PHYSICOCHEMICAL PROPERTIES OF THE DIGESTIVE SYSTEM OF

WORKER TERMITES COPTOTERMES CURVIGNATHUS

By

SIA SIAW LANG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

Month and year of Viva Voce

 COPYRIGHT

All material contained within the thesis, including without limitation text, logos, icons,

photographs and all other artwork, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use may be made of any material contained within the thesis

for non-commercial purposes from the copyright holder. Commercial use of material

may only be made with the express, prior, written permission of Universiti Putra

Malaysia.

Copyright  Universiti Putra Malaysia

ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the Degree of Master of Science

PHYSICOCHEMICAL PROPERTIES OF THE DIGESTIVE SYSTEM OF

WORKER TERMITES COPTOTERMES CURVIGNATHUS

By

SIA SIAW LANG

Month and Year of Viva Voce

Chair: Assoc Prof. Dr. Patricia King Jie Hung, PhD

Faculty: Agriculture and Food Sciences

Extensive research efforts have been dedicated to describing the termites’ ability to

sustain solely on wood diet. Termites have evolved different enzymatic and non-

enzymatic strategies to utilize this plentiful but robust plant material that comprises of

cellulose, hemicellulose and lignin. Many studies focus on enzymatic machinery that

engaged in wood degradation. In this study, the physicochemical properties of the

termite Coptotermes curvignathus’s digestive system were characterized to elucidate

ecological niches-based strategy used by termite to sustain their oligonitrotrophic

lifestyle. The insect has three main regions in their digestive system: foregut, midgut

and hindgut, with 0.75  0.08 mm, 4.16  0.52 mm and 7.27  0.69 mm in length

respectively. Based on gut metabolites analysis, uric acid was found to be the most

iii
concentrated compound in all gut compartments. The N atom of uric acid has been

speculated for many years might be recycled back to termites through the action of

uricolytic gut microbes. For more than 70 years this provocative hypothesis remained

untested in C. curviganathus. In this study, the metabolite study revealed that (i)

termites contain uric acid, but they do not void the purine and (ii) the hindgut of C.

curvignathus harbors bacteria capable of producing uric acid. Twenty one cellulolytic

microbes that were isolated from C. curvignathus digestive system all displayed ability

to produce uric acid. C. curvignathus had an average of seven Malpighian tubules

attached between the midgut and the anterior hindgut. The organ transported the

nitrogenous waste to hindgut that can be dissimilated by gut bacteria leads to

assimilation of 15N into termite tissues. The ability to store uric acid would

compensate the wood-eating termites, which are oligonitrotrophic with only 0.03-0.15%

N in their diets. The second most abundant organic acid was propionic acid. Other

organic acid, antibiotic and antioxidant compounds such as salicylic acid, butyric acid,

vancomycin hydrochloride and quercetin were also found in the fluid of C.

curvignathus. From antioxidant test, hindgut fluid of C. curvignathus exhibited the

highest antioxidant activities, followed by midgut and the foregut fluids had the lowest

antioxidant activity. The result agrees with the metabolites analysis, where the ascorbic

acid and uric acid was found mainly in hindgut. The findings of this study provide

insight into the gut structure, biochemistry and cellulolytic microbial diversity that

enable us to explain how C. curvignathus sustain on wood.

iv
 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Sarjana Sains

CIRI-CIRI FISIKOKIMIA SISTEM PENCERNAAN ANAI-ANAI PEKERJA

COPTOTERMES CURVIGNATHUS

Oleh

SIA SIAW LANG

Bulan dan Tahun Viva Voce diadakan

Pengerusi: Prof. Madya Dr. Patricia King Jie Hung, PhD

Fakulti: Pertanian dan Sains Makanan

Usaha penyelidikan yang meluas telah didedikasikan untuk menggambarkan

kemampuan anai-anai bermandiri semata-mata pada diet kayu. Termite telah

mengembangkan strategi enzimatik dan bukan enzimatik yang berlainan untuk

memanfaatkan bahan tumbuhan ini yang banyak tetapi susah dicernakan, di mana

terdiri daripada selulosa, hemiselulosa dan lignin. Banyak kajian menumpukan pada

jentera enzimatik yang terlibat dalam degradasi kayu. Dalam kajian ini, ciri-ciri

fizikokimia sistem pencernaan Coptotermes curvignathus dicirikan untuk menjelaskan

strategi berasaskan kepelbagaian ekologi yang digunakan oleh anai-anai untuk

mengekalkan gaya hidup oligonitrotropik mereka. Serangga ini mempunyai tiga

kawasan utama dalam sistem pencernaan mereka: usus depan, usus tengah dan usus

v
belakang, masing-masing 0.75 ± 0.08 mm, 4.16 ± 0.52 mm dan 7.27 ± 0.69 mm.

Berdasarkan analisis metabolit usus, asid urik didapati adalah kompaun paling

terkonsentrasi dalam semua usus. Atom N asid urik telah dijangkakan selama

bertahun-tahun mungkin dikitar semula kepada anai-anai melalui tindakan mikrob

usus urikolitiknya. Selama lebih daripada 70 tahun, hipotesis provokatif ini masih

belum diuji dalam C. curviganathus. Dalam kajian ini, kajian metabolit menunjukkan

bahawa (i) anai-anai mengandungi asid urik, tetapi mereka tidak membuang purin ini

dan (ii) bakteria yang dipencilkan daripada usus C. curvignathus mampu

menghasilkan asid urik. Dua puluh satu mikrob selulosa yang diasingkan daripada

sistem pencernaan C. curvignathus semuanya menunjukkan keupayaan menghasilkan

asid urik. C. curvignathus mempunyai purata tujuh tiub Malpighian yang mengunjur

di antara usus tengah dan anterior usus belakang. Organ ini mengangkut sisa nitrogen

ke usus belakang anai-anai di mana yang boleh disimilasikan oleh bakteria usus dan

kemudian diasimilasikan dalam bentuk 15N ke dalam tisu anai-anai. Keupayaan untuk

menyimpan asid urik akan memberi manfaat kepada anai-anai yang oligonitrotropik

ini kerana makanan mereka hanya mengandungi 0.03-0.15% N. Asid organik yang

kedua paling banyak adalah asid propionik. Asid organik lain, sebatian antibiotik dan

antioksidan seperti asid salisilat, asid butrik, vankomisin hidroklorida dan quersetin

juga terdapat dalam cecair C. curvignathus. Dari ujian antioksidan, usus belakang C.

curvignathus menunjukkan aktiviti antioksidan tertinggi, diikuti oleh usus tengah dan

usus depan mempunyai aktiviti antioksidan paling rendah. Hasil ini bersetuju dengan

analisis metabolit, di mana asid askorbik dan asid urik banyak didapati terutamanya di

usus belakang. Penemuan kajian ini memberikan gambaran mengenai struktur usus,

kepelbagaian biokimia dan kepelbagaian mikroba selulosa yang membolehkan kita

memahami bagaimana C. curvignathus bermandiri dengan makanan kayu.

vi
 ACKNOWLEDGEMENT

First and foremost, I am thankful to God for blessing me with the mentality and
physically strength to finally completion of this thesis. Following is a number of people
who have assisted me in finalizing my education that I wish to express my highly
gratitude to.

I would like to express my sincere appreciation to my main supervisor, Assoc Prof. Dr.
Patricia King Jie Hung, my co-supervisors Assoc Prof. Dr. Ong Kian Huat and Assoc
Prof. Dr. Shahrul Razid Bin Sarbini for their guidance and assistance along my MSc
research.

I am also grateful to all the laboratory mates for their times and efforts who have
participated in assisting me with termite sampling and data analyzing.

I am sincerely felt grateful to my family who has showed unconditional love and
supports to me all the time to complete this research.

Thank you all.

vii
I certify that a Thesis Examination Committee has met on (date of viva voce) to
conduct the final examination of Sia Siaw Lang on her thesis entitled
“Physicochemical Properties of the Digestive System of Worker Termites
Coptotermes Curvignathus” in accordance with the Universities Putra Malaysia
[P.U.(A) 106] 15 March 1998. The Committee recommends that the students be
awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

_________________________________

Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:

viii
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:

Patricia King Jie Hung, PhD

Associate Professor
Faculty of Agriculture and Food Sciences
Universiti Putra Malaysia
(Chairman)

Ong Kian Huat, PhD

Associate Professor
Faculty of Agriculture and Food Sciences
Universiti Putra Malaysia
(Member)

Shahrul Razid Bin Sarbini, PhD

Associate Professor
Faculty of Agriculture and Food Sciences
Universiti Putra Malaysia
(Member)

_________________________________

Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:

ix
Declaration by graduate student

I hereby confirm that:

 this thesis is my original work;
 quotations, illustrations and citations have been duly referenced;
 this thesis has not been submitted previously or concurrently for any other degree
at any other institutions;
 intellectual property form the thesis and copyright of thesis are fully-owned by
Universiti Putra Malaysia, as according to the Universiti Putra Malaysia (Research)
Rules 2012;
 written permission must be obtained from supervisor and the office of Deputy
Vice-Chancellor (Research and Innovation) before thesis is published (in the form
of written, printed or in electronic form) including books, journals, modules,
proceedings, popular writings, seminar papers, manuscripts, posters, reports,
lecture notes, learning modules or any other materials as stated in the Universiti
Putra Malaysia (Research) Rules 2012;
 there is no plagiarism or data falsification/fabrication in the thesis, and scholarly
integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies)
Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research)
Rules 2012. The thesis has undergone plagiarism detection software.

Signature: ____________________________ Date: _____________________

Name and Matric No.: Sia Siaw Lang (GS33318)

x
Declaration by Memebers of Supervisory Committee

This is to confirm that:

 the research conducted and the writing of this thesis was under our supervision;
 supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: ________________________
Name of
Chairman of Associate Professor
Supervisory Dr. Patricia King Jie Hung
Committee: ________________________

Signature: ________________________
Name of
Chairman of Associate Professor
Supervisory Dr. Ong Kian Huat
Committee: ________________________

Signature: ________________________
Name of
Chairman of Associate Professor
Supervisory Dr. Shahrul Razid Bin Sarbini
Committee: ________________________

xi
 LIST OF TABLES

Table Page

2.1 The nutritional content of edible insects and other animals 27

based on 100 gram serving

4.1 Gut measurements in length and diameter (mm) of 41

Coptotermes curvignathus workers

4.2 Comparison of C. curvignathus gut length measurements 41

(mm) and number Malpighian tubules with other families

4.3 Concentration of chemical compounds detected in main 43

gut compartments of C. curvignathus worker

4.4 Absorbance of standard compound (Quercetin) 50

4.5 Total flavonoid content in different gut fluids of 50

C. curvignathus

4.6 Bacterial isolates from different gut compartments 52

4.7 Uric acid production (mg/dL) from cellulolytic 54

microorganisms after different incubation periods

xii
 LIST OF FIGURES

Figure Page

3.1 Intestinal tract of worker Coptotermes curvignathus 31

4.1 Digestive tract of Coptotermes curvignathus worker 40

4.2 Free radical scavenging activity of gut fluids in 48

different compartment

4.3 Standard curve of Quercetin 49

4.4 Cellulase activity screening of the gut fluids with 51

cellulase Aspergillus niger served as positive control

xiii
 TABLE OF CONTENTS

Page
ABSTRACT iii
ABSTRAK v
ACKNOWLEDGEMENTS vii
APPROVAL viii
DECLARATION x
LIST OF TABLES xii
LIST OF FIGURES xiii

CHAPTER
1 INTRODUCTION 1

2 LITERATURE REVIEW 6
2.1 Biology of Termites 6
2.1.1 Life Cycle and Caste System 6
2.1.2 Feeding Ecology 8
2.1.3 Types of Termites 10

2.2 Termite Gut Compartmentation 10

2.2.1 Foregut 11
2.2.2 Midgut 11
2.2.3 Hindgut 12
2.2.4 Malphigian Tubules 13

2.3 Physiochemical Conditions in the Gut 13

2.3.1 Oxygen Status and Redox Conditions 14
2.3.2 Hydrogen partial pressure 15
2.3.3 Intestinal pH 16

2.4 Degradation of Lignocellulose in Termites 16

2.4.1 Structural of Lignocellulose and 17
Enzymatic Properties
2.4.2 Cellulolytic Systems of Termites 19

2.5 Symbiotic Digestion between Termites and Gut 21

Microorganisms
2.5.1 Termite and Symbiont Enzyme 21
2.5.2 Diversity of Gut Symbionts 22
2.5.3 Metabolic Activities of Gut Symbionts 24

2.6 Potentials of Termite in Food and Medicine 26

2.6.1 Nutritional Values of Termites 26
2.6.2 Use of Termites as Medicine 28

xiv
 3 MATERIALS AND METHODS/METHODOLOGY 30
3.1 Sampling of Termites 30
3.2 Extraction of Gut Fluids 30
3.3 Measurement of the Digestive System 30
3.4 Chromatographic Determination of Gut Metabolites 31
3.4.1 Chemicals and Reagents 31
3.4.2 Preparation of Standard Solutions 32
3.4.3 Preparation of Sample Solutions 32
3.4.4 HPLC Instrumentation and Chromatographic 32
Conditions
3.4.5 Statistical Analysis 33
3.5 Antioxidant Assays 33
3.5.1 Preparation of Standard Solutions and 33
Chemicals
3.5.2 DPPH Radical-Scavenging Activity 34
3.5.3 Determination of Total Flavonoid Content 35
3.6 Plate Screening for Cellulolytic Activity 35
3.7 Isolation and Identification of Cellulolytic 36
Microorganisms
3.7.1 Isolation of the Cellulolytic Microorganisms 36
3.7.2 DNA Extraction 36
3.7.3 Identification by Polymerase Chain Reaction 37
(PCR) and 16S rRNA Sequence
3.8 Quantification of Uric Acid Contents 38

4 RESULTS AND DISCUSSION 39

4.1 Physical Characterization of Coptotermes Curvignathus 39
Gut Structure
4.2 Chromatographic Determination of Gut Metabolites 42
4.3 Antioxidant across the Digestive system 48
4.3.1 DPPH radical-scavenging activity 48
4.3.2 Determination of Total Flavonoid Content 49
4.4 Mapping of Cellulase Activity to Gut Structure 51
4.5 Cellulolytic Microorganisms 52
4.6 Uric Acid Producing Microorganisms 53

5 SUMMARY, CONCLUSION AND 55

RECOMMENDATIONS FOR FUTURE RESEARCH

REFERENCES/BIBLIOGRAPHY 56
BIODATA OF STUDENT 70

xv
 CHAPTER 1

INTRODUCTION

Coptotermes curvignathus belongs to the family of Rhinotermitidae, the most family

of lower termites found in Malaysian forests (Tho, 1992; Collins, 1988; Thapa, 1981).

C. curvignathus is a subterranean termite that causes detrimental impact on economic

gain of agricultural sectors, especially the plantation of oil palm (Elaeis guineensis)

on the peat soils in Malaysia (Chan et al., 2011; Lim and Bit, 2001). C. curvignathus

is unusual among termites in being able to attack and feed actively on the healthy

living woody plants. The aspects of digestive system are common to all termites.

However, there are some notable differentiations between the phylogenetically lower

and higher taxa according to variations in feeding behaviours. Characterization of the

digestive system of C. curvignathus would consider following the pattern of

previously studied wood-feeding termites.

Like other insects thriving on lignocellulosic diets, foregut, midgut and hindgut are

the three distinct compartments in the digestive tracts of worker termites. Foregut is

an esophageal tract that includes the salivary glands and proventriculus.

Proventriculus is a funnel-shaped masticating organ formed with teeth-like structures

in the upper part that grinds mechanically to reduce the size of ingested wood particles

prior enters to the midgut (Watanabe and Tokuda, 2010). Whereas, salivary glands

produce cellulolytic enzymes to predigest the lignocellulose particles (Nakashima et

al., 2002; Lo et al., 2000; Watanabe et al., 1997). Midgut is usually narrow, columnar

and microvilli-lined tube. It is generally the main secretion site for digestive enzymes

1
and nutrient absorption in insects. An enteric valve at the end of the midgut functions

in inhibiting the passage of endogenous cellulase into the hindgut. A number of

Malpighian tubules found attached at the posterior end of the midgut and anterior of

the hindgut. Malpighian tubules are crucial to wood-feeding termites like C.

curvignathus, especially in absorbing the nitrogenous waste and later excrete to the

hindgut for nitrogen recycling (Breznak and Brune, 1994). The hindgut is the largest

site of cellulose digestion that harbors the most diverse microbial community includes

Archae, Bacteria and Eukaryotes (protists), compared to the relatively little

microorganisms found in foregut and midgut. Higher termites develop more complex

hindgut compartment than lower termites, with a mixed segment in between the

midgut and hindgut.

