25% of wood dry weight. Therefore,

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120 the termite symbionts are vital to effective wood digestion. In addition, while the endogenous termite enzymes may be of use in industrial digestion of amorphous cellulose, the symbiont enzymes should prove far more valuable in the digestion of cellulose and xylan in general. There is some redundancy with regards to amor phous cellulose digestion in the termites we have investigated, as both the termites and their symbionts produce endoglucanases and betaglucosidases. This could compli cate cellulase inhibition efforts. The enzymes responsible for digestion of crystalline cellulo se and xylan are largely produced by the hindgut symbionts, with little contribution from the termite itself. A lthough this removes one level of redundancy, the presence of multiple symbiont species pr oducing differing enzymes may still complicate cellulase and xylanase inhibition efforts. However, these very redundancies could make termites a robust system to adapt for the industria l degradation of cellulosic waste. Compared to cellulose, xylan is often overlooked as a starting compound for the production of alternative fuels. However, xylan may comprise up to 25% of wood dry weight, depending on the species, as well as a signifi cant portion of other plan t materials used for alternative fuel production. The te rmites and termite symbionts inve stigated in this dissertation may provide enzymes for the degradation of xylan in this process. The balance of endogenous versus symbiont cel lulolytic activi ties appears to change in response to diet in both species, most likely by changes in the hindgut protozoan communities as well as the termite enzyme expression. This capacity for adaptation and partial balancing between the termite and its symbionts could make termite control by means of cellulase inhibition more difficult, but may eventually yi eld a mechanism to increase efficiency in industrial cellulose degradation.

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121 The xylanolytic activities of the two termite species investigated changed to accommodate dietary xylan content, most likely by change s in the hindgut symbiont communities. These termites capacity for xylan digestion allows th em to gain more energy from a wood diet, and their adaptability allows them to efficiently utilize diets of varying xylan content. This adaptability could make termite xylanase inhi bition difficult, but it may provide a means for effective xylan degradation on an industrial s cale using living termites or termite symbionts. The enzymatic mechanisms of w ood digestion in these subterranean termite pests are both complex and effective. A greater understandi ng of these mechanisms may open new avenues in safer termite control, and may certainly improve processes for recycling cellulosic waste and development of alternative fuels.

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123 Han S. J., Yoo Y. J. and Kang H. S. (1995) Characterization of a bi functional cellulase and its structural gene. J. Biol. Chem. 270 26012-26019. Hogan M.E., Schulz M.W., Slaytor M., Czolu R. T. and OBrien R.W. (1988) Components of termite and protozoal cellulases from the lower termite, Coptotermes lacteus Froggatt. Insect Biochem. 18 45-51. Inoue T., Murashima K., Azuma J.-L., Sugimoto A. and Slaytor M. (1997) Cellulose and xylan utilization in the lower termite Reticulitermes speratus J. Insect Physiol. 43 235-242. Inoue T., Moriya S., Okhuma M. and Kudo T. (2005) Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus Gene. 349 67-75. Lewis J. L. and Forschler B. T. (2004) Protist communities from four cas tes and three species of Reticulitermes (Isoptera: Rhinotermitidae). Ann. Entomol. Soc. Am. 97 1242-1251. Mannesmann R. (1972) Relationship between di fferent wood species as a termite food source and the reproduction ra te of termite symbionts. Z. Angew. Entomol. 72 116-128. Mishra S. C. (1991) Carbohydrases in the gut of the termite Coptotermes heimi (Wasm.) (Rhinotermitidae), their origin, distri bution and evolutionary significance. Ann.Entomol 9 41-46. Mishra S. C. and Sen-Sarma P. K. (1979) Studies on deterioration of wood by insects. III. Chemical composition of faecal matter, nest ma terial and fungus comb of some Indian termites. Mater. Organismen 14 1-14. Moriya S., Dacks J. B., Takagi A., Noda S., Ohkuma M., Doolittle W. F. and Kudo T. (2003) Molecular phylogeny of three oxymonad genera: Pyrsonympha Dinenympha and Oxymonas J. Eukaryot. Microbiol., 50 190-197. Nakashima K., Watanabe H. and Azuma J.-I. (20 02a) Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of th e wood-feeding termite Coptotermes formosanus Cell. Mol. Life Sci. 59 1554-1560. Nakashima K., Watanabe H., Saitoh H., Tokuda G. and Azuma J.-I. (2002b) Dual cellulosedigesting system of th e wood-feeding termite, Coptotermes formosanus Shiraki. Insect Biochem. Molec. 32 777-784. Noirot C., and Darlington J. (2002) Termite nest s: architecture, regulation and defence. In Termites: evolution, sociality, symbioses, ecology, eds. T. Abe, D. E. Bignell, and M. Higashi, pp. 121-139. Kluwer Academic Publishers, Boston.