Generally, lignocellulose consists of cellulose (20–50 %), hemicellulose (15–35 %)

and lignin (18–35 %). Wood-feeding termites are reported able to digest 65–99%

wood-cellulose and hemicellulose, and 5–83% lignin within 24 hours under natural

conditions (Ke et al., 2011). High efficiency of wood digestion in termites especially

lower termites, is accomplished by its endogenous endoglucanases that are mostly

expressed in salivary glands and midgut (Zhang et al., 2011), as well as symbiotic

cellulases that are produced by the hindgut cellulolytic protozoa (Zhou et al., 2007;

Nakashima et al., 2002). In addition, mastication in proventriculus would disrupt the

protective lignocellulose structure to expose the cellulose fibers that buried in lignin

and hemicelluloses for termite endogenous enzymes to function efficiently. Some

studies have suggested or reported the alteration in the chemical structure of lignin

throughout the whole termite gut environment (Geib et al., 2008; Katsumata et al.,

2007). Both C. formosanus (Shiraki) and R. flavipes (Kollar) showed the disruption

2
of wood-lignin is initiated in the foregut and continued in the midgut (Li et al., 2012;

Ke et al., 2010).

In the phylogenetically lower termites, initial structural changes of lignocellulose

already occurred at the mastication step in foregut and continuous modifications by

enzyme digestion in midgut. Cellulolytic protozoa resided in the anoxic hindgut

continue their fermentation processes by breaking down of carbohydrates to short-

chain fatty acids, particularly hydrogen, carbon dioxide and acetate that are eventually

reabsorbed by the host and flagellates as their energy sources (Mathew et al., 2011;

Nakashima et al., 2002). Other short chain fatty acids of the hindgut metabolism such

as propionate and butyrate also present in small quantities. Bacteroidetes that play

important roles in acetogenesis, methanogenesis, nitrogen fixation and cellulose-

derived monosaccharide fermentation as reported in lower termites Reticulitermes

flavipes (Breznak and Brune, 1994) was found present abundantly in the gut fluid of

C. curvignathus (King et al., 2014. Some of the gut bacteria were reported capable of

scavenging oxygen to maintain anaerobic conditions of hindgut and also preventing

invasion of foreign bacteria into the hindgut (Veivers et al., 1982). Since nitrogen is

scarce in wood, some symbiotic bacteria such as Lactobacillales and Clostridiales as

well as spirochetes are capable in fixing atmospheric nitrogen to synthesize essential

amino acids and vitamins. Besides, these intestinal microorganisms degrade

nitrogenous waste products (e.g. uric acid) into acetate and ammonia (Brune, 2013;

Brune and Ohkuma, 2011; Potrikus and Breznak, 1981). Moreover, symbiotic

interactions among different microbial populations would enable them to manage

their metabolisms in a way that adapt rapidly to the changing circumstances in the gut

ecosystem. The spatial distribution of microbial populations and their metabolic

3
functions would be influenced by the physicochemical gradients across different gut

regions such as oxygen, hydrogen, redox potential and pH (Brune, 2013; Brune and

Friedrich, 2000).

Despite of feeding on living plants rich in xenobiotic and detoxification compounds,

termites C. curvignathus are believed to have a strong antioxidant system to overcome

plant defense mechanisms, either by antioxidant compounds or antioxidant enzymes.

Previous study have shown that C. curvignathus consist of potential enzymes such as

cytochrome P450s monooxygenases, glutathione S-transferase, carboxylesterase,

UDPglucuronyltransferases and N-acetyltransferase that are likely crucial for

detoxification of reactive oxygen species (ROS) produced by xenobiotics (Charles et

al., 2014). Termite Reticulitermes speratus (Tasaki et al., 2017) showed uric acid

functions as antioxidant compounds against the accumulated ROS in the body. In

particular, over-generation of ROS during normal metabolic activities would cause

oxidative damage to biomolecules, such as nucleic acid, proteins and lipids, result in

aging and reduction in the lifespan. In addition, it is remained to be determined

whether the antioxidant ability of lower termites such as R. speratus and present

studied C. curvignathus depends on their gut symbionts.

Thus, it is necessary to discover variety interactions of the gut metabolites, gut

symbionts, antioxidants and enzyme activities across distinct gut structure that can

advance a greater understanding of the digestive system of termite C. curvignathus.

4
Objectives

The objectives of this study are to characterize the physicochemical properties of the

digestive system of worker termite Coptotermes curvignathus including physical

characterization of the gut structure, quantification of the chemical compounds,

determination of antioxidant properties, screening of cellulase activity, isolation of

cellulolytic microorganisms, and quantification of uric acid that are found in the gut

fluids.

5
 CHAPTER 2

LITERATURE REVIEW

2.1 Biology of Termites

Termites are known as eusocial insects that are classified under order of Isoptera. They

live in highly organized colonies with overlapping generations. They are mostly found

in the tropical forests (Eggleton 2000) and some are distributed throughout the

subtropical and temperate regions. Termites feed on wide range of living, dead or

decaying plant materials as well as soil that rich with organic matters. Termites are

known capable to feed on highly content of cellulose and lignocellulose by depending

on the gut symbionts such as protozoa (flagellate protists), fungi and prokaryotes

during digestion. Whenever termites are locating food sources or tunneling activities,

they are ecologically act as “ecosystem engineers” by breaking down organic matter

which will influence the properties of soil structure, availability of soil nutrient and

help in recycling of waste material from decomposition of organic matter (Bignell et

al., 2011; Wickings and Grandy, 2011; Donovan et al., 2001). However, termites also

act as serious pests that cause negative impacts on agricultural productions, forestation

and even attack certain household commodities (Li, et al., 2011).

2.1.1 Life Cycle and Caste System

Generally, a termite nest and colony are initiated by the primary reproductives, king

and queen. They are the adult termites with fully developed wings that responsible in

reproduction. The male reproductive or king goes for dispersal flights and mating with

the queen. Once mating, these pair sheds their wings and mark the onset of the life

6
cycle of termites. The male reproductive or king has important role to actively moving

around the nest and interacting with the queen and other castes (Maistrello and Sbrenna,

1999). The primary role of the female reproductive or queen is to produce eggs. Eggs

are accommodated in the gradually enlarged abdomen and about 3000 eggs may be

laid per day (Thompson, 2000). These yellowish-white eggs are incubated about 50-

60 days until the nymphs hatch out in the form of neonates. They go through a number

of moultings before develop into different forms of castes, a worker, a soldier or a

reproductive (king or queen). A termite colony reaches its maximum size with more

than 60,000-200,000 workers. These workers can further changed into sterile soldiers

or remain as sterile workers. Both workers and soldiers are wingless, lack of eyes,

ranging 6-8 mm in length and pale cream in color. Worker castes are responsible for

all the labours required in the nest, such as food storage, shelter maintenance and

digestion of cellulose for feeding other castes through a process called trophallaxis.

The process involves the exchange of partially digested food among castes from mouth

to mouth (stomodeal feeding) or from anus to mouth (proctodeal feeding). Mostly the

workers build tunnels and galleries in searching and harvesting of the food rather than

foraging in open and unprotected areas in order to escape predation (Bignell et al.,

2011). Workers communicate through semiochemicals and release trail of pheromones

during food searching outside of their colonies (Costa-Leonardo, 2009). Soldier castes

are characterized by their distinct head capsules. They have mandibles in defending

colony against invaders.

Among the three main castes: reproductives (king and queen), soldiers and workers,

the queen can reach a lifespan of even 30 years (Keller, 1998), which is 10 to 100 times

longer than the workers (Tasaki et al., 2017). The caste system of the termites can vary

7
according to different species. For example, in some termite families such as

Mastotermitidae, Kalotermitidae and Termopsidae, the worker castes are replaced by

the “false worker”, pseudergates (Roisin, 2000). Some closely related genera and

species are clearly distinctive through the characteristics of polymorphisms between

soldier and worker castes. For examples, termites Macrotermes sp. and Odontotermes

sp. have both dimorphic workers and soldiers; Hospitalitermes sp. has dimorphic

workers but monomorphic soldiers; whereas Ancistrotermes pakistanicus consist of

monomorphic workers but dimorphic soldiers (Tho, 1992).

2.1.2 Feeding Ecology

Termites are mostly herbivores and detritivorous. Their food sources generally range

from dead wood, soil, grass, cultivated fungi and litter layers, and they are capable in

decaying 50-100% of dead plant materials and biomass in the tropical regions (Bignell

and Eggleton, 2000). They can be differentiated into groups based on their food

sources and feeding habits, such as wood-feeders, soil-feeders, soil-wood interfacing

feeders, grass-feeders or litter-feeders. Termites are mostly found belong to wood-

feeder group. They are mainly feed on woody litter, including the dead branches on

the trees. The wood may be taken by Coptotermes, Schedorhinotermes and

Microcerotermes dubius on living trees, Kalotermitidae on dead wood, or

Nasutitermitinae, Macrotermitinae and some Termitinae on fungus-attacked wood

(Collins, 1984). For soil-feeders, termites feed on mineral soils, with some selection

of silt and clay fractions. They are commonly found in tropical rain forest (Wood,

1976), such as the soil-feeders in South-east Asian regions are dominated by

Apicotermitinae, Termitinae and Nasutitermitinae (Abe, 1987). Termites of soil-wood

interface feeders group feed on highly decomposed wood and soil under logs or soil

8
mixed with leaf litters. These feeders are only found in Termitinae, Nasutitermitinae

an Apocotermitinae. This group of worker castes was found to have darker body

(Eggleton et al., 1997; 1996). Grass-feeders are another important feeding group,

which feed on dried dead grass and sometimes may also take dung and vertebrate

corpses. They are mostly belonging to the family of Hodotermitinae, Termites that

feed on leaves litter and small woody materials are litter-feeders, including

Macrotermitinae, Apicotermitinae, Termitinae and some Nasutitermitinae. Litter

feeders always transport their food sources to and store temporarily in the nest

(Eggleton et al., 1997; 1996). The feeding groups can be overlapped in some species

(Bignell and Eggleton, 2000) and their feeding habits are highly influenced by

different atmospheric factors, such as temperature, moisture, relative humidity, lignin

and hardness content of the plant substrates. Temperature is known to be one of the

vital factors for the survival, reproduction, growth and development of insects. The

numbers of subterranean termites, Reticulitermes spp. are found to be higher in hot

climate and warmest winters in Southeast and California regions compared to USA

(Quarles, 2007). In temperate regions, termites Reticulitermes hesperus have lower

temperature requirement for their sustenance (Smith and Rust, 1993). Coptotermes

formosanus are found to have high feeding rate and survival rate at temperature of

30 °C (Nakayama et al., 2005; Fei and Henderson, 2002). Water-bound trees in high

humid areas are more heavily infested by Formosan subterranean termites, in the fact

that wood fibres could be getting soften and easy to be masticated (Henderson, 2008;

Delaplane and LaFage, 1989).

9
2.1.3 Types of Termites

Based on the feeding ecology, termites in the order of Isoptera are divided between

higher and lower termite species. Higher termites belong to the family Termitidae

which comprise 80% of all termite species. Lower termites comprise of the remaining

20% termite species which are the six families from Mastotermitidae, Kalotermitidae,

Hodotermitidae, Termopsidae, Rhinotermitidae and Serritermitidae (Berlanga et al.,

2011).

The key of this division is the particular nature of their gut symbiotic partners. Lower

termites prefer feed on wood as well as fungus-infected wood due to protein richness

in fungi, but higher termites have broader diets that feed on soil, grass, leaves, roots

and faeces (Radek, 1999). As the lower termite families feed exclusively on wood,

they form relationships with both eukaryotic and prokaryotic symbionts in their

digestive system for the digestion of cellulosic materials to occur. Although salivary

glands has been reported to produce endogenous cellulase in Recticulitermes sp.

(Watanabe et al., 1997), there is the presence of flagellated protozoa in the hindgut as

the main digestive site in facilitating the breakdown of cellulosic compounds (Inoue

et al., 2000). However in higher termites Termitidae, although they produce their own

cellulose enzymes, they are lacking of flagellates in the guts and only depend on

bacteria in cellulose digestion (Breznak and Brune, 1994).

2.2 Termite Gut Compartmentation

Workers termite are the major group in a colony to perform the lignocelluloses

digestion and feeding to other castes. Like other insects thriving on lignocellulosic

10
diets, the worker gut generally comprised of three distinct compartments: foregut,

midgut and hindgut.

2.2.1 Foregut

Foregut is an esophageal tract that includes the salivary glands and proventriculus.

Salivary glands function in producing cellulolytic enzymes and contribute to

predigestion of the surface of wood particles, such as endo--1,4-glucanase (EG)

which has been shown in other lower termites, Reticulitermes flavipes (Lo et al., 2000;

Watanabe et al., 1997) and Coptotermes formosanus (Nakashima et al., 2002).

Whereas, proventriculus, a funnel-shaped masticating organ formed with teeth-like

structures in the upper part, acts in mechanical grinding and reducing the size of

ingested wood fragments prior enter to the midgut (Watanabe and Tokuda, 2010).

Mastication in proventriculus would disrupt the protective lignocellulose structure to

expose the cellulose fibers that buried in lignin and hemicelluloses for termite

endogenous enzymes to function efficiently. In addition, lignin degradation

capabilities in termite guts have been verified in previous studies (Geib et al., 2008;

Katsumata et al., 2007; Breznak and Brune, 1994). Both C. formosanus (Shiraki) and

R. flavipes (Kollar) showed the disruption of wood-lignin is initiated in the foregut and

continued in the midgut (Ke et al., 2010).

2.2.2 Midgut

Midgut is usually a narrow, columnar and microvilli-lined tube. It is the main site of

digestive enzymes and nutrients absorption as taken place in most insects. In the

midgut of C. formosanus (Shiraki), an immunohistochemistry method showed the

endogenous cellulase was distributing in the endoperitrophic space and some on the

11
microvilli lining (Fujita et al., 2010). An endo-ectoperotrophic circulation of digestive

enzymes seems to be found in both lower and higher termites (Terra and Ferreira, 2012;

Nakashima et al., 2002; Terra and Ferreira, 1994). Soluble sugars such as glucose and

cellobiose could pass through and absorbed through the midgut wall. An enteric valve

at the end of the midgut helps to inhibit the passage of endogenous cellulase into the

hindgut. Previous studies showed some lignocelluloses degradation or modification is

detected in the midgut of termites under oxygenated environment (Ke et al., 2010).

Higher oxygen concentration was found in the midgut region of wood-feeding termites

(Ke et al., 2010) compared to other gut regions, in which the lignin oxidation could

have been promoted (Scharf and Tartar, 2008; Breznak and Brune, 1994). Phenol

oxidase and esterase are the enzymes involved in lignin degradation and

hemicelluloses solubilization. Both the enzymes are detected in termite gut instead of

derived from symbionts (Karl and Scharf, 2015; Tartar et al., 2009; Scharf and Tartar,

2008).

2.2.3 Hindgut

Hindgut is the largest region that harbors numerous microorganisms, includes Archae,

Bacteria and Eukaryotes (protists). Earlier studies stated the volume of hindgut can be

up to one-third of the body weight for lower termites. Most cellulose digestion and

microbial fermentation processes take place in hindgut compartment under anaerobic

conditions. Anoxic conditions within the hindgut are maintained through oxygen

consumption of bacteria that residing near to the gut wall. Hence, there is a micro-oxic

region around its periphery (Brune et al., 1995b). Aromatic-degrading bacteria are

found present residing on or near to the aerobically epithelium hindgut which might

12
support the degradation of lignin and phenolic compounds in the anoxic hindgut

(Watanabe et al., 2003; Brune et al., 1995).

2.2.4 Malphigian Tubules

Malpighian tubules are the structures in the excretory system of insects and terrestrial

arthropods. The Malpighian tubules in termites are usually attached at the posterior

end of the midgut and anterior of the hindgut. Like insects, the main function of the

Malpighian tubules is to maintain internal environment through the removal of

unnecessary substances that present in hemolymph while retaining or reabsorbing

useful substances for the body (Pannabecker, 1995). This organ is crucial to wood-

feeding termites especially in absorbing the nitrogenous waste and then excretes to the

hindgut for nitrogen recycling (Breznak and Brune, 1994).

2.3 Physiochemical Conditions in the Gut

Over the course of evolution, the hindguts of all termites have increased in length and

volume. The hindgut in lower termites consists of dilated “hindgut paunch” which

gradually smaller into the colon and ends in the rectal compartment (Noirot, 1995).

While higher termites especially soil-feeders, show further elongation and some

compartmentalization of the hindguts (Noirot, 2001; 1995). Therefore, the

enlargement proctodeal increase the habitation time of the digesta that lead to longer

exposure to the activities of the intestinal microbiota. Furthermore, host factors and

microbial activities cause physicochemical gradients that construct diverse

microenvironmental conditions in each gut compartment. Since microbes are not

randomly distributed within the gut, variable oxygen, hydrogen, redox potential, and

13
intestinal pH has to be present for any other metabolites as source and sink are spatially

separated (Brune and Friedrich, 2000).