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124 Noirot C., and Noirot-Timothee C. (1969) The digestive system. In Biology of Termites, eds. K. Krishna and F. M. Weesner, pp. 49-88. Academic Press, New York. Ohkuma M., Ohtoko K., Iida T., Tokura M., Mori ya S., Inoue, T., Usami R., Horikoshi K. and Kudo T. (2000) Phylogenetic id entification of hypermastigotes, Pseudotrichonympha Spirotrichonympha Holomastigotoides and parabasalian symbionts in the hindgut of termites. J. Eukaryot. Microbiol. 47 249-259. Ohtoko K., Ohkuma M., Moriya S., Inoue T., Us ami R., and Kudo T. (2000) Diverse genes of cellulase homologues of glycosyl hydrolase fa mily 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus. Extremophiles 4 343-349. Parsiegla G., Reverbel-Leroy C., Tardif C., Bela ich J. P., Driguez H. and Haser R. (2000) Crystal structures of the cellulase Cel48F in complex with inhibitors and substrates give insights into its pr ocessive action. Biochem. 39 11238-11246. Pettersen R. C. (1984) The chemical composition of wood. Adv. Chem. Ser 207 57-126. Potrikus C. J. and Breznak J. A. (1980) Anaerobic degradation of uric ac id by gut bacteria of termites. Appl. Environ. Microb. 40 125-132. Potter M. F. (2004) Termites. In Handbook of pest control, 9th ed., eds. A. Mallis, S. A. Hedges and D. Moreland, pp. 217-361. GI E Media, Inc., Richfield. SAS Institute. (2001) SAS Institute, Cary, N.C. Smith J. A. and Koehler P. G. (2007) Cha nges in Reticulitermes flavipes (Isoptera: Rhinotermitidae) Gut Xylanoly tic Activities in Response to Dietary Xylan Content. Ann. Entomol. Soc. Am. 100 568-573. Sineiro J., Dominguez H., Nunez M. J. and Lema J. M. (1997) Inhibition of cellulase activity by sunflower polyphenols. Biotechnol. Lett. 19 521-524. Timell T. E. (1964) Wood Hemicelluloses. Part I. Adv. Carbohyd. Chem. Bi. 19 247302. Waller D. A. and La Fage J. P. (1987) Food quality and foraging response by the subterranean termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). B. Entomol. Res. 77 417-424. Watanabe H., Noda H., Tokuda G. and Lo N. (1998) A cellulase gene of termite origin. Nature 394 330-331.

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125 Watanabe H., Nakashima K., Saito H. a nd Slaytor M. (2002) New endo-beta-1,4glucanases from the parabasalian symbionts, Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites. Cell Molec. Life Sci. 59 19831992. Wenzel M., Schonig I., Bercht old M., Kampfer P. and Koni g H. (2002) Aerobic and facultatively anaerobic cellylolytic bacteria from the gut of the termite Zootermopsis angusticollis J. Appl. Micribiol. 92 32-40. Whistler R.L. and Chen C.C. (1991) Hemicelluloses. In Wood structure and composition eds. M. Lewin and I. S. Goldstein pp. 287-320. International Fiber Science and Technology Series, Vol. II. Marcel Decker, Inc., New York. Yamin M. A. (1979) Flagellates of the orders Trichmonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi and Foa reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodoterm itidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology 4 5-119. York W. S., Qin Q. and Rose J. K. C. (2004) Proteinaceous inhibitors of endo-glucanases. Biochim. Biophys. Acta 1696 223-233. Zhou X., Smith J. A., Oi F. M., Koehler P. G., Bennett G. W. and Scharf M. E. (2007) Correlation of cellulase gene e xpression and cellulolytic activ ity throughout the gut of the termite Reticulitermes flavipes Gene 395 29-39.

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126 BIOGRAPHICAL SKETCH Joseph Anthony Smith was born in Bremerton, Washington, October 14, 1977, the youngest of six children. After graduating fr om Olympic High School in 1993, he entered the baccalaureate program at Brigham Young University in Provo, Utah. He earned a Bachelor of Science degree in zoology with an entomology emphasis in 1996. After entering the doctoral program in the University of Washington Department of Zoology in 2000, he transferred to the University of Florida Department of Entomology and Nematology in 2002. He married Jessica Awad in August of 2006, and has been involved in a variety of research, teaching and extension projects during the pur suit of his PhD in entomology.