2.3.1 Oxygen Status and Redox Conditions

A continuous flux of oxygen in the hindgut boundary is driven by the steep oxygen

gradients between the oxic gut epithelium and the anoxic gut contents. Oxygen (O2) is

found penetrating 50-200 µm into the boundary of hindgut lumen in all termites,

allowing only the central portion of the dilated compartments anoxic (Kappler and

Brune, 1999; Schmitt-Wagner and Brune, 1999; Brune, 1998; Ebert and Brune, 1997).

All hindgut microbiota and those crossing the oxic-anoxic boundary occasionally are

certainly expose to oxygen in either continuously or within limited period (Brune et

al., 2000). This explains the presence of aerotolerant, facultatively aerobic or

obligately aerobic bacteria being cultivated from the oxygen-limited hindgut of R.

flavipes (Tholen et al., 1997).

Colonies that are isolated from the gut homogenates of Zootermopsis angusticollis and

incubated under hypoxic conditions with 2% oxygen were twice higher than the

colonies that isolated under aeration (Graber and Breznak, 2005). Controlled oxygen

fluxes without exceeding the capacity of oxygen removal would preserve

Methanobrevibacter species metabolically active in dense cell suspension (Tholen et

al., 1997). The metabolic effects of oxygen on termite gut suggested that the

continuous influx of oxygen in anoxic condition of the hindgut might shift the

fermentations toward more oxidized products (Ebert and Brune, 1997).

14
Fermenting bacteria such as lactic acid bacteria shifts the fermentation pathways from

lactate or propionate to acetate production during oxygen reduction (Bauer et al., 2000;

Tholen et al., 1997). Moreover, although the fermentative processes in the hindgut

increase the removal of oxygen in the gut boundary, even the smaller size of termite

Anoplotermes pacifus (Termitidae) is not limited to have gut symbiotic digestion but

with different fermentation products compared to other termites (Bauer et al., 2000).

2.3.2 Hydrogen Partial Pressure

The major intermediate lignocellulose degradation by the community of termite

hindgut is the molecular hydrogen (H2), which is contributed abundantly by the

community of Spirochaeta spp. in higher termites (Leschine et al., 2006) and

Treponema azotonutricium isolated from the lower termite Zootermopsis angusticollis

(Graber et al., 2004).

In the study of the gut of Nasutitermes corniger termites with microsensor

measurements, the dilated hindgut paunch which is in anoxic condition showed the

highest bacterial communities with high accumulation of H2 partial pressures up to

12kPa (Köhler et al., 2012). Whereas, hydrogen partial pressures in the anterior

hindgut of N. corniger are found to have the same range as R. flavipes (Ebert and

Brune, 1997). A strong hydrogen sink is present according to the existence of steep H2

gradient in the dilated hindgut paunch of N. corniger. This is in the agreement with the

large accumulation amount of H2 within the gut lumen but significant rates of

hydrogen emission by living termites (Sugimoto et al., 1998).

15
2.3.3 Intestinal pH

The intestinal pH in the midgut is near to neutrality and that of the hindgut content is

in the range of 6.0 to 7.5 for both lower and higher termites (Bignell and Anderson,

1980). In exception of the pH values that found in soil-feeding higher termites, such

as Odontotermes obesus as high as 10.4 to 12 (Noirot-Timothee, 1969).

In both lower termite R. flavipes and higher termites Nasutitermes nigriceps and

Microcerotermes parvus, the midgut and hindgut showed the pH values of around 7

(Brune et al., 1995). However, the anterior hindguts of the higher termites showed the

tendency towards stronger alkalinity along their axes, reaching the pH values of greater

than 10 at the posterior region.

This observation meets with the proposal of Bignell and colleagues (1983) suggesting

the increment of potassium ion concentrations that contributes to alkalinity conditions

in midgut-hindgut regions of soil-feeding higher termites was probably secreted by the

mesenteral lining of the mixed segment.

2.4 Degradation of Lignocellulose in Termites

Lignocellulose is formed directly from plant photosynthesis, and primarily contains

cellulose, hemicellulose, and lignin. Lignocellulose biomass usually consists of 35-50%

cellulose, 20-35% hemicellulose, and 10-25% lignin. These three components form

are not uniformly distributed within the cell walls. Various types of species, tissues

and maturity of the plant cell wall have different structure and quantity of these plant

cell wall components. Therefore, in termites, both the host and symbiont enzymes play

a vital role in lignocellulose degradation.

16
2.4.1 Structural of Lignocellulose and Enzymatic Properties

Cellulose is the main component of lignocellulose. It is a polysaccharide comprising

of a linear polymer of glucose linked through β-1,4-glycosidic linkages. The hydrogen

bonds between different layers of the polysaccharides are the factors to high stability

and resistance to degradation of crystalline form cellulose. The crystalline forms are

tightly packed to prevent even the smallest active molecules in breaking down the

structure. Thus, the β-1,4-glycosidic linkages are cleaved by cellulase.

Cellulase consists of three main types of enzymes such as endoglucanase (1,4-β-D-

glucan-4-glucanohydrolases, EC 3.2.1.4), exoglucanase (1,4-β-D-glucan

cellobiohydrolases, EC 3.2.1.74) and β-glucosidase (EC 3.2.1.21) (Morana et al.,

2011). Cellulase hydrolyzes cellulose and produce glucose, cellobiose and other oligo-

saccharides as primary products. Endoglucanases function in the cleavage of

amorphous sites in the cellulose chain into different lengths of oligosaccharides

production. Exoglucanases release cellobiose or glucose by attacking the ends of

cellulose fibers. And β-glucosidases hydrolyze cellobiose and other cello-oligomers

from the non-reducing ends to release the glucose monomers.

The second most abundant component of lignocellulose is hemicellulose. Unlike

cellulose, hemicellulose has a random and amorphous structure. It is comprised of a

branched chain of several heteropolymers, including five-carbon sugars (such as D-

xylose and L-arabinose) and 6-carbon sugars (D-galactose, D-glucose, and D-

mannose), and uronic acid. Hemicelluloses are imbedded in the plant cell walls

through linking cellulose fibres into microfibrils and cross-linking with lignin to form

a complex bonding that provides strength to the structural.

17
Hydrolysis of hemicellulose is catalyzed by hemicellulases. Hemicellulases function

in exposing cellulose to the action of cellulases for more accessibility towards

depolymerization. Xylanases (EC 3.2.1.8) is one of the components of hemicellulose

that plays important roles in the digestion. The complete hydrolysis of hemicellulose

requires the synergistic action of other accessory hemicellulolytic enzymes including

β-xylosidases (EC 3.2.1.37), β-mannanases (EC 3.2.1.78), α-L-arabinases (EC

3.2.1.99), α-L-arabinofuranosidases (EC 3.2.1.55), α-glucoronidases (EC 3.2.1.131)

and feruloyl esterases (EC 3.2.1.73).

Lignin is an amorphous aromatic polymer of phenolic compounds with strong

intramolecular bonding that makes it more difficult to hydrolyze than cellulose and

hemicellulose. It enhances the strength properties of the plant tissue and the individual

fibres, strength to the cell wall and resistance against insects and pathogens (Rubin,

2008). Typically softwoods contain 30±5% lignin whereas hardwoods generally have

lower lignin content with 25±5%.

The main lignin-degrading enzymes are laccases (E.C. 1.10.3.2) and peroxidases:

lignin peroxidase (E.C. 1.11.1.14) and manganese peroxidase (E.C. 1.11.1.13). There

are a few less significant oxidative ligninolytic enzymes including diaryl propane

oxygenases, versatile peroxidases, and dye-decolorizing peroxidases. Additionally,

some accessory enzymes, such as oxidases and reductases mediate in ligninolytic

activity of the major enzymes. Cleavage of lignin advances the accessibility of

cellulases and hemicellulases and hence improves the efficiency of lignocellulose

degradation (Placido and Capareda, 2015).

18
2.4.2 Cellulolytic Systems of Termites

Degradation of lignocellulose in termites depends on a dual system that requires

accomplishment by both the host and its gut symbionts (Brune, 2014; Nakashima et

al., 2002). Besides, mechanical digestion through mandibles and proventriculus

together with exposure to gut enzymatic action maximize the degradation of

lignocellulose. Studies have shown that lignocellulose hydrolysis in termites was

facilitated by the various enzyme-producing symbiotic microorganisms including

protistans, archaels, bacteria and fungi (Ni and Tokuda, 2013; Ohkuma, 2003).

In lower termites, the cellulolytic process begins in the foregut, where wood fragments

are ground by the gizzard into smaller particles for about 10 to 20 µm in diameter.

Hydrolysis of cellulose begins with release of endoglucanases by the salivary glands

into the foregut (Brune 2014; Zhou et al., 2007; Watanabe et al., 1997). In the midgut,

the presence of high concentrated endoglucanases further breaks down the cellulose

particles. Lastly, the protistan flagellates harboured in the hindgut produce the three

major types of cellulases (endoglucanases, exoglucanases, and β-glucosidases) as well

as hemicellulases that work together in hydrolyzing the crystalline cellulose,

hemicellulose and other ingestion fragments. Brune (2014) stated that the wood

particles enter the hindgut are immediately taken into the food vacuoles of the protistan

flagellate, results in the bacterial microorganisms in lower termites seem not to have a

lead role in lignocellulose hydrolysis.

Differences in termite caste and development stages show various expression of

cellulolytic enzymes in the termite gut (Fujita et al., 2010). All the identified termite

endogenous endoglucanases that have been reported belong to the glycosyl hydrolases

19
GH 9 family (Scharf 2015; Zhang et al. 2012; Leonardo et al., 2011; Watanabe and

Tokuda 2010; Teather and Wood 1982). Whereas, characterization of the protistan

endoglucanases seemed belong to a set of essential enzyme of GHs 5, 7, and 45 (Scharf

2015). The host β-glucosidases so far were found belong to the family of GH 1

(Tokuda et al., 2002). Some findings concerning hemicellulases have reported

xylanases are from the host GH 11 and protist symbionts GH 45. Several ligninolytic

enzymes such as laccases, peroxidases, phenol oxidases, aldo-keto reductases and

esterases, have also been identified in termites (Sethi et al., 2013; Coy et al., 2010).

By contrast, in higher termites, lack of eukaryotic symbionts results in the dependent

entirely of the prokaryotic microbial community in the hindgut for lignocellulose

degradation. Instead of endoglucanase secretion by the salivary glands, the enzymatic

production in the midgut epithelium is more significant to cellulose digestion than in

lower termites (Brune 2014). Therefore, the cellulose hydrolysis is performed mainly

by termite and bacterial endosymbiont endoglucanases secreted by the midgut and the

hindgut, respectively (Tokuda et al., 2004).

There are also many cellulases, xylanases and other glycoside hydrolases produced in

higher termites. The hindgut of Nasutitermes corniger, a higher termite has identified

almost a quarter of the 886 proteins were enzymes and 36 were glycoside hydrolases

through metaproteomics examination to assist in cellulose degradation (Burnum et al.,

2011). Endoglucanases have been reported in both termite and bacterial GH 9; whereas,

GH 5 and 45 are originated only from bacterial. The β-glucosidases (GH 1) have been

detected mainly in the salivary glands and midgut in majority of Nasutitermes sp than

in other species, this activity occurs mostly in the hindgut (Uchima and Arioka, 2012;

20
Wang et al., 2012; Slaytor 2000). There are two isolations of xylanases (GH 10 and

11) from Nasutitermes sp. and Globitermes brachycerastes bacterial symbionts,

respectively (Brennan et al. 2004). A basidiomycete fungus in the subfamily

Macrotermitinae that is found less common but with obvious association is cultivated

inside the nest and showed contributions in lignocellulose degradation (Sapountzis et

al., 2016).

2.5 Symbiotic Digestion between Termite and Gut Microorganisms

Like other insects thriving on a lignocellulose diet, lower termites survive on

lignocellulose through a complex symbiosis relationship with prokaryotic and

eukaryotic symbionts; whereas, nutritional mutualism in higher termites has been

reduced to the symbiosis with prokaryote (Eutick et al., 1978). Generally, termites

provide favourable regions of the alimentary tract for the symbionts. And in return,

the symbionts digest the dietary components into microbial fermentation products,

which are then eventually reabsorbed by the intestinal epithelia. The gut symbionts

digest the lignocellulose through fermentation of the soluble carbohydrates (e.g.,

glucose) in producing acetate, H2, and carbon dioxide (CO2). Termites earn a

nutritional resource by thriving on the metabolic products of its gut symbionts. Such

exchange reveals a mutual symbiotic relationship that benefits both the host and

symbionts.

2.5.1 Termite and Symbiont Enzymes

In nourishing on the high lignin and poor in nitrogen diet, enzymes are essential for

both host and gut symbionts to catalyze its breakdown and supplement termite

nutritional deficiencies such as amino acids, vitamins, and sterols. Several highly

21
active enzymes that are contributed by termites include endogenous cellulases (β-1,4-

endoglucanase and β-glucosidase) and lignin detoxifiers (such as aldo-keto reductase,

laccase, catalase, cytochrome p450s) (Sethi et al., 2013; Scharf et al., 2010; Zhou et

al., 2010). In lower termites, hindgut protistans also contribute several crucial glycosyl

hydrolases such as GHFs 5, 7, 45 to help in cellulolytic activity (Sethi et al., 2013;

Todaka et al., 2010; Ohtoko et al., 2000) which are important in hydrogen cycling

(Inoue et al., 2007, 2005). Both protists and bacteria own hemicellulases (Tartar et al.,

2009; Inoue et al., 1997), at the same time termite endogenous cellulases also have

been shown to have hemicellulase activity (Karl and Scharf, 2015; Scharf et al., 2011,

2010).

2.5.2 Diversity of Gut Symbionts

There is a diverse ecosystem in the gut symbionts of lower termite. These gut

symbionts are known in capabilities of carbohydrate metabolism, methanogenesis,

nitrogen cycling, amino acid biosynthesis and hydrogen turnover to supply

deficiencies of the host. In addition to the contributions of individual organisms,

synergistic collaboration the host fraction (foregut, midgut, and salivary glands) and

the symbiont fraction (hindgut) of the termite digestive system have been shown

(Scharf et al., 2011).

The xylophagous flagellate harboured in hindgut is the major source of cellulose and

hemicellulose hydrolysis of subterranean termites. It has been reported in Konig and

co-worker (2002) that the strictly anaerobic flagellates can occupy over 90% of the

dilated paunch volume. Oximonada and Parabasalia are the two protozoan lineages of

lower termites (Yamin, 1979).

22
While protists responsible more in lignocellulolytic activity, the prokaryotic

community play more diverse roles in the termite gut. Spirochetes are the most

noticeable bacterial group found in lower termite guts. They have diverse capabilities

in metabolic processes, including acetogenesis, nitrogen fixation, and degradation of

lignin phenolics (Graber and Breznak, 2004; Lilburn et al., 2001). Several spirochetes

species from lower termite guts showed their metabolic capabilities and cooperation

potential within the community as a whole (Dröge et al., 2006; Graber and Breznak,

2005, 2004; Graber et al., 2004; Salmassi and Leadbetter, 2003; Lilburn et al., 2001;

Leadbetter et al., 1999).

The bacteria that are closely related with gut flagellates as intracellular endosymbionts

is another major component of lower termite microorganisms (Noda et al., 2009; Stingl

et al., 2005). These bacterial endosymbionts found within protist cells are found in

four phyla: Elusimicrobia, Bacteroidetes, Proteobacteria, and Actinobacteria

(Strassert et al., 2012; Stingl et al., 2005). These groups are found to have capabilities

in fermenting glucose, synthesizing amino acids, producing cofactors, fix nitrogen,

and recycling nitrogenous wastes (Zheng et al., 2015; Strassert et al., 2012; Ohkuma

and Brune, 2011; Hongoh et al., 2008a, b).

Methanobrevibacter is a methanogenic archaeal genus associated with termite gut

flagellates. It contributes methane to the gut environment using plentiful amounts of

hydrogen that is present in the gut lumen as a product of cellulose metabolism (Hongoh

and Ohkuma, 2011; Tokura et al., 2000; Shinzato et al., 1999). This creates a three-

party symbiosis: prokaryotes within protozoa within termites. Representatives

Methanobacteriaceae are also known to associate with the microaerobic termite gut

23
lining (Brune, 2011; Ohkuma et al., 1999; Leadbetter and Breznak, 1996).

Methanobrevibacter spp. was found attached to the gut epithelium of the

termite Reticulitermes flavipesseem to tolerate exposure to O2 (Leadbetter and

Breznak, 1996). Therefore, association with the flagellate endosymbionts, the large

amount of methane produced in termite digestion can be credited to archaea that

associated with the hindgut lining (Brune, 2011; Hongoh and Ohkuma, 2011).

2.5.3 Metabolic Activities of Gut Symbionts

Generally, the polysaccharide of wood or cellulose particles taken up by the major

metabolic groups, flagellates, are oxidized to acetate and carbon dioxide (CO2) while

the reducing equivalents are released as H2 (Brune and Stingl, 2005). Besides the role

of the gut protists in cellulose degradation, the gut bacteria are crucial in reductive

acetogenesis from H2 plus CO2 and nitrogen fixation for the nutrition requirement of

the host termites.

It is known that most of the lower termites, especially wood-feeding termites

demonstrated a rather higher H2 emission capability than higher termites such as soil-

feeding and fungus-feeding termites (Pester and Brune, 2007; Sugimoto et al., 1998;

Brauman et al., 1992). There is enormous accumulation of hydrogen in the hindgut of

Reticulitermes flavipes, Reticulitermes virginicus and Coptotermes formosanus (Cao

et al., 2010; Inoue et al., 2007; Ebert and Brune, 1997). In the metabolic reactions

involved in lignocellulose degradation in termite hindguts, hydrogen acts as the key

intermediate linking the fermentative breakdown of carbohydrates with

methanogenesis and homoacetogenesis (Brune, 2014). .

24
The productions of H2 and CO2 from the cellulose fermentation are converted by the

gut bacteria through reductive acetogenesis to acetate, which accounts for up to one-

third of the carbon and energy demand of the host termite (Breznak and Switzer, 1986).

Presence of homoacetogenesis is found in a large number of termite species including

wood-feeding, fungus-cultivating and soil-feeding termites (Breznak, 1994; Brauman

et al., 1992; Breznak and Kane, 1990). Gut fermenting bacteria more prefer

acetogenesis over methanogenesis as an H2 sink in termites (Breznak and Brune, 1994).

Termites that feed on soil and have fungi in their gut seem to perform methanogenesis

more often than acetogenesis. Higher termites that just feed on wood prefer

acetogenesis (Brune, 1998). From what has been studied so far, formate is formed in

the hindguts of many termites, which then accumulates, oxidizes to carbon dioxide, or

reduced to acetate (Brune, 2014).

On the other hand, nitrogen (N2) is an essential component in protein and nucleotide

biosynthesis and the production of other essential metabolites necessary for growth

and development of insects. Many xylophagous termites are intensely limited of

nitrogen (Colins, 1983). Abundance of N2 in the atmosphere can only be used by

certain organisms, such as microorganisms that supply inorganic form of nitrogen to

their hosts via symbiotic or non-symbiotic interactions. Termites are also able to obtain

some metabolic nitrogen via nitrogenous waste recycling by bacterial symbionts

(Hongoh et al., 2008b; Dillon and Dillon, 2004; Potrikus and Breznak, 1980b). The

feeding behaviour of proctodeal trophallaxis for termites to access the hindgut

microorganisms directly increases in frequency during limitation level of nitrogen. The

nitrogenous products probably take place in the foregut and midgut as indicated by the

activities of lysosome and protease (Fujita et al., 2004).

25
Similarly, in early studies, microbial nitrogen fixation has been reported in several

insects, including gut microorganisms associated with termites. Some termite species

make use of diazotrophic bacteria to fix atmospheric N2 to overcome the severe

shortage of dietary nitrogen (Sapountzis et al., 2016). Several bacterial endosymbionts

that have the ability to fix N2 have also been found in the gut of lower termites

(Peterson and Scharf, 2006). Fungus-growing termites (subfamily Macrotermitinae)

host a fungal exosymbiont (genus Termitomyces) that aid in digestion of the main food

source for the termites. This has been thought to prevent the need for N2-fixation by

bacterial symbionts (Sapountzis et al., 2016).

2.6 Potentials of Termite in Food and Medicine

2.6.1 Nutritional Values of Termites

Worldwide, there are 43 termite species serving as human food and domestic animal

feed (Figueiredo et al., 2015). In the past, insects have been consumed as an important

food source for humans in many regions and cultures all over the world. Until today

entomophagy remains a part of different food cultures in several developing countries

across America, Africa, and Asia (Chakravorty et al., 2011). Food and Agriculture

Organization, FAO (2012) reported more than two billion people practice

entomophagy around the world. There are over 1500 species of edible insects have

been recorded in 300 ethnic groups from 113 countries (Lokeshwari et al., 2010). The

common edible insects are beetles (31%), caterpillars (18%), ants, bees, and wasps

(14%), grasshoppers, crickets and locust (13%), cicadas, leafhoppers, plant hoppers

and bugs (10%), dragonflies (3%), termites (3%), flies (2%) and other insects (5%)

(Zielińska et al., 2015). They are generally rich in protein and contain lipids of easily

digestible fatty acid composition, moderate amounts of carbohydrates, good source of

26
iron, B-vitamins and valuable minerals. Termites are reported as the most consumed

among other insect species in the world (Chung and Yu, 2010). The less developed

nations facing malnutrition are known to harvest the termites as food for meeting

protein requirements in their diets. In Africa, the tribes harvest the alates that are rich

in fat and protein. The queens are highly delicious and hunted followed by the soldiers

(Figueiredo et al., 2015).

The importance of nutrient resources from edible insects are often served as traditional

foods among indigenous peoples. Many researchers from different countries have

conducted studies on nutrient analysis for various insects. Table 2.1 show the

comparison of nutritional composition among edible insects and other conventional

foods (Lokeshwari et al., 2010). The highest values of energy content have been

recorded in the termites Macrotermes subhyanlinus followed by weevil

Rhynchophorus phoenicis, which both are also the well-represented edible insects in

the reviews of nutrient composition by Payne and colleagues (2015).

Table 2.1. The nutritional content of edible insects and other animals based on
100 gram serving
Energy Protein Iron Thiamine Riboflavin Niacin
Animal
(Kcal) (g) (mg) (mg) (mg) (mg)
Termites
(Macrotermes 613 14.2 0.75 0.13 1.15 0.95
subhyanlinus)
Catepillar
370 28.2 35.5 3.67 1.91 5.2
(Usata terpsichore)
Weevil
(Rhynchophorus 562 6.7 13.1 3.02 2.24 7.8
phoenicis)
Beef
219 27.4 3.5 0.09 0.23 6.0
(Lean ground)
Fish
170 28.5 1.0 0..08 0.11 3.0
(Broiled cod)
Adapted from Lokeshwari et al., (2010)

27
2.6.2 Use of Termites in Medicine

Many species of insect and insect-derived substances have been used in medicinal

resources. Science has proven that insects consist of immunological, antibacterial

(antiseptic), anesthetic (painkiller), anti-rheumatic (relive aching), and diuretic

properties in their bodies. China and Korea are among the countries that widely use

insects as traditional medicine for the treatment of several illnesses. For example,

caterpillars are especially rich in B1, B2 and B6 needed in tiny amounts for normal

growth and health. Chinese caterpillar fungus Cordyceps sinensis contains the fungal

mycelium and sporophore which able to strengthen immunity and endurances.

Caterpillars of Bombyx mori infected by the white muscardine fungus Beauveria

bassiana are used in the treatment of strokes in the Republic of Korea (Chakravorty et

al., 2011).

Termites are also commonly used insects in traditional medicinal purposes around the

world, with the most widely used in the countries of Africa. In African medicinal

systems, the paste of termite and mound is boiled and applied onto the external wounds,

and also to treat internal hemorrhages through ingestion (Hoare, 2007). Termites are

known to be used in the treatment of various diseases that affect humans, such as heart

conditions (Costa-Neto, 2005), tuberculosis (Costa-Neto and Motta, 2008), anemia

and diarrhea (Senthikumar et al., 2008). Termites Nasutitermes macrocephalus are not

only traditionally used in Brazil in the treatment of asthma, hoarseness and sinusitis,

but also widely used in folk medicine in India (Chaves et al. 2017). In Nigeria, termite

Macrotermes nigeriensis is used on pregnant women with wounds and sickness

(Figueiredo et al. 2015). Additionally, some peptides like espinigerine and termicine

have been isolated from Pseudocanthotermes spiniger (Figueiredo et al., 2015) and

28
the Japanese subterranean termite, Reticulitermes speratus (Kolbe) are found to exhibit

antibiotic and antifungal activities.

There is a fungus Xylaria nigripes (Klotzsch) derived from the termite nest that has

been used as traditional Chinese medicine in fermented broth to extract ethyl acetate

for insomnia and depression treatment (Chang et al., 2017). Besides, some cases of

bronchitis, wounds, colds, flu, rheumatism and other conditions are reported to be

treated through inhalation of the incinerated termite soil and also the use of tea-crushed

insects.

29
 CHAPTER 3

MATERIALS AND METHODS

3.1 Sampling of termites

Coptotermes curvignathus were baited from the site in Woodman Oil Palm Plantation,

namely Block 16 (N 030100.6, E 1125251.7) that covered 39.41 ha of palm trees,

at Semanok Tatau, Bintulu, Sarawak. For termite baiting system, fresh rubber woods

(Hevea brasiliensis) were installed below the ground that close to termite infected

palm trees. After two to three months, the bait woods were collected and stored under

dark condition in the laboratory prior gut extraction.

3.2 Extraction of termite gut

Surface of worker termites was sterilized with 70% ethanol and rinsed with sterilized

distilled water. Termites were chilled and the whole guts were extracted with fine-

tipped sterile forceps by holding the thorax part while grasping the extreme posterior

part of the body simultaneously. The whole gut was then separated into foregut,

midgut and hindgut for further analysis of the individual compartments.

3.3 Measurements of the digestive system

The whole guts were obtained from the sterilized worker termites. Length and

diameter of foregut, midgut and hindgut were measured using Nikon SMZ1000 stereo

zoom microscope. The measurements were carried out with NIS-Elements

microscope imaging software.

30
Figure 3.1. Intestinal tract of worker Coptotermes curvignathus. The gut regions
are abbreviated as follows: FG, foregut; MG, midgut; HG, hindgut.

3.4 Chromatographic determination of gut metabolites

3.4.1 Chemicals and reagents

Chemicals used as reference standards include propionic acid, butyric acid, uric acid,

quercetin, and vancomycin hydrochloride, which were purchased from Sigma Aldrich

(Germany); ascorbic acid was from HmbG (Germany); pyrocatechol was from Merck

(Germany); salicylic acid and sulphuric acid were from Fisher (United Kingdom).

Acetonitrile was of high-performance liquid chromatography (HPLC) grade,

purchased from Merck and the other chemicals used were all of analytical grade.

Preparation of ultrapure water was obtained from an Arium 611 water purification

system (Sartorius, Germany).

31
3.4.2 Preparation of standard solutions

Standard stock solutions of the reference were prepared and diluted quantitatively to

obtain solutions with a range of known concentrations. All the standard solutions were

filtered through a 0.22 m MS PTFE filter membrane prior to HPLC injection. The

known concentrations of the stock solutions were then performed in triplicates to

construct the calibration curves.

3.4.3 Preparation of sample solutions

Whole guts of the fifty workers were dissected and separated into foregut, midgut and

hindgut. Different gut compartments were pooled separately in 1.5 mL

microcentrifuge tubes and homogenized with sterile micropestle in 0.5 mL Ultrapure

water. The solutions were shaken and centrifuged for 10 min at 20,000 g. The

supernatants were filtered at 0.22 m MS PTFE syringe filter into autosampler vials.

3.4.4 HPLC instrumentation and chromatographic conditions

Automated high-performance liquid chromatography (HPLC) system (JASCO, Japan)

was used. The system was equipped with an intelligent sampler (AS-2059 Plus), a

quaternary gradient pump (PU-2089 Plus), a column thermostat (CO-2060 Plus), and

a UV/VIS detector (UV-2075 Plus). The system was operated with EZChrom Elite

Version 3.1.6 software. Chromatographic separation was performed on a Hypersil

Gold C8 column (100 mm  4.6 mm ID  5 m particle size; Thermo Scientific). The

mobile phase used consisted of a mixture of 0.015 N sulphuric acid in Ultrapure water

(solvent A) and acetonitrile (solvent B). The mobile phase was filtered and degassed

by sonication before used. The elution program was performed using a 20-min

gradient, which initial solvent consisted of 100% A maintained for 4 min, changed

32
over next 12 min to 50% A:50% B, and backed to 100% A for 4 min. The column

temperature was kept at 35C. The flow rate was kept at 0.50 ml/min. The wavelength

for UV/VIS detector was set at 215 nm. An amount of 5 L termite gut fluids were

injected from autosampler vial into the chromatograph. Presence of different

chromatographic peaks was identified by comparing the retention times with the

reference standards.

3.4.5 Statistical analysis

The HPLC assays were carried out in triplicate at least. Datas were calculated as

means  standard deviations using Microsoft Office Excel 2007. Analysis of variance

and Tukey test were performed at the level of P-value <0.05 to indicate the

significance of the different experimental group.

3.5 Antioxidant assays

3.5.1 Preparation of standard solutions and chemicals

DPPH (1,1-diphenyl-2-picryl hydrazyl) solution was prepared by dissolving 0.5 mg

of DPPH (Sigma-Aldrich) in 10 mL methanol (Merck). The solution was kept in

darkness and left to stable for 30 min prior to the measurement of the antioxidant

activity. L(+)-ascorbic acid (HmbG Chemicals), butylated hydroxyanisole (BHA,

Merck), DL--tocopherol (Calbiochem) and quercetin (Sigma-Aldrich) were

prepared in methanolic solutions. Preparation of 2% aluminium chloride (AlCl3,

Merck) was done by dissolving 0.2 g in 10 mL distilled water.

33
3.5.2 DPPH radical-scavenging activity

The stable DPPH radical was used to determine the free radical scavenging activity

of the different gut compartments according to the method of Dorman et al. (2004).

Ascorbic acid, BHA and -tocopherol were used as positive controls whereas DPPH

solution was used as negative control. An amount of 20 L gut fluids (50 guts/0.5 mL)

was added to 0.1 mL methanolic solution of DPPH (0.05 mg/mL). The mixture was

shaken vigorously and incubated in darkness at room temperature. After 30 min,

absorbance was measured at 517 nm using an ELISA microplate reader (Sunrise,

TECAN). Each sample was done in triplicate. Mean of the three absorption readings

were taken to calculate the capability of the different gut compartment fluids in

scavenging the DPPH radical using the following equation:

DPPH scavenging effect (%) = Absorbance -ve - Absorbance sample  100

Absorbance -ve

34
3.5.3 Determination of total flavonoid content

Total flavonoid content of different gut compartments was measured according to

aluminium chloride method (Ranković et al., 2011). A 25 L of 2% aluminium

trichloride (AlCl3) in methanol was mixed with 25 L gut fluids (50 guts/0.5 mL).

The mixture was shaken vigorously and left incubated at room temperature for 10 min.

The absorbance was measured at 415 nm against a blank sample without AlCl3 using

an ELISA microplate reader (Sunrise, TECAN). Quercetin was used as the standard.

Quercetin solution was prepared by dissolving 3 mg quercetin in 1 mL methanol and

subsequently diluted to various concentrations of 2.4, 1.8, 1.2 and 0.6 mg/mL.

Calibration curve of quercetin was plotted by measuring the absorbance of the

dilutions at 415 nm with ELISA microplate reader (Figure 4.3). Total flavonoid

contents were expressed as microgram quercetin equivalent by referring to the

standard curve of quercetin.

3.6 Plate screening for cellulolytic activity

Media containing 1 % carboxymethylcellulose (CMC) sodium salt (Calbiochem) and

1.5 % agar noble (DifcoTM) were prepared in sterile 90 mm Petri dishes. A total of 15

guts from each gut compartments were pooled in 250 L Ultrapure water respectively.

Five microliters of each gut fluids were pipetted onto CMC agar plate. Cellulase

Aspergillus niger was used as positive control and sterilized water as negative control.

Three replicates of the CMC agar plates were incubated for 2 hours at 37 C. The

plates were stained with 0.1 % Congo red dye for 15 min, followed by destaining with

1.0 M NaCl solutions for 15 min (Teather and Wood, 1982). Enzyme activity was

evaluated through observations of the clear zone formed.

35
3.7 Isolation and Identification of Cellulolytic Microorganisms

3.7.1 Isolation of Cellulolytic Microorganisms

Fifteen worker termites were degutted into foregut, midgut and hindgut under sterile

condition. Different gut compartments were homogenized in 0.5 mL of Ultrapure

water respectively. The suspensions were mixed with 4.5 mL of 1.0 % CMC broth

media. A serial dilution of the mixture was performed up to 10-12 and incubated for 3

days at 37 C. After incubation, 1.0 mL of the cultures was spread on nutrient agar

plates and incubated for 24 hours at 37 C. Single colonies were obtained by several

sub-culturing under the sterile conditions for further analysis of DNA extraction and

uric acid concentration measurement.

3.7.2 DNA Extraction

Pure isolates were grown overnight in nutrient broth. The broth cultures were

centrifuged at 20,000 rpm for 2 min at 20 C. Supernatants were discarded and washed

with Ultrapure water for two to three times. DNA was extracted according to the

protocol of Qiagen DNeasy Tissue Kit. The DNA concentrations in the extracts were

quantified by agarose gel electrophoresis.

36
3.7.3 Identification by Polymerase Chain Reaction (PCR) and 16S ribosomal

RNA Gene Sequence

The DNA extracts were amplified in a thermocycler (MJ Mini Personal Thermal

Cycler, Bio-Rad). Two bacterial universal primers were used for amplifying the 16S

rRNA genes, forward 27F (5-AGAGTTTGATCMTGGCTCAG-3) and reverse

1492R (5-TACGGYTACCTTGTTACGACTT-3). Each PCR mixture with a final

volume of 50 L contained the following components: 38.6 L sterile Ultrapure water,

5.0 L 10 PCR buffer [500 mM KCl, 100 mM Tris-HCl, pH 8.3], 1.5 L MgCl2 (50

mM), 1.0 L of each primer (10 M), 2.0 L deoxynucleoside triphosphate (2mM

dNTPs), 0.5 L isolated DNA (20-50 ng), and 0.4 L Taq DNA polymerase (5U/L).

PCR profile was started by an initial denaturation at 95 C for 2 min. Denaturing step

continued at 95 C for 30 s, followed by annealing at 50 C for 1 min and primer

extension at 72 C for 4 min for a total of 18 thermal cycles. The thermal cycles

completed with final extension at 72 C for 10 min.

For analysis of the PCR products, 1 L aliquots were loaded onto 1.0 % agarose gel

in 1.0 TAE buffer (pH 8.0) and electrophoresis was carried out for 1 h at 100 V. Gels

were stained with ethidium bromide (10 mg/mL), destained with distilled water and

observed under UV light. Three replicates of the PCR products were re-amplified and

quantified. DNA bands were excised from the gel with sterile scalpels and placed in

microcentrifuge tubes for purification according to QIAquick Gel Extraction Kit

protocol. Purified PCR products were sent to 1st Base company for sequencing

analysis. The results of 16S rRNA sequences were analyzed by BLASTN program

(National Center for Biotechnology Information, NCBI).

37
3.8 Quantification of Uric Acid Contents

Uric Acid Assay Kit (Abnova) was used to detect presence of uric acid concentrations

among the cellolytic isolates. Blank control and standard (10 mg/dL uric acid) were

provided. Working reagents were freshly prepared by mixing 10 volumes of Reagent

A, 1 volume Reagent B and 1 volume Reagent C. The mixture was left equilibrate to

room temperature prior to assay. Fresh broth cultures were prepared by growing the

cellulolytic isolates in nutrient broths for an overnight. The broth cultures were

centrifuged at 10,000 rpm for 5 min to remove insoluble particles. A 5 L of the blank,

standard and the cell culture supernatant were transferred in triplicates wells of a clear

bottom 96 well plates. An amount of 200 L working reagent was added and tapped

lightly to mix. Uric acid concentrations were measured after 24, 48 and 72 h of

incubations at room temperature. Optical density (OD) was read at 590 nm on ELISA

microplate reader (Sunrise, TECAN). The readings were used to calculate the uric

acid concentration as the following formula:

OD Sample  OD Blank  10 mg/dL

OD Standard  OD Blank

38
 CHAPTER 4

RESULTS AND DISCUSSION

4.1 Physical Characterization of Coptotermes Curvignathus Gut Structure

The gut of Coptotermes curvignathus were divided into foregut, midgut and hindgut.

The length measurements for each segment were taken (Figure 4.1). Hindgut was the

longest segment (7.27  0.69 mm) of the digestive system in C. curvignathus, which

accounted approximately 60% of its total length. Midgut was the second longest in

length in the system, 4.16  0.52 mm (approximately 34%) and the foregut was the

shortest, 0.75  0.08 mm (approximately 6%). The foregut of C. curvignathus was

relatively shorter than other reported termite species (Table 4.2). The Kalotermitidae

termites have the longest foregut length as compared to other termites, especially

Neotermes bosei, which has foregut proportionally to 24% of the total gut length.

The hindgut of C. curvignathus was about 60% the total length of its gut. This

indicates the hindgut is the major absorption segment in digestive system. However,

the longest hindgut of termite was reported in wood-feeding higher termite,

Nasutitermes takasagoensis workers, approximately 70 % of the total gut length,

where the foregut and midgut constitute only 8 and 22 % respectively (Tokuda, 2001).

Two wood-feeder species of the neotropical genus Tauritermes also have

proportionally longer hindgut region, about 61 % of their total gut length.

39
 Foregut Midgut Hindgut

Figure 4.1. Digestive tract of Coptotermes curvignathus worker.

In C. curvignathus, hindgut was proportionally the longest region as compared to

foregut and midgut. In addition, hindgut was relatively wider in the diameter as

compared to foregut and midgut. At its widest diameter, it was measured up to 0.70 

0.14 mm whereas the uniform diameter of midgut was measured at 0.50  0.07 mm

and the foregut was at 0.50  0.09 mm (Table 4.1). Wider in diameter would allow

more anaerobes to reside in hindgut. Thus indicate fermentation or anaerobic

metabolism was the major activity in hindgut. On the other hand, less anaerobe would

be found in foregut and midgut, because the oxygen will penetrate through the gut

membrane and due to smaller diameter, the anaerobic zone in the lumen would be

very limited or lacking. The highly oxygenated condition would promote lignin

oxidation (Ke et al., 2010; Li et al., 2012) and allow lignocelluloses to be degraded

further in subsequent gut segment.

40
 Table 4.1. Gut measurements in length and diameter (mm) of C. curvignathus
workers
Gut compartments Length Diameter

Foregut 0.75  0.08 0.50  0.09

Midgut 4.16  0.52 0.50  0.07

Hindgut 7.27  0.69 0.70  0.14

Each value is expressed as mean  standard deviation (n = 15).

Table 4.2. Comparison of C. curvignathus gut length measurements (mm) and

number of Malpighian tubules with other families
Number of
Species Foregut Midgut Hindgut Malpighian References
tubules

Rhinotermitidae
Coptotermes
curvignathus 0.750.08 4.160.52 7.270.69 7
(n=15)

Kalotermitidae
Tauritermes
Godoy,
taurocephalus 1.800.16 2.400.30 6.600.59 8
2004
(n=30)
Tauritermes
sp. Godoy,
1.800.13 2.300.27 6.600.55 8
2004
(n=30)
Neotermes Hariprabow
bosei 3.320.57 3.860.61 6.560.91 Not reported o et al.,
(n=10) 2006

Nasutitermitidae
Nasutitermes
takasagoensis Approx. Approx. Approx. Tokuda,
Not reported
0.90 2.50 8.10 2001
(n=5)

41
There was an average of seven Malpighian tubules attached between the midgut and

the anterior hindgut region of C. curvignathus. The number was slightly lower than

Tauritermes spp. Tauritermes spp. have eight Malpighian tubules (Godoy, 2004)

compared to seven in C. curvignathus. The Malpighian tubules function as the

excretory system in many insect species, include termites.

4.2 Chromatographic Determination of Gut Metabolites

HPLC was used to determine the compounds presented in the C. curvignathus

digestive system. Compounds that were detectable throughout the digestive tract of C.

curvignathus are shown in Table 4.3. Uric acid was found to be the most concentrated

compound in all of the termite’s gut compartments. Uric acid was detected right from

the beginning of the digestive tract. This indicates C. curvignathus extraordinary

ability in extracting and storage uric acid. Concentration of uric acid further increased

approximately 15-fold in midgut. Uric acid is probably stored in the fat body of

termite’s midgut which has significant function as nitrogen reservoir (Coasta-

Leonardo et al., 2013). Retaining uric acid rather than voiding it into feces has been

reported in other termite species such as Reticulitermes flavipes (Potrikus and Breznak,

1981) and Mastotermes darwiniensis (Coasta-Leonardo et al., 2013). Malpighian

tubules are attached at the foremost part of the hindgut (Figure 4.1) and are known

actively in transporting fat body uric acid to the hindgut, which is believed to be the

main reason that caused the continuous increment of uric acid in the hindgut of C.

curvignathus. The concentration of uric acid was the highest in hindgut among the gut

segments. This indicates hindgut could be the major uric acid storage site. The uric

acid would be further recycled by uricolytic bacteria into carbon, nitrogen and energy

(Potrikus and Breznak, 1981; 1980a; 1980b). Since the diet of termites is usually short

42
supply in nitrogen, recycling of uric acid nitrogen in termites is a strategy for nutrient

conservation.

Table 4.3. Concentration of chemical compounds detected in main gut

compartments of C. curvignathus worker
Estimated concentration (mM)
Compounds
Foregut Midgut Hindgut

Ascorbic acid – – 0.194  0.008 a

Butyric acid – 18.114  2.473 a 16.650  6.270 a

Propionic acid 2.733  0.086 c 28.612  4.086 b 48.496  4.468 a

Pyrocatechol 0.014  0.001 c 0.528  0.084 a 0.386  0.045 b

Quercetin 0.360  0.085 a 0.686  0.108 a 0.377  0.306 a

Salicylic acid 0.010  0 b 0.094  0.014 a 0.076  0.005 a

Uric acid 36.135  1.071c 511.210  78.865 b 752.872  56.110 a

Vancomycin HCl 0.001  0 b 0.010  0.002 a 0.008  0.002 a

Data are shown as mean  standard deviation (n = 3).

Means with different letters within a row are significantly different (p < 0.05).
“  ”, not detected.

Presence of pyrocatechol, quercetin and salicylic acid showed that there are changes

in the chemical structure of lignin during passage through the entire gut system, which

was initiated in the foregut and continued in the midgut and hindgut. These three

compounds were initially detected in foregut, and found abundantly in midgut and

slightly decreased in the hindgut (Table 4.3). Mastication in foregut changes the

structural of lignin in most of the phylogenetically lower termites (Ke et al., 2012).

Many wood-feeding termites including C. formosanus (Shiraki) have been reported

to have the capability in metabolizing aromatic compounds efficiently such as lignin

which begin at the foregut and intensified in the midgut regions (Ke et al., 2011).

43
Decomposition of lignin is continued via enzyme digestion when entering midgut.

This explained the 10-fold increment of propionic acid in midgut. Amount of

pyrocatechol, salicylic acid and vancomycin HCI also significantly increased in

midgut and reduced slightly when entering hindgut. This indicates the lignocellulosic

materials were continuously digested in the midgut, and hindgut was the major

nutrients absorption site. An immunohistochemistry study was done on the midgut of

C. formosanus (Shiraki) and showed evidence that there are distributions of

endogenous cellulase in the endoperitrophic space and on the microvilli lining (Fujita

et al., 2010), in both lower and higher termites (Terra and Ferreira, 1994; Nakashima

et al., 2002; Terra and Ferreira, 2012). These enzymes help in decomposition the

wood diets in termites. Digestion products include soluble sugars such as glucose and

cellobiose could pass through and absorbed through the midgut wall.

Indication of high lignin modification in midgut of C. curvignathus was consistence

with the physical characteristic of the midgut. As shown in Table 4.1, the midgut of

C. curvignathus was relatively small in diameter as compared to hindgut; it means the

midgut mainly constituted of aerobic region. In fact, studies have shown the wood-

feeding termites contain higher oxygen concentration in midgut as compared to other

gut regions (Ke et al., 2010). This is important features as pertaining to its function in

digestion. Lignin, which is the main component that protects the lignocellulosic

material from decomposition, can only be modified in aerobic condition. Studies have

supported that lignin oxidation is promoted in the midgut (Geib et al., 2008; Scharf

and Tartar, 2008 Katsumata et al., 2007; Breznak and Brune, 1994). Phenol oxidase

and esterase act in aerobic condition to modified lignin and solubilize hemicellulose

(Karl and Scharf, 2015; Tartar et al., 2009; Scharf and Tartar, 2008). In addition,

44
actinobacteria that are known to have the ability in producing wide range of catabolic

enzymes such as peroxidases to depolymerize lignin or degrading lignin derivatives

(Tian et al., 2014), was found abundantly in the intestinal tract of C. curvignathus

(King et al., 2014). This helps the breakdown of lignin. In termite R. flavipes, lignase

genes particularly laccases and peroxidases were identified in the salivary gland and

foregut tissues, which had implied lignin degradation to occur in the front part of the

digestive system (Scharf and Tartar, 2008). The protective lignocelluloses structure is

disrupted further via mastication in proventriculus (Watanabe and Tokuda, 2010).

These would expose the cellulose fibers that are previously buried in lignin and

hemicelluloses thus would be accessible by the termite endogenous enzymes for

further decomposition. This explained the surge in concentration for most of the

chemical compounds such as propionic acid, butyric acid, uric acid, pyrocatechol,

quercetin, salicylic acid and vancomycin HCl in midgut region (Table 4.3).

The presence of vancomycin HCl in the digestive fluids of C. curvignathus is

supporting the finding of high percentage of Streptomyces in the gut of C.

curvignathus (King et al., 2014). Streptomyces, member of the Actinomycetes has

been reported to produce bioactive secondary metabolites compound, vancomycin

hydrochloride (Chaudhary et al., 2013; Levine, 2006; Moellering, 2006; Berdy, 2005).

Streptomyces niverotuber that was isolated from termite’s gut is capable in producing

bioactive compound with wide spectrum of biological activities against human

pathogen (Khucharoenphaisan et al., 2012).

Pyrocatechol (Table 4.3) was found in the highest concentration in midgut.

Pyrocatechol, also known as catechol is an important building block in the cuticle of

45
an insect. Catechol strengthens the cuticle layer by cross linking the chitin to other

structural proteins. In termite, the cuticle layer lining on top of the epithelial cell layer

of foregut and hindgut (Engel and Moran, 2013). This offers protection to the

epithelial cell layer of the foregut and hindgut against direct exposure to

microorganisms or toxins. These physical barriers between epithelium and lumen are

good examples for tolerance mechanisms, because the termites do not have to reduce

the bacterial load in the gut as most of the bacteria are beneficial to their digestive

system, but they reduce the impact of the bacteria on the host.

From Table 4.3, the midgut can be regarded as the major production site for several

metabolites such as butyric acid, pyrocatechol, quercetin, and salicylic acid. However,

several metabolites can only be detected or reached their highest concentration in

hindgut, which were the ascorbic acid, propionic acid and uric acid. This means

decomposition of wood diet continues in hindgut. Ascorbic acid or commonly known

as vitamin C was only detectable in the hindgut of C. curvignathus. Most intestinal

tract of insects is unable to synthesize ascorbic acid internally although a trace amount

is still required in their nutrition for growth and development purposes. The

requirement perhaps is fulfilled by certain gut microbial symbionts that able to

synthesize it (Kramer and Seib, 1982). Ascorbic acid is a highly antioxidative

compound that removes free radicals to prevent tissue damages and acts as a cofactor

for various enzymatic hydroxylation reactions (Mathews et al., 1997). Actinomycetes

are microbes that capable of synthesizing antioxidants apart from capabilities in

degrading cellulose and lignin (Chaudhary et al., 2013). These microbes have been

isolated from the gut of various species of termite including C. curvignathus (King et

al., 2014; Upadhyay, 2011).

46
Propionate and butyrate were among the fermentable products that were detectable in

high concentration in the hindgut region. Both compounds could be derived from

lactate fermentation by Bacteroides strains (Schultz and Breznak, 1979). In the gut of

C. curvignathus, Bacteroides were reported as the most abundant microbes (King et

al., 2014). Many studies have evidenced that Bacteriodetes are the most dominant

phyla in natural ligninolytic environments such as termite gut and rumen (Wu and He,

2013; Kudo, 2009). They display excellence cellulolytic activity with high biomass

conversion rate.

Butyric acid was the third major metabolites found in the gut of C. curvignathus

(Table 4.3). Clostridium species, another common gut microbe in C. curvignathus

(King et al., 2014) has the ability to utilize glucose and xylose as carbon source for

butyric acid production. Clostridia are facultative anaerobes (Rieu-Lesme et al., 1996)

and are reported to be associated with the midgut epithelium and colonize on the

midgut wall (Tokuda et al., 2000). This explains high concentration of butyric acid at

the midgut and continuous increase in concentration in the hindgut of C. curvignathus.

Although Lactobacillales is regarded as normal microflora in the gut of C.

curvignathus (King et al., 2014), there is no lactic acid compound detectable in the

gut. This may be because of the lactate presences only in a trace amount and is quickly

broken down into other metabolites. Previous studies show that the lactate produced

by Streptococcus lactis, residing in the gut of R. flavipes will be fermented by

Bacteroides to propionate and acetate (Schultz and Breznak, 1979; Schultz and

Breznak, 1978). Thus the lactic acid formed in the gut of C. curvignathus could be

47
rapidly broken down by Bacteroides, which is the major constituent of C.

curvignathus gut microflora (King et al., 2014).

4.3 Antioxidant across the Digestive System

4.3.1 DPPH radical scavenging activity

The result of the antioxidant study based on DPPH radical scavenging method is

shown in Figure 4.2. Purified antioxidants such as ascorbic acid, butylated

hydroxyanisole (BHA) and -tocopherol were used as standard. The hindgut fluid of

C. curvignathus exhibited the highest antioxidant activities, followed by midgut and

the foregut fluids had the lowest antioxidant activity. The result is in agreement with

Table 3, where the ascorbic acid is mainly found in hindgut.

100 95.25 93.14 95.33

90
DPPH radical scavenging (%)

80
70
60 55.59

50 46.62

40
30
20.36
20
10
0
Foregut Midgut Hindgut Ascorbic acid α-Tocopherol BHA

Figure 4.2. Free radical scavenging activity of gut fluids in different

compartment

48
4.3.2 Determination of Total Flavonoid Content

The amount of total flavonoid was determined using quercetin as a standard

compound. Total flavonoid was expressed as mM quercetin equivalent. A standard

quercetin graph with R2 = 0.9956 was obtained and its equation was y = 126.55x +

0.5597, where y is the reading of absorbance at 510 nm and x equals to the total

flavonoid content in the gut fluids of C. curvignathus (Figure 4.3). Table 4.4 showed

the mean absorbance of a spectrum concentration of quercetin. The total flavonoid

content of the studied gut fluids of C. curvignathus is shown in Table 4.5. The result

showed that the hindgut fluids and midgut fluids were 8.1 and 2.1 mM of quercetin

equivalent. Foregut fluids showed less than 2 mM of quercetin equivalent, which was

below the detectable limit of the used method.

2
1.8
1.6
1.4
Absorbance

y = 126.55x + 0.5597
1.2
R2 = 0.9956
1
0.8
0.6
0.4
0.2
0
0 2 4 6 8 10
Concentration (mM)

Figure 4.3. Standard curve of Quercetin

49
 Table 4.4. Absorbance of standard compound (Quercetin)
Absorbance (Mean)
Concentration (mM)
max = 510nm
2 0.765
4 1.115
6 1.340
8 1.574
10 1.801

Table 4.5. Total flavonoid content in different gut fluids of C. curvignathus

Total flavonoid
Different gut compartments
(mM quercetin equivalent)

Foregut - 1.2 *

Midgut 2.1  0.071

Hindgut 8.1  0.045

Each value is expressed as mean  standard deviation (n = 5).

*
Below detection limit (< 2 mM)

The results of DPPH free radical scavenging activities and total flavonoid contents

throughout the gut compartments revealed that C. curvignathus contains high

antioxidant properties. Uric acid which was most prevailing metabolites in the gut of

C. curvignathus, especially hindgut, could act as antioxidant. A study using Japanese

termite R. speratus shows that uric acid could effectively suppress reactive oxygen

species, ROS in the body of R. speratus (Tasaki et al., 2017). Moreover,

administration of uric acid increased the survival of termites during deficiencies in

uric acid production. Uric acid is also known as one of the major antioxidants to have

scavenging capacity against oxygen radicals in human and insects (Matsuo and

Ishikawa, 1999; Ames et al., 1981). Strong antioxidant defense mechanisms might

explain the extraordinary long lifespan of the highly eusocial insects such as ants,

termites, honeybees and wasps, especially the worker and queen castes.

50
Apart from uric acid, ascorbic acid is also known to possess antioxidant properties.

Presence of ascorbic acid in the hindgut of C. curvignathus enhanced the antioxidant

capacities of the gut compartment. Besides, pyrocatechol which contains phenolic

compounds also could have contributed to the antioxidant activities in C. curvignathus.

Pyrocatechol which belongs to the ortho position of phenol hydroxyl group, was

found to be the best and the most stable antioxidant properties against superoxide

radicals (Velika and Kron, 2013).

4.4 Mapping of Cellulase Activity to Gut Structure

All three main gut segments of C. curvignathus showed presence of cellulase activity

that can be observed as a clearing zone on the CMC agar plate stained with congo red

(Figure 4.4). Zhang and colleagues (2011) showed that wood digestion starts in the

mouth of termites, where endogenous endoglucanases are secreted from salivary

glands in foregut to initiate the reaction. Cellulase activity occurred in all segments

and this is in agreement with the metabolites result shown in Table 4.3.

R1 R2 R3

Figure 4.4. Cellulase activity screening of the gut fluids with cellulase Aspergillus
niger served as positive control

51
4.5 Cellulolytic Microorganisms

There were 21 cellulolytic microorganisms isolated from the foregut, midgut and

hindgut compartment. Their DNAs were extracted, amplified and sequenced. Based

on 16S rRNA sequencing analysis, these isolates were identified mainly as Bacillus

spp. as shown in Table 4.6.

Table 4.6. Bacterial isolates from different gut compartments

Name of the isolate Phylum/Class Identities of isolates
Foregut
F1 Firmicutes Bacillus sp.
F2 Firmicutes Bacillus sp.
F3 Firmicutes Bacillus sp.
F4 Proteobacteria Burkholderia sp.
Midgut
M1 Firmicutes Bacillus sp.
M2 Proteobacteria Escherichia sp.
M3 Proteobacteria Escherichia sp.
M4 Firmicutes Bacillus sp.
M5 Firmicutes Bacillus sp.
M6 Firmicutes Bacillus sp.
M7 Firmicutes Bacillus sp.
M8 Proteobacteria Escherichia coli
M9 Proteobacteria Dyella sp.
Hindgut
H1 Firmicutes Bacillus sp.
H2 Firmicutes Bacillus sp.
H3 Firmicutes Bacillus sp.
H4 Firmicutes Bacillus sp.
H5 Firmicutes Bacillus sp.
H6 Proteobacteria Escherichia sp.
H7 Proteobacteria Escherichia sp.
H8 Firmicutes Bacillus sp.
F=foregut; M=midgut; H=hindgut.

52
Overall, the BLAST results showed high degree of similarity, ranging from 99 to

100 %, with E-value equalled to “ 0 ” when BLASTN to annotated sequences

deposited in the NCBI databases. Most of the isolated cellulolytic microorganisms

from each gut compartment were members of Firmicutes, followed by Proteobacteria.

At genus level, Bacillus was the dominant genera.

4.6 Uric Acid Producing Microorganisms

The isolated cellulolytic microorganisms displayed ability to gradually increase uric

acid concentration after 72 hours of incubation in nutrient broth (Table 4.7). Previous

studies show that uric acid gradually accumulated within termite bodies after being

captivated in a laboratory for a period of time (Potrikus and Breznak, 1980c).

Accumulation of uric acid in R. speratus workers was found to increase the

antioxidant activities and suppress the ROS levels in the body of termites after

captivated (Tasaki et al., 2017). Tasaki and colleagues explained that when termites

are outside of the colony with more aerobic conditions, termites might experience

with elevated oxidative stress that caused reduction of intestinal anaerobes and

decelerated uric acid degradation. Thus, uric acid could be produced by facultative

anaerobes, such as the isolated cellulytic microbes from the gut of C. curvignathus to

increase the survival of their host in laboratory condition.

53
Table 4.7. Uric acid production (mg/dL) from cellulolytic microorganisms after
different incubation periods.
Incubation Periods 24 hours 48 hours 72 hours
Foregut
F1. Bacillus sp. 2.182  0.064 b 2.626  0.121 b 3.507  0.113 a
F2. Bacillus sp. 1.959  0.080 b 2.270  0.146 b 3.452  0.397 a
F3. Bacillus sp. 2.301  0.154 b 2.326  0.135 b 3.385  0.165 a
F4. Burkholderia sp. 2.346  0.051 ab 2.293  0.059 b 2.536  0.026 a
Midgut
M1. Bacillus sp. 1.881  0.102 b 1.942  0.053 b 3.115  0.151 a
M2. Escherichia sp. 1.840  0.045 b 1.921  0.068 b 2,904  0.180 a
M3. Escherichia sp. 1.845  0.004 b 2.254  0.037 b 3.053  0.174 a
M4. Bacillus sp. 1.835  0.021 a 2.336  0.116 a 2.304  0.298 a
M5. Bacillus sp. 1.815  0.141 b 2.090  0.117 b 4.535  0.133 a
M6. Bacillus sp. 1.815  0.024 c 2.200  0.091 b 2.880  0.064 a
M7. Bacillus sp. 1.729  0.035 c 2.101  0.084 b 3.151  0.058 a
M8. Escherichia coli 2.053  0.164 b 2.401  0.176 b 3.583  0.246 a
M9. Dyella sp., 1.184  0.078 a 2.168  0.173 a 2.695  0.592 a
Hindgut
H1. Bacillus sp. 2.063  0.085 c 2.438  0.068 b 3.297  0.024 a
H2. Bacillus sp. 1.979  0.079 c 2.422  0.050 b 3.105  0.149 a
H3. Bacillus sp. 2.096  0.095 b 2.706  0.102 ab 3.343  0.232 a
H4. Bacillus sp. 1.990  0.023 c 2.792  0.067 b 3.285  0.089 a
H5. Bacillus sp. 1.977  0.103 b 3.108  0.332 b 5.791  0.551 a
H6. Escherichia sp. 1.820  0.055 a 2.280  0.078 a 3.099  0.525 a
H7. Escherichia sp. 1.898  0.134 b 2.637  0.090 a 2.856  0.040 a
H8. Bacillus sp. 1.852  0.016 c 2.744  0.085 b 3.373  0.098 a
Data are shown as mean  standard errors (n = 3).
Means with different letters within a row are significantly different (p < 0.05).

54
 CHAPTER 5

SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE

RESEARCH

In summary, this study has unveiled the distribution of metabolic compounds in the

intestinal tract to understanding the metabolism in termite C. curvignathus. However,

the characterization of overall gut fluid compounds in the termite is still meager. More

detail approach of the chemical compounds is required to evidence them. The

specificity of certain compounds found in different compartments seems to provide

insights into the digestive processes of C. curvignathus, involving presence of the

distinct fermenting microorganisms and physiocochemical conditions of the gut

environment that affect the rate of plant polymer hydrolysis.

55
 REFERENCES

Abe, T. (1987). Evolution of life types in termites. In Kawano, S., Connell, J.H. and
Hidda, T. (Ed.), Evolution and coadaption in biotic communities (pp. 125-148).
University of Tokyo Press, Tokyo.

Ames, B. N., Cathcart, R., Schwiers, E., and Hochstein, P. (1981). Uric acid provides
an antioxidant defense in humans against oxidant- and radical-caused aging and
cancer: A hypothesis. Proceedings of the National Academy of Sciences of the
United States of America, 78(11): 6858-6862.

Bauer, S., Tholen, A., Overmann, J., and Brune, A. (2000). Characterization of
abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-
feeding termites by molecular and culture-dependent techniques. Archives of
Microbiology, 173: 126-173.

Berdy, J. (2005). Bioactive microbial metabolites. The Journal of Antibiotics, 58(1):

1-26.

Berlanga, M., Paster, B. J., Grandcolas, P. and Guerrero, R. (2011). Comparison of the
gut microbiota from soldier and worker castes of the termite. International
Microbiology, 14: 83-93.

Bignell, D. E. (2011). Morphology, physiology, biochemistry and functional design of

the termite gut: an evolutionary wonderland. In Bignell, D. E., Roisin, Y., and
Lo, N. (Ed.), Biology of Termites: A Modern Synthesis (pp. 375-412). Dordrecht:
Springer.

Bignell, D. E. and Eggleton, P. (2000). Termites in ecosystems. In T. Abe, D. E.

Bignell, and M. Higashi (Eds.), Termites: Evolution, sociality, symbioses,
ecology (pp. 363-388). Dordrecht: Kluwer Academic Publishers.

Bignell, D. E., and Anderson, J. M. (1980). Determination of pH and oxygen status in

the guts of lower and higher termites. Jorunal of Insect Physiology, 26: 183-188.

Bignell, D. E., Oskarsson, H., Anderson, J. M., Ineson, P., and Wood, T. G. (1983).
Structure, microbial association and function of the so-called “mixed segment”
of the gut in two soil-feeding termites, Procubitermes aburiensis and Cubitermes
severus (Termitidae, Termitinae). Journal of Zoology London, 201: 445-480.

Brauman, A., Kane, M. D., Labat, M., and Breznak, J. A. (1992). Genesis of acetate
and methane by gut bacteria of nutritionally diverse termites. Science, 257: 1384-
1387.

Brennan, Y., Callen, W. N., Christoffersen, L., Dupree, P., Goubet, F., Healey, S.,
Hernandez, M., Keller, M., Li, K., Palackal, et al., (2004). Unusual microbial
xylanases from insect gut. Applied and Environmental Microbiology, 70: 3609-
3617.
Breznak J. A. and Switzer, J. M. (1986). Acetate synthesis from H2 plus CO2 by
termite gut microbes. Applied and Environmental Microbiology, 52(4): 623-630.

56
Breznak, J A. and Kane, M. D. (1990). Microbial H2/CO2 acetogenesis in animal guts:
nature and nutritional significance. FEMS Microbiology Review, 87: 309-314.

Breznak, J. A. (1994). Acetogenesis from carbon dioxide in termite guts. In Drake, H.

L. (Ed.), Acetogenesis (pp. 303-330). Chapman and hall, New York.

Breznak, J. A. and Brune, A. (1994). Role of microorganisms in the digestion of

lignocelluloses by termites. Annual Reviews of Entomology, 39: 453-487.

Brune, A. (1998). Termite guts: the world’s smallest bioreactors. Trends in

Biotechnology, 16: 16-21.

Brune, A. (2013). Symbiotic associations between termites and prokaryotes. In E.

Rosenberg et al., (Ed.), The Prokaryotes – Prokaryotic Biology and Symbiotic
Associations (pp. 545-577). Springer-Verlag Berlin Heidelberg.

Brune, A. (2014). Symbiotic digestion of lignocellulose in termite guts. Nature

Reviews Microbiology, 12: 168-180.

Brune, A. and Friedrich, M. (2000). Microecology of the termite gut: structure and
function on a microscale. Current Opinion in Microbiology, 3: 263-269.

Brune, A. and Ohkuma, M. (2011). Role of termite gut microbiota in symbiotic

digestion. In D.E. Bignell et al., (Ed.), Biology of Termites: A Modern Synthesis
(pp. 439-475). Springer Science+Business Media B.V.

Brune, A. and Stingl, U. (2005). Prokaryotic symbionts of termite gut flagellates:

phylogenetic and metabolic implications of a tripartite sysmbiosis. In Overmann,
J. (Ed.) Molecular basis of symbiosis. Springer, New York.

Brune, A., Emerson, D. and Breznak, J. A. (1995). The termite gut microflora as an
oxygen sink: microelectrode determination of oxygen and pH gradients in guts
of lower and higher termites. Applied and Environmental Microbiology, 61:
2688-2695.

Brune, A., Emerson, D. and Breznak, J. A. (1995a). The termite gut microflora as an
oxygen sink: microelectrode determination of oxygen and pH gradients in guts
of lower and higher termites. Applied and Environmental Microbiology, 61:
2681-2687.

Brune, A., Frenzel, P., and Cypionka, H. (2000). Life at the oxic-anoxic interface:
microbial activities and adaptation. FEMS Microbiology Review, 24: 691-710.

Brune, A., Miambi, E., and Breznak, J. A. (1995b). Roles of oxygen and the intestinal
microflora in the metabolism of lignin-derived phenylpropanoids and other
monoaromatic compounds by termites. Applied and Environmental
Microbiology, 61: 2688-2695.

57
Burnum, K. E., Callister, S. J., Nicora, C. D., Purvine, S. O., Hugenholtz, P., Warnecke,
F., Scheffrahn, R. H., Smith, R. D., and Lipton, M. S. (2011). Proteome insights
into the symbiotic relationship between a captive colony of Nasutitermes
corniger and its hindgut microbiome. ISME Journal, 5: 161-164.

Cao, Y.Q., Sun, J-Z, Rodriguez, J. M. and Lee, K. C. (2010). Hydrogen emission by
three wood-feeding subterranean termite species (Isoptera: Rhinotermitidae):
Production and characteristics. Insect Science, 17: 237-244.

Chakravorty, J., Ghosh, S., and Meyer-Rochow, V. B. (2011). Practices of

entomophagy and entomotherapy by members of the Nyishi and Galo tribes, two
ethnic groups of the state of Arunachal Pradesh (North-East India). Journal of
Ethnobiology and Ethnomedicine, 7: 5.

Chan, S. P., Bong, J. C. F. and Lau, W. H. (2011). Damage pattern and nesting
characteristic of Coptotermes curvignathus (Isoptera: Rhinotermitidae) in oil
palm on peat. American Journal of Applied Science, 8(5): 420-427.

Chang, J. C., Hsiao, G., Ling, R. K., Kuo, Y. H., Ju. Y. M., and Lee, T. H. (2017).
Bioactive constituents from the termite nest-derived medicinal fungus Xylaria
nigripes. Journal of Natural Products, 80(1): 38-44.

Charles, S., Hung, P.K.J., Fah, J.B.C., Huat, O., Bakar, F.D.A., Murad, A.M.A., Tsang,
A., and Mahadi, N.M. (2014). Detoxification related genes in gut of Coptotermes
curvignathus. Entomology, Ornithology and Herpetology, 3: 117.

Chaudhary, H. S., Soni, B., Shrivastava, A. R., and Shrivastava, S. (2013). Diversity
and versatility of actinomycetes and its role in antibiotic production. Journal of
Applied Pharmaceutical Science, 3(8 Supplement 1): S83-S94.

Chaves, T. P., Policarpo, I. S., Coutinho, H. D. M., Alves, R. R. N., Vasconcellos, A.,
Medeiros, A. C. D. (2017). Correlatio between bioaccumulated
phytocompounds and bioactivities in Nasutitermes microcephalus (Silvestri,
1903). (Isoptera: Termidae) and its nest extract against multi-resistant bacterial
strains. Indian Journal of Traditional Knowledge, 16: 583-587.

Chung, H. Y. and Yu, Z. (2010). Food and function. Royal Society of Chemistry, 1: 1-
132.

Coasta-Leonardo, A. M., Casarin, F. E., and Lima, J. T. (2009). Chemical

communication in Isoptera. Nerotropical Entomology, 38(1): 1-6.

Coasta-Leonardo, A. M., Laranjo, L. T., Janei, V., and Haifig, I. (2013). The fat body
of termites: Functions and stored materials. Journal of Insect Physiology, 59:
577-587.

Collin, N. M. (1984). The termites (Isoptera) of the Gunung Mulu National Park, with
a key to the genera known from Sarawak. Sarawak Museum Journal, 30: 65-87.

58
Collins, N. M. (1988). Termites. In Cranbrook (Ed.), Key Environments Malaysia.
Oxford: Pergamon Press.

Corona, M., Hughes, K. A., Weaver, D. B., Robinson, G. E. (2005). Gene expression
patterns associated with queen honey bee longevity. Mechanisms of Ageing and
Development, 126(11): 1230-1238.

Costa-Neto, E. M. (2005). Entomotherapy, or the medicinal use of insects. Journal of

Ethnobiology and Ethnomedicine, 25: 93-114.

Costa-Neto, E. M. and Motta, P. C. (2008). Animal species traded as ethnomedical

resources in the federal district, central west region of Brazil. The Open
Complementary Medicine Journal, 2: 24-30.

Coy, M. R., Salem, T. Z., Denton, J. S., Kovaleva, E. S., Liu, Z., Barber, D. S., et al.
(2010). Phenol-oxidizing laccases from the termite gut. Insect Biochemistry and
Molecular Biology, 40: 723-732.

de Figueiredo, R. E., Vasconcellos, A., Policarpo, I. S., and Alves, R. R. (2015). Edible
and medicinal termites: a global overview. Journal of Ethnobiology and
Ethnomedicine, 11: 29.

Delaplane, K. S., and LaFage, J. P. (1989). Foraging tenacity of Reticulitermes flavipes

and Coptotermes formosanus (Isoptera: Rhinotermitidae). Sociobiology, 16:
183-189.

Dillon, R. J. and Dillon, V. M. (2004). The gut bacteria of insects: nonpathogenic

interactions. Annual Reviews of Entomology, 49: 71-92.

Donovan, S. E., Eggleton, P., and Bignell, D. E. (2001). Gut content analysis and a
new feeding group classification of termites. Ecology Entomology, 26: 356-366.

Dröge, S., Fröhlich, J., Radek, R., and König, H. (2006). Spirochaeta Coccoides sp.
nov., a novel cocoid spirochete from the hindgut of the termite Neotermes
Castaneus. Applied and Environmental Microbiology, 72: 392-397.

Ebert, A. and Brune, A. (1997). Hydrogen concentration profiles at the oxicanoxic

interface: a microsensor study of the hindgut of the wood-feeding lower termite
Reticulitermes flavipes (Kollar). Applied and Environmental Microbiology, 63:
4039-4046.

Eggleton, P. (2000). Global patterns of termite diversity: In Abe, T., Bignell, D. E.,
and Higashi, M. (Ed.), Termites: evolution, sociality, symbioses, ecology (pp.
25-51). Kluwer Academic Publishers, Dordrecht.

Eggleton, P., Bignell, D. E., Sands, W. A., Mawdsley, N. A., Lawton, J. H., Wood, T.
G. and Bignell, N. C. (1996). The diversity, abundance and biomass of termites
under differing levels of disturbance in the Mbalmayo Forest Reserve, southern
Cameroon. Philosophical Transaction of the Royal Society London B. 351: 51-
68.

59
Eggleton, P., Homathevi, R., Jeeva, D., Jones, D. T., Davies, R. G. and Maryati, M.
(1997). The species richness and composition of termites (Isoptera) in primary
and regenerating lowland dipterocarp forest in Sabah, East Malaysia. Ecotropica.
3: 119-128.

Engel, P. and Moran, N. A. (2013). The gut microbiota of insects – diversity structural
and function. FEMS Microbiology Review, 37: 699-735.

Eutick, M. L., Veivers, P. O’Brien, R. W., and Slaytor, M. (1978). Dependence of the
higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lateus
on their gut flora. Journal of Insect Physiology, 24: 363-368.

Fei, H., and Henderson, G. (2002). Formosan subterranean termite (Isoptera:

Rhinotermitidae) wood consumption and worker survival as affected by
temperature and soldier proportion. Environmental Entomology, 31, 509-514.

Fujita, A. (2004). Lysozymes in insects: what role do they play in nitrogen metabolism?
Physiological Entomology, 299: 305-310.

Fujita, A., Hojo, M., Aoyagi, T., Hayashi, Y., Arakawa, G., Tokuda, G., and Watanabe,
H. (2010). Details of the digestive system in the midgut of Coptotermes
formosanus, Shiraki. The Japan Wood Research Society, 56: 222-226.

Fujita, A., Shimizu, I., and Abe, T. (2001). Distribution of lysozyme and protease, and
amino acid concentration in the guts of a wood-feeding termite, Reticulitermes
speratus (Kolbe): possible digestion of symbiont bacteria transferred by
trophallaxis. Physiological Entomology, 26: 116-123.

Geib, S. M., Fillety, T. R., Hatcher, P. G., Hoover, K., Carlson, J. E., Jimenez-Gasco,
M. D., Nakagawa-Izumi, A., Sleighter, R. L., and Tien, M. (2008). Lignin
degradation in wood-feeding insects. Proceedings of the National Academy of
Sciences of the United States of America, 105(35): 12932-12937.

Godoy, M. C. (2004). Gut structure of two species of the neotropical genus

Tauritermes Krishna (Isoptera: Kalotermitidae). Neotropical Entomoglogy,
33(2): 163-167.

Graber, J. R. and Breznak, J. A. (2004). Physiology and nutrition of Treponema

primitia, an H2/CO2 acetogenic spirochete from termite hindguts. Applied and
Environmental Microbiology, 70: 1307-1314.

Graber, J. R. and Breznak, J. A. (2005). Folate cross-feeding supports symbiotic

homoacetogenic spirochetes. Applied and Environmental Microbiology, 71:
1883-1889.

Graber, J. R., Leadbetter, J. R., and Breznak, J. A. (2004). Description of Treponema

azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes
isolated from termite guts. Applied and Environmental Microbiology, 70: 1315-
1320.

60
Hariprabowo, L. E., Raffiudin, R. and Prawasti, T. S. (2006). Alimentary canal
anatomy and histology of the worker termite Neotermes bosei. Biotropia, 13(2):
99-110.
Henderson, G. (2008). The termite menace in New Orleans: Did they cause the
floodwalls to tumble? American Entomologist, 54: 156-162.

Hoare, A. L. (2007). The use of non-timber forest products in the Congo Basin:
Constraints and opportunities (pp.1–54). London: The rainforest foundation.

Hongoh, Y. and Ohkuma, M. (2011). Termite gut flagellates and their methanogenic
and eubacterial symbionts. In Hackstein J. H. P. (Ed.), Microbiology
Monographs: (Endo) Symbiotic Methanogenic Archaea (pp. 55-79). Berlin:
Springer-Verlag.

Hongoh, Y., Sharma, V. K., Prakash, T., Noda, S., Toh, H., Taylor, T. D., Kudo, T.,
Sakaki, Y., Toyoda, A., Hattori, M., and Ohkuma, M. (2008a). Complete
genome of the uncultured Termite Group 1 bacteria in a single host protist cell.
Proceedings of the National Academy of Sciences, 105(14): 5555-5560.

Hongoh, Y., Sharma, V. K., Prakash, T., Noda, S., Toh, H., Taylor, T. D., Kudo, T.,
Sakaki, Y., Toyoda, A., Hattori, M., and Ohkuma, M. (2008b). Genome of an
endosymbiont coupling N2 fixation to cellulolysis within protist cells in termite
gut. Science, 322: 1109-1109.

Inoue, J. L., Saita, K., and Kudo, T., et al., (2007). Hydrogen production by termite
gut protists: characterization of iron hydrogenases of parabasalian symbionts of
the termite Coptotermes Formosanus. Eukaryote Cell, 6: 1925-1932.

Inoue, T., Kitade, O., Yoshimuta, T., and Yamaoka, I. (2000). Symbiotic associations
with protists. In Abe, T., Bignell, D. E., and Higashi, M. (Ed.) Termites:
ecolution, sociality, symbiosis, ecology (pp. 275-288). Kluwer, Dordrecht.

Inoue, T., Moriya, S., Ohkuma, M., and Kudo, T. (2005). Molecular cloning and
characterization of a cellulase gene from a symbiotic protest of the lower termite
Coptotermes formosanus. Gene, 349: 67-75.
Inoue, T., Musrashima, K., Azuma, J-I, et al., (1997). Cellulose and xylan utilization
in the lower termite Reticultimes speratus. Journal of Insect Physiology, 43: 235-
242.
Kappler, A. and Brune, A. (1990). Influence of gut alkalinity and oxygen status on
mobilization and size-class distribution of humic acids in the hindgut of soil-
feeding termites. Applied Soil Ecology, 13: 219-229.

Karl, Z. J. and Scharf, M. E. (2015). Effects of five diverse lignocellulosic diets on

digestive enzymes biochemistry in the termite Reticulitermes flavipes. Archives
of Insect Biochemistry and Physiology, 1-15.

Katsumata, K. S., Jin, Z. and Hori, K. (2007). Structural changes in lignin of tropical
woods during digestion by the termite, Cryptotermes brevis. The Japan Wood
Research Society, 53: 419-426.

61
Ke, J., Laskar, D., Gao, D., and Chen, S. (2012). Advanced biorefinery in lower
termite-effect of combined pretreatment during the chewing process.
Biotechnology for Biofuels, 5(1): 11.

Ke, J., Singh, D. and Chen, S. L. (2011). Aromatic compound degradation by the
wood-feeding termite Coptotermes formosanus (Shiraki). International
Biodeterioration and Biodegradation, 65: 744-756.

Ke, J., Sun, J. Z., Nguyen, H. D., Singh, D., Lee, K. C., Beyenal, H., and Chen, S. L.
(2010). In-situ oxygen profiling and lignin modification in guts of wood-feeding
termites. Insect Science, 17: 277-290.

Keller, L. (1998). Queen lifespan and colony characteristics in ants and termites.
Insectes Sociaux, 45: 235-236.

Khucharoenphaisan, K., Sripairoj, N. and Sinma, K. (2012). Isolation and

identification of Actinomycetes from termite’s gut against human pathogen.
Asian Journal of Animal and Veterinary Advances, 7(1): 68-73.

King, P. J. H., Mahadi, N. M., Bong, J. C. F., Ong, K. H., and Hassan, O. (2014).
Bacterial microbiome of Coptotermes curvignathus (Isoptera: Rhinotermitidae)
reflects the coevolution of species and dietary pattern. Insect Science, 21: 584-
596.

Köhler T., Dietrich C., Scheffrahn R. H., Brune A. (2012). High-resolution analysis of
gut environment and bacterial microbiota reveals functional compartmentation
of the gut in wood-feeding higher termites (Nasutitermes spp.). Applied and
Environmental Microbiology, 78: 4691-4701.

Kramer, K. J. and Seib, P. A. (1982). Ascorbic acid and the growth and development
of insects. In P. A. Seib and B. M. Tolbert (Ed.), Ascorbic Acid: Chemistry,
Metabolism and Uses (pp. 275-291). Advances in Chemistry Series No. 200,
American Chemical Society, Washington, DC.
Kudo, T. (2009). Termite-microbe symbiotic system and its efficient degradation of
lignocelluloses. Bioscience, Biotechnology and Biochemistry, 73(12): 2561-
2567.

Leadbetter, J. R. and Breznak, J. A. (1996). Physiological ecology of

ofMethanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp.
nov., isolated from the hindgut of the termite Reticulitermes flavipes. Applied
and Environmental Microbiology, 62:3620-3631.

Leadbetter, J. R., Schmidt, T. M., Graber, J. R., and Breznak, J. A. (1999).

Acetogenesis from H2 plus CO2 by spirochetes from termite guts. Science,
283:686-689.

Leonardo, F. C., da Cunha, A. F., da Silva, M. J., Carazzolle, M. F., Costa-Leonardo,

A. M., Costa, F. F., et al. (2011). Analysis of the workers head transcriptome of
the Asian subterranean termite, Coptotermes gestroi. Bulletin of Entomological
Research, 101: 383-391.

62
Leschine, S., Paster, B., and Canale-Parola, E. (2006). Free-living saccharolytic
spirochetes: the genus Spirochaeta. In Dworkin, M., Falkow, S., Rosenberg, E.,
Schleifer, K-H., Stackebrandt (Ed.), The prokaryotes 3rd ed. Vol. 7 (pp. 195-210).
Springer, New York, NY.

Levine, D. P. (2006). Vancomycin: A history. Clinical Infectious Diseases,

42(Supplement 1): S5-12.

Li, H. J., Lu, J. R. and Mo, J. C. (2012). Physiochemical lignocelluloses modification

by the formosan subterranean termite Coptotermes formosanus Shiraki (Isoptera:
Rhinotermitidae) and its potential uses in the production of biofuels.
BioResources, 7(1): 675-685.

Li, H-F., Su, N. Y., Wu, W. J., and Hsu, E-L. (2011). Termite Pests and Their Control
in Taiwan. Sociobiology, 57(3): 575-586.

Lilburn, T. G., Kim, K. S., Ostrom, N. E. et al., (2001). Nitrogen fixation by symbiotic
and free-living spirochetes. Science, 292: 2495-2498.

Lim, K.H. and Bit. S. (2001). Termite infestation on oil palms planted on deep peat in
Sarawak: Tradewinds experience. Proceedings of the 2001 PIPOC International
Palm Oil Congress, Agriculture Conference, Kuala Lumpur, Malaysia, 20-22
August, pp. 355-368.

Lo, N., Tokuda, G., Watanabe, H., Rose, H., Slaytor, M., Maekawa, K., Bandi., C. and
Noda, H. (2000). Evidence from multiple gene sequences indicates that termites
evolved from wood-feeding cockroaches. Current Biology, 10: 801-804.

Lo, N., Tokuda, G., Watanabe, h., Rose, H., Slaytor, M., Maekawa, K., Bandi, C., and
Noda, H. (2000). Evidence from multiple gene sequences indicated that termites
evolved from wood-feeding cockroaches. Current Biology, 10(12): 801-804.
Lokeshwari, R. K. and Shantibala, T. (2010). A Review on the Fascinating World of
Insect Resources: Reason for Thoughts. Psyche, Volume 2010, Article ID
207570, 11 pages.

Maistrello, L., and Sbrenna, G. (1999). Behavioral differences between male and
female replacement reproductives in Kalotermes flavicollis (Isoptera,
Kalotermitidae). Insectes Sociaux, 46: 186-191.

Mathew, G. M., Ling, S. J., Chang, J. J. and Huang, C. C. (2011). DGGE detection
and screening of lignocellulolytic bacteria from the termite gut of Coptotermes
formosanus. Malaysia Journal of Microbiology, 7(4): 201-209.

Mathews, M. C., Summers, C. B. and Felton, G. W. (1997). Ascorbate peroxidase: A

novel antioxidant enzyme in insects. Archives of Insect Biocehmistry and
physiology, 34: 57-68.

Matsuo, T. and Ishikawa, Y. (1999). Protective role of uric acid against photooxidative
stress in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Japanese
Society of Applied Entomology and Zoology, 34(4): 481-484.

63
Moellering, R. C. Jr. (2006). Vancomycin: A 50-year reassessment. Clinical Infectious
Diseases, 42(Supplement 1): S3-4.

Nakashima, K., Watanabe, H., Saitoh, H., Tokuda, G., and Azuma, J. I. (2002). Dual
cellulose-digesting system of the wood-feeding termite, Coptotermes
Formosanus Shiraki. Insect Biochemistry and Molecular Biology, 32: 777-784.

Nakayama, T., Yoshimura, T., and Imamura, Y. (2005). Feeding activities of

Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe) as
affected by moisture content of wood. Journal of Wood Science, 51, 60-65.

Ni, J. and Tokuda, G. (2013). Lignocellulose-degrading enzymes from termites and

their symbiotic microbiota. Biotechnology Advances, 31(6): 838-850.

Noda, S., Hongoh, Y., Sato, T., Ohkuma, M. (2009). Complex coevolutionary history
of symbiotic Bacteroidales bacteria of various protists in the gut of
termites. BMC Evolutionary Biology, 9:158.

Noirot, C. (1995). The gut of termites (isopteran): comparative anatomy, systematics,

phylogeny. I. Lower termites. Annales-Societe Entomologique de France, 31:
197-226.

Noirot, C. (2001). The gut of termites (Isoptera). Comparative anatomy, systematics,

phylogeny. II. Higher termites (Termitidae). Annales-Societe Entomologique de
France, 37: 431-471.

Noirot, C. and C. Noirot-Timothée, 1969. The digestive system. In: K. Krishna and
F. M. Weesner (Eds.) Biology of Termites (pp. 49-88). New York: Academic
Press.

Ohkuma, M., Noda, S., and Kudo, T. (1999). Phylogenetic diversity of nitrogen
fixation genes in the symbiotic microbial community in the gut of diverse
termites. Applied and Environmental Microbiology, 65: 4926-4934.

Ohtoko, K., Ohkuma, M., Moriya, S., et al., (2000). Diverse genes of cellulase
homologues of glycosyl hydrolase family 5 from the symbiotic protists in the
hindgut of the termite Reticulitermes speratus. Extremophiles, 4: 343-349.

Pannabecker, T. (1995). Physiology of the Malpighian tubule. Annual Reviews of

Entomology, 40: 493-510.

Payne, C. L., Scarborough, P., Rayner, M., and Nonaka, K. (2015). A systematic
review of nutrient composition data available for twelve commercially available
edible insects, and comparison with reference values. Trends in Food Science
and Technology, 47: 69–77.

Pester, M. and Brune, A. (2007). Hydrogen is the central free intermediate during
lignocellulose degradation by termite gut symbionts. ISME Journal, 1:551-565.

64
Peterson, B. F. and Scharf, M. E. (2006). Lower termite associations with microbes:
synergy, protection, and interplay. Frontiers in Microbiology, 7: 422.

Placido, J., and Capareda, S. (2015). Ligninolytic enzymes: A biotechnological

alternative for bioethanol production. Bioresource Bioprocess, 2: 23.

Potrikus, C. J. and Breznak, J. A. (1980a). Anaerobic degradation of uric acid by gut

bacteria of termites. Applied and Environmental Microbiology, 40(1): 125-132.

Potrikus, C. J. and Breznak, J. A. (1980b). Uric acid-degrading bacteria in guts of

termites [Reticulitermes flavipes (Kollar)]. Applied and Environmental
Microbiology, 40(1): 117-124.

Potrikus, C. J. and Breznak, J. A. (1980c). Uric acid in wood-eating termites. Insect

Biochemistry, 10: 19-27.

Potrikus, C. J. and Breznak, J. A. (1981). Gut bacteria recycle uric acid nitrogen in
termites: A strategy for nutrient conservation. Proceedings of the National
Academy of Sciences of the United States of America, 78(7): 4601-4605.

Quarles, W. (2007). Global warming means more pests. The IPM Practitioner, 24, 1-
8.

Radek, R. (1999). Flagellates, bacteria, and fungi associated with termites: diversity
and function in nutrition – a review. Ecotropica, 5: 183-196.

Ranković, B. R., Kosanić, M. M., Stanojković. (2011). Antioxidant, antimicrobial and

anticancer activity of the lichens Cladonia furcate, Lecanora atra and Lecanora
muralis. BMC Complementary and Alternative Medicine, 11: 97.

Rieu-Lesme, F., Dauga, C., Morvan, B., Bouvet, O. M. M., Grimont, P. A. D., and
Dore, J. (1996). Acetogenic coccoid spore-forming bacteria isolated from the
rumen. Research in Microbiology, 147: 753-764.

Roisin, Y. (2000). Diversity and evolution of caste patterns. In T. Abe, D. E. Bignell,

and M. Higashi (Eds.), Termites: Evolution, sociality, symbioses, ecology (pp.
95-119). Dordrecht: Kluwer Academic Publishers.

Rubin, E. (2008). Genomics of cellulosic biofuels. Nature, 454: 841-845.

Salmassi, T. M. and Leadbetter, J. R. (2003). Molecular aspects of CO2-reductive

acetogenesis in cultivated spirochetes and the gut community of the termite
Zootermopsis angusticollis. Microbiology, 149: 2529-2537.

Sapountzis, P., de Verges, J., Rousk, K., Cilliers, M., Vorster, B. J., and Poulsen, M.
(2016). Potential for nitrogen fixation in the fungus-growing termite symbiosis.
Frontiers in Microbiology, 7: 1993.

Scharf, M. E. (2015). Termites as targets and models for biotechnology. Annual

Reviews of Entomology. 60: 77–102.

65
Scharf, M. E. and Tartar, A. (2008). Termite digestomes as sources for novel
lignocellulases. Biofuels, Bioproducts and Biorefining, 2: 540-552.

Schmitt-Wagner, D. and Brune, A. (1999). Hydrogen profiles and localization of

methanogenic activities in the highly compartmentalized hindgut of soil-feeding
higher termites (Cubitermes spp.). Applied and Environmental Microbiology,
65: 4490-4496.

Schultz, J. E. and Breznak, J. A. (1978). Heterotrophic bacteria present in hindgutsof

wood-eating termites [Reticulitermes flavipes (Kollar)]. Applied and
Environmental Microbiology, 35(5): 930-936.

Schultz, J. E. and Breznak, J. A. (1979). Cross-feeding of lactate between

Streptococcus lactis and Bacteroides sp. isolated from termite hindguts. Applied
and Environmental Microbiology, 37(6): 1206-1210.

Senthilkumar, N., Barthakur, N. D., and Rao, L. (2008). Bioprospecting with the
reference to medicinal insects and tribes in India: an overview. Indian Forester
Journal, 12: 1575-1591.

Sethi, A., Slack, J., Kovaleva, E. B., Buchman, G. W., and Scharf, M. E. (2013).
Lignin-associated metagene expression in a lignocellulose-digesting termite.
Insect Biochemistry and Molecular Biology, 43(1): 91-101.

Shinzato, N., matsumoto, T., Yamaoka, I, Oshima, T., and Yamagishi, A. (1999).
Phylogentic diversity of symbiotic methanogens living in the hindgut of the
lower termite Reticulitermes speratus analyzed by PCR and in situ hybridization.
Applied and Environmental Microbiology, 65: 837-840.

Slaytor, M. (2000). Energy metabolism in the termite gut and its gut microbiota. In
Abe, T., Bignell, D. E., and Higashi, M. (Ed.), Termites: evolution, sociality,
symbiosis, ecology (pp. 307-332). Kluwer, Dordrecht.

Smith, J. L. and Rust, M. K. (1993). Influence of temperature on tunneling, feeding

rates, and oxygen requirements of the western subterranean termite,
Reticulitermes hesperus (Isoptera: Rhinotermitidae). Sociobiology, 21: 225-236.

Stingl, U. Radek, R., and Brune, A. (2005). “Endomicrobia”: cytoplasmic symbionts

of termite gut protozoa form a separate phylum of prokaryotes. Applied and
Environmental Microbiology, 71: 1473-1479.

Strassert, J. F. H., Kohler, T., Wienemann, T. H. G., Ikeda-Ohtsubo, W., Faivre, N.,
Franckenberg, S., et al., (2012). ‘Candidatus Ancillula trichonymphase’, a novel
lineage of endosymbiotic Actinobacteria in termite gut flagellates of the genus
Trichonympha. Environmental Microbiology, 14: 3259-3270.

Sugimoto, A., T. Inoue, I. Tayasu, L. Miller, S. Takeichi, and T. Abe. 1998. Methane
and hydrogen production in a termite-symbiont system. Ecologcal Research,
13:241-257.

66
Tartar, A., Wheeler, M. M., Zhou, X., Coy, M. R., Boucias, D. G. and Scharf, M. E.
(2009). Parallel metatranscriptome analyses of host and symbiont gene
expression in the gut of the termite Reticulitermes flavipes. Biotechnology for
Biofuel, 2: 25.

Tasaki, E., Sakurai, H., Nitao, M., Matsuura, K. and Iuchi,Y. (2017). Uric acid, an
important antioxidant contributing to survival in termites. PLoS ONE, 12(6):
e0179426.

Teather, R.N. and Wood, P.J. (1982). Use of Congo Red-Polysaccharide interactions
in enumeration and characterization of cellulolytic bacteria from the bovine
rumen. Applied Environmental Microbiology, 43: 777-780.

Terra, W. R. and Ferreira, C. (1994). Insect digestive enzymes: properties,

compartmentalization and function. Comparative Biochemistry and Physiology,
109B(1): 1-62.

Terra, W. R. and Ferreira, C. (2012). Biochemistry and molecular biology of digestion.

In L. I. Gilbert (Ed.), Insect Molecular Biology and Biochemistry (pp. 365-418).
Academic Press/Elsevier, London.

Thapa, R. S. (1981). Termites of Sabah (East Malaysia). Sabah Forest Record. 12: 1-
374.

Tho, Y. P. (1992). Termites of Peninsular Malaysia. Malayan Forest Records No. 36.
Forest Research Institute of Malaysia: 224.

Tholen, A. Schink, B., and Brune, A. (1997). The gut microflora of Reticulitermes
flavipes, its relation to oxygen, and evidence for oxygen-dependent acetogenesis
by the most abundant Enterococcus sp. FEMS Microbial Ecology, 24: 137-149.

Thompson, G. (2000). Termites (Tropical topics newsletter No. 64, pp. 1-8). Tropical
Savanna CRC, Environmental Protection Agency. The State of Queensland,
Australia.

Tian, J. H., Pourcher, A. M. and Bouchez, T. (2014). Occurrence of lignin degradation

genotypes and phenotypes among prokaryotes. Applied Microbiology and
Biotechnology, 98: 9527-9544.

Todaka, N., Lopez, C. M., and Inoue, T., et al., (2010). Heterologous expression and
characterization of an endoglucanase from a symbiotic protist of the lower
termite, Reticulitermes speratus. Applied Microbiology and Biotechnology, 160:
1168-1178.

Tokuda, G., Lo, N., Watanabe, H., Arakawa, G., Matsumoto, T., and Noda, H. (2004).
Major alteration of the expression site of endogenous cellulases in members of
an apical termite lineage. Molecular Ecology, 13: 3219-3228.

67
Tokuda, G., Nakamura, T., Murakami, R., and Yamaoka, I. (2001). Morphology of the
digestive system in the sood-feeding termite Nasutitermes takasagoensis
(Shiraki) [Isoptera: Termitidae]. Zoological Science, 18: 869-877.

Tokuda, G., Yamaoka, I. and Noda, H. (2000). Localization of symbiotic Clostridia in

the mixed segment of the termite Nasutitermes takasagoensis (Shiraki). Applied
and Enviromental Microbiology, 66(5): 2199-2207.

Tokura, M., Ohkuma, M., and Kudo, T. (2000). Molecular phylogeny of methanogens
associated with flagellated protists in the gut and with the gut epithelium of
termites. FEMS Microbial Ecology, 33: 3219-3228.

Uchima, C. A. and Arioka, M. (2012). Expression and one-step purification of

recombinant proteins using an alternative episomal vectore for the expression of
N-tagged heterologous proteins in Pichia pastoris. Bioscience, Biotechnology
and Biochemistry, 76: 368-371.

Upadhyay, R. K. (2011). Symbiotic and non-symbiotic micro flora of termite gut: a

unique nonhuman agricultural system that can recycle photo-synthetically fixed
carbon and nutrients. Journal of Pharmacy Research, 4(4): 1161-1166.

Veivers, P. C., O’Brien, R. W. and Slaytor, M. (1982). Role of bacteria in maintaining

the redox potential in the hindgut of termites and preventing entry of foreign
bacteria. Journal of Insect Physiology, 28(11): 947-951.

Veliká, B. and Kron, I. (2013). Antioxidant properties of phenols against superoxide

radicals. Chemical Monthly, 144: 1287-1290.

Wang, Q., Qian, C., Zhang, X. Z., Liu, N., Yan, X., and Zhou, Z. (2012).
Characterization of a novel thermostable β-glucosidase from a metagenoic
library of termite gut. Enzyme and Microbial Technology, 51: 319-324.

Watanabe, H. and Tokuda, G. (2010). Cellulolytic System in Insects. Annual Review

of Entomology, 55: 609-632.

Watanabe, H., Nakamura, M., Tokuda, G., Yamaoka, I., Serivener, A. M., and Noda,
H. (1997). Site of secretion and properties of endogenous endo-β-1,4-glucanase
components from Reticulitermes speratus (Kolbe), a Japanese Subterranean
termite. Insect Biochemistry and Molecular Biology, 27(4): 305-313.

Watanabe, Y., Shinzate, N, and Fukatsu, T. (2003). Isolation of actinoycetes from

termites’ guts. Bioscience, Biotechnology, and Biochemistry, 67: 1797-1801.

Wickings, K., and Grandy, A. S. (2011). The oribatid mite Scheloribates moestus
(Acari: Oribatida) alters litter chemistry and nutrient cycling during
decomposition. Soil Biology and Biochemistry, 43: 351-358.

68
Wood, T.G. (1976). The role of termites (Isoptera) in decomposition processes. In:
Anderson, J. M. and MacFayden, A. (Ed.) The Role of Terresterial and Aquatic
Organisms in Decomposition Processes (pp. 145-168). Blackwell Scientific
Publications, Oxford.

Wu, Y. R. and He, J. (2013). Characterization of anaerobic consortia coupled lignin

depolymerization with biomethane generation. Bioresource Technology, 139: 5-
12.

Yamin, M. A. (1979). Termite flagellated. Sociobiology, 4: 1-119.

Zhang, D., Lax, A. R., Bland, J. M. and Allen, A. B. (2011). Characterization of a new
endogenous endo-β-1,4-glucanase of Formosan subterranean termite
(Coptotermes formosanus). Insect Biochemistry and Molecular Biology, 41:
211-218.

Zhang, Q., Zhang, W., Lin, C. Y., Xu, X. L., and Shen, Z. C. (2012). Expression of an
Acidothermus cellulolyticus endoglucanase in transgenic rice seeds. Protein
Expression and Purification, 82: 279-283.

Zheng H., Dietrich C., Radek R., Brune A. (2015). Endomicrobium proavitum, the first
isolate of Endomicrobia class. Nov. (phylum Elusimicrobia) – an
ultramicrobacterium with an unusual cell cycle that fixes nitrogen with a Group
IV nitrogenase. Environmental Microbiology, 18: 191-204.

Zhou, X., Smith, J. A., Oi, F. M., Koehler, P. G., Bennett, G. W. and Scharf, M. E.
(2007). Correlation of cellulase gene expression and cellulolytic activity
throughout the gut of the termite Reticulitermes flavipes. Gene, 395: 29-39.

Zielińska, E., Baraniak, B., Karaś, M., Rybczyńska, K., and Jakubczyk, A. (2015).
Selected species of edible insects as a source of nutrient composition. Food
Research International, Volume 77, January, pp. 460-466.

69
 BIODATA OF STUDENT

My name is Sia Siaw Lang from Sibu, Sarawak. I graduated from Universiti Malaysia

Sabah (UMS) with a Bachelor of Agriculture Science in Livestock Production in year

2011. During my degree semester break, I used to join fellow coursemates for

internships in agriculture sector, to gain more knowledge either on machineries or

animals. After graduate, I continue pursuing postgraduate program at UPM Bintulu

Campus in the microbial research field. Throughout my research program, I assisted

supervisor in student laboratory work, at the same time I am able to gain experiences.

Currently I am working as a music educator since I wait for the completion of my

master program. Besides that, I am in the midst of considering to pursue another degree

in music field after successfully complete my UPM master degree.

